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Research Article Genetic Diversity Analysis of Genotype 2 Porcine Reproductive and Respiratory Syndrome Viruses Emerging in Recent Years in China Lei Zhou, Xiaorong Yang, Yuan Tian, Shuoyan Yin, Gang Geng, Xinna Ge, Xin Guo, and Hanchun Yang Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of Agriculture, College of Veterinary Medicine and State Key Laboratory of Agrobiotechnology, China Agricultural University, No. 2 Yuanmingyuan West Road, Haidian District, Beijing 100193, China Correspondence should be addressed to Hanchun Yang; [email protected] Received 5 November 2013; Accepted 7 January 2014; Published 25 February 2014 Academic Editor: Raymond Rowland Copyright © 2014 Lei Zhou et al. is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by its extensive genetic diversity. Here we analyzed 101 sequences of NSP2 hypervariable region, 123 ORF3 sequences, and 118 ORF5 sequences from 128 PRRSV-positive clinical samples collected in different areas of China during 2008–early 2012. e results indicated that the amino acid identities of the three genes among these sequences were 87.6%–100%, 92.5%–100%, and 77%–100%, respectively. Meanwhile, 4 novel patterns of deletion and insertion in NSP2 region or GP5 were first found. e phylogenetic analysis on these 3 genes revealed that the Chinese PRRSV strains could be divided into three subgroups; majority of genes analyzed here were clustered in subgroup 3 with multiple branches; the strains with 30-aa deletion in NSP2-coding region were still the dominant virus in the field. Further phylogenetic analysis on four obtained complete genomic sequences showed that they were clustered into different branches with the Chinese corresponding representative strains. Our analyses suggest that the genetic diversity of genotype 2 PRRSV in the field displays a tendency of increasing in recent years in China, and the 30-aa deletion in NSP2-coding region should be no longer defined as the molecular marker of the Chinese HP-PRRSV. 1. Introduction Porcine reproductive and respiratory syndrome (PRRS) char- acterized as reproductive failure in sow and respiratory disorder in all-age pigs [1] is regarded as one of the major concerns for disease controlling in pig farms [2–5]. e first outbreak of PRRS in Western Europe and North America was almost concurrently documented during the late 1980s and early 1990s [6, 7]. Within the succeeding years, PRRS was an endemic disease in North America, Europe, and Asia [7–11]. Since then, PRRS has become the most economically devastating disease for global pig industry [4, 5]. e causal agent, porcine reproductive and respiratory syndrome virus (PRRSV), is classified into the order Nidovi- rales, family Arteriviridae, together with equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) [12, 13]. According to the genetic diversity, PRRSV can be divided into two genotypes: type 1 (European) PRRSV with prototype Lelystad and type 2 (North American) PRRSV with prototype VR- 2332. Although the two types of PRRSV can cause similar syndrome to the infected pigs, they share only 55%–70% nucleotide and 50%–80% amino acid similarity in their various genes [14]. e single positive-strand RNA genome of PRRSV is approximately 15 kb in length, encoding at least 10 open reading frames (ORF) [15–18]. e ORF1a and ORF1b encode replication-related polymerase proteins, which can be autoproteolytically cleaved into at least 13 nonstructural proteins (NSP) [19–22]. And the rest of ORFs 2 to 7 encode viral structural proteins [15, 17, 23, 24]. Among them, the largest nonstructural protein gene—NSP2, ORF3 encoding minor glycosylated structural protein—GP3, and ORF5 that encodes major envelope protein—GP5 are oſten selected for variation investigation and phylogenetic analyses for their Hindawi Publishing Corporation BioMed Research International Volume 2014, Article ID 748068, 13 pages http://dx.doi.org/10.1155/2014/748068

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Research ArticleGenetic Diversity Analysis of Genotype 2 PorcineReproductive and Respiratory Syndrome Viruses Emerging inRecent Years in China

Lei Zhou Xiaorong Yang Yuan Tian Shuoyan Yin Gang GengXinna Ge Xin Guo and Hanchun Yang

Key Laboratory of Animal Epidemiology and Zoonosis of Ministry of AgricultureCollege of Veterinary Medicine and State Key Laboratory of Agrobiotechnology China Agricultural UniversityNo 2 Yuanmingyuan West Road Haidian District Beijing 100193 China

Correspondence should be addressed to Hanchun Yang yanghanchun1caueducn

Received 5 November 2013 Accepted 7 January 2014 Published 25 February 2014

Academic Editor Raymond Rowland

Copyright copy 2014 Lei Zhou et alThis is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Porcine reproductive and respiratory syndrome virus (PRRSV) is characterized by its extensive genetic diversity Here we analyzed101 sequences of NSP2 hypervariable region 123 ORF3 sequences and 118 ORF5 sequences from 128 PRRSV-positive clinicalsamples collected in different areas of China during 2008ndashearly 2012 The results indicated that the amino acid identities of thethree genes among these sequences were 876ndash100 925ndash100 and 77ndash100 respectively Meanwhile 4 novel patterns ofdeletion and insertion in NSP2 region or GP5 were first foundThe phylogenetic analysis on these 3 genes revealed that the ChinesePRRSV strains could be divided into three subgroups majority of genes analyzed here were clustered in subgroup 3 with multiplebranches the strains with 30-aa deletion in NSP2-coding region were still the dominant virus in the field Further phylogeneticanalysis on four obtained complete genomic sequences showed that they were clustered into different branches with the Chinesecorresponding representative strains Our analyses suggest that the genetic diversity of genotype 2 PRRSV in the field displays atendency of increasing in recent years in China and the 30-aa deletion in NSP2-coding region should be no longer defined as themolecular marker of the Chinese HP-PRRSV

1 Introduction

Porcine reproductive and respiratory syndrome (PRRS) char-acterized as reproductive failure in sow and respiratorydisorder in all-age pigs [1] is regarded as one of the majorconcerns for disease controlling in pig farms [2ndash5] The firstoutbreak of PRRS in Western Europe and North Americawas almost concurrently documented during the late 1980sand early 1990s [6 7] Within the succeeding years PRRSwas an endemic disease in North America Europe and Asia[7ndash11] Since then PRRS has become the most economicallydevastating disease for global pig industry [4 5]

The causal agent porcine reproductive and respiratorysyndrome virus (PRRSV) is classified into the order Nidovi-rales familyArteriviridae togetherwith equine arteritis virus(EAV) lactate dehydrogenase-elevating virus (LDV) andsimian hemorrhagic fever virus (SHFV) [12 13] According

to the genetic diversity PRRSV can be divided into twogenotypes type 1 (European) PRRSVwith prototype Lelystadand type 2 (North American) PRRSV with prototype VR-2332 Although the two types of PRRSV can cause similarsyndrome to the infected pigs they share only 55ndash70nucleotide and 50ndash80 amino acid similarity in theirvarious genes [14]The single positive-strand RNA genome ofPRRSV is approximately 15 kb in length encoding at least 10open reading frames (ORF) [15ndash18] The ORF1a and ORF1bencode replication-related polymerase proteins which canbe autoproteolytically cleaved into at least 13 nonstructuralproteins (NSP) [19ndash22] And the rest of ORFs 2 to 7 encodeviral structural proteins [15 17 23 24] Among them thelargest nonstructural protein genemdashNSP2 ORF3 encodingminor glycosylated structural proteinmdashGP3 and ORF5 thatencodes major envelope proteinmdashGP5 are often selected forvariation investigation and phylogenetic analyses for their

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014 Article ID 748068 13 pageshttpdxdoiorg1011552014748068

2 BioMed Research International

genetic diversities [25 26]The genetically extensive variationwith geneticantigenic diverse strains in the field is regardedas an important reason for vaccination failure and occasionaloutbreaks of more severe forms of PRRS [21 26]

Since the first outbreak of PRRS in China was docu-mented at the end of 1995 [27] this disease has been accom-panying the Chinese swine industry [28] Considering Chinahas the largest number of pig farms with diversity of size anddifferent levels of biosecurity control and management theeconomical cost caused by PRRS in China should be higherthan that in theUnited States whichwas estimated to be $664million per year [4 29] Especially in 2006 a large-scale out-break caused by the highly pathogenic PRRSV (HP-PRRSV)was characterized by prolonged high fiver rubefaction on theskin and increased morbidity and mortality in all ages ofpigs resulting in unprecedented damage to theChinese swineindustry [25 30 31]Thephylogenetic analyses have indicatedthat the causative pathogen HP-PRRSV was evolved by agradual variation and accumulation progress of genomechanges from the early Chinese domestic strain [25 26] Inthe following years the HP-PRRSV has been becoming thedominant strains in the field [25] In the year 2011 the Chi-nese HP-PRRSV-derived commercial vaccines which wereattenuated by serial passaging on the MARC-145 cells wereapproved to put on the domesticmarket In the same year theEuropean PRRSV isolates were first reported in China [32]Considering the risk of potential reversion to virulence andrecombination the two events increased the complexity ofPRRSV epidemic situation in China which will attract moreattention on the molecular epidemiology analysis

In this study we phylogenetically analyzed the PRRSVNSP2 hypervariable (HV) region andORF3 and ORF5 geneswhich were directly amplified from the clinical samples col-lected from various pig farms around the pig-producing areasof China during the period from 2008 to early 2012 Simul-taneously we described the complete genomic sequences offour new Chinese PRRSV isolates including one strain fromTibet mini-pig and three sharing novel characteristic geneticvariations and compared their genetic characterization withprevious strains Finally a phylogenetic tree based on the full-length genomic sequence is conducted in order to analyze theevolutionary relationship of these strains

2 Materials and Methods

21 Sample Collection and Geographic Distribution Duringthe period from 2008 to early 2012 128 clinical samplesincluding lung brain spleen lymph node and sera whichwere positive for PRRSV by conventional laboratory detec-tion and diagnosis were collected from pig farms distributedin 18 regions of China These samples were further used forPRRSV isolation or NSP2 HV region and ORF3 and ORF5genes amplification and sequencing

22 RNA Extraction and RT-PCR Amplification andSequencing Total RNA was extracted from 250 120583L oftissue homogenates or serum by using TRIzol LS reagent(Invitrogen Corporation Auckland NY USA) Then reversetranscription was performed by using M-MLV reverse

transcriptase (Promega Madison WI USA) and specificantisense primers (Table 1) Resulting cDNA was amplifiedby using PrimeSTARHS DNA polymerase (TaKaRa Biotech-nology Co Dalian China) in the following process 34 cyclesof denaturation at 98∘C for 12 s annealing at 56∘C for 10 s andextension at 72∘C for 1minkbThe PCR products were exam-ined by gel electrophoresis and purified by using AgaroseGel DNA Extraction Kit (BioDev Co Beijing China) andthen subjected to BGI (Beijing China) for sequencing

23 Cells and Virus MARC-145 cells were grown at 37∘Cin Dulbeccorsquos minimum essential medium (DMEM) supple-mented with 10 fetal bovine serum (FBS) and antibioticsThe pulmonary alveolar macrophage (PAM) cells were pre-pared as described previously [33] andmaintained in 10FBS1640 medium Serum or supernatant of tissue homogenatesfrom PRRSV-positive samples were used to inoculate theMARC-145 cells or PAM for PRRSV isolation

24 Full-Length Genomic Sequencing of PRRSV IsolatesFourteen pairs of primers for genotype 2 PRRSV (Table 1)covering the full-length genomes were designed based onJXwn06 (Accession number EF641008) Each fragment of theisolates was amplified and cloned into pEASY-Blunt vector(Transgen Tech Co Beijing China) as described previously[34] The 51015840 and 31015840 ends region was amplified using 51015840 and31015840 full RACE kit (TaKaRa Dalian China) according to themanufacturerrsquos instructions The PCR products or plasmidwith cloned PRRSV fragments was subjected to BGI (BeijingChina) for sequencing

25 Sequence Alignment and Phylogenetic Analysis Thenucleotide and deduced amino acid sequences were alignedby ClustalW in software Lasergene (DNASTAR Inc Madi-son WI USA) to determine sequence homology And phy-logenetic and molecular evolutionary analyses were con-ducted using MEGA version 5 (Tamura Peterson Peter-son Stecher Nei and Kumar 2011) along with multiplesequences of representative PRRSV available in GenBankfrom various countries and areas (Supplementary Table S1see Table S1 in Supplementary Material available online athttpdxdoiorg1011552014748068)

3 Results

31 Number of NSP2 HV Fragment and ORF3 and ORF5Genes Amplified from PRRSV-Positive Clinical Samples Thefragments of NSP2 HV region and ORF3 and ORF5 genesamplified fromPRRSV-positive samples were sequencedTheresults showed that totally 101 NSP2 123 ORF3 and 118ORF5 sequenceswere successfully obtained from 128 PRRSV-positive samples collected during the period from 2008 toearly 2012 (Table 2)

32 Sequence Alignment and Phylogenetic Analysis of NSP2HVRegion TheamplifiedNSP2HV region exhibited varioussizes in length (Table 2) Nucleotide and deduced amino acidsequences analysis revealed that 86 out of 101 NSP2 HV

BioMed Research International 3

Table 1 Primers used for amplification and sequencing of PRRSV genome and NSP2 HV region and ORF3 and ORF5 genes

Name Primer sequence Locationlowast Length (bp)51015840-Outer-R TTTCACTTCATCCCCACCAC 800 81951015840-Inner-R CCCCGTTCATAAACTTGTAGAG 542 563W1F ATGACGTATAGGTGTTGGCTCT 1W1R TACTCTTTCAGGAAGGGTGG 1575 1575W2F ACGCTCTGGTGCGACTACTA 1362W2R AGGTTGTTCGGTTGTCTGATT 2253 892W3F CCTCCGTGGCGCAACAAGTCTTG 2115W3R CGATGATGGCTTGAGCTGAGTAT 3178 1064W4F TGAGCCTCTGGATTTGTCTGC 2949W4R GGCGATCTCATTAGGAGCAGTT 4329 1381W5F TGCTTAGGCTTGGCATTGTTG 4214W5R ACGGTGTTCAGTGAGGGCTTT 5564 1351W6F ACTAACATTGCTGGTCTCGTCA 5350W6R AAGGAAATCCAAGTCCTCGTC 6750 1401W7F TTGTGACCTCGCCAGTCCCAGTG 6500W7R CCAAAGCGTGCCATCAATCCC 7922 1423W8F GGTTGATGGTGGTGTTGTGCT 7787W8R GTCTTCTTTGGGTCCGTCTGG 9226 1440W9F TGGTCACCCTCATGGCCTTCT 9038W9R CAAATACATAGCAATGGGAGTCAAA 10323 1286W10F TTCCTGGATGAAGCGGCGTAT 10194W10R AACTCGGATGTATGAGGCGTAG 11573 1380W11F GGTGCTGGAAAGTGATGTTGG 11440W11R AAAGCGGGCATACCGTGTAAT 12774 1335W12F AGTGGTTTGGATGTGGTGGCT 12400W12R TGTTGTTGTTGCTGGCGTTGA 13803 1404W13F ATGTGCGACTGCTTCATTTCA 13599W13R TTTGCTGCTTGCCGTTGTTAT 14826 1228W14F TCCACTACGGTCAACGGCACAT 14702W14R GGATCCGGTACCTCTAGATCAGA 63731015840race adaptor-T GGATCCGGTACCTCTAGATCAGATTTTTTTTTTTTTTTTTTNsp2-F CCTCCGTGGTGCAACAAATCTTG 2115Nsp2-R CGATGATGGCTTGAGCTGAGTAT 3178 10641154ORF3-F CAGGGTCAAATGTAACCATAGTG 12506ORF3-R GGCAAGAAGAAAGCATGAGGAG 13457 952ORF5-F AGCCTGTCTTTTTGCCATTCT 13654ORF5-R CTTTTGTGGAGCCGTGCTATC 14335 682ORF7F TGATAACCACGCATTTGTCG 14668ORF7R GCCATTCACCACACATTCTTC 15228 561lowastThe location is according to the genomic sequences of JXwn06 (GenBank Accession number EF641008)

region sequences had the same length of 1014 nucleotide (nt)containing the same 30-aa deletion at aa 482 and aa 533ndash561as JXwn06 and other HP-PRRSV strains compared with thetype 2 prototype VR-2332 and the Chinese early strains TheLN1101 and GZ1101 showed two novel deletion patterns intheir NSP2 regions whose nucleotide sequences length was1050 nt and 1095 nt respectivelyThe other 13NSP2 sequenceswere 1104 nt in length same as those of VR-2332

Pairwise comparisons showed that those 86 sequenceswith 30-aa deletions in NSP2 shared 876ndash100 amino acid

similarity with each other And their amino acid similaritieswith JXwn06 ranged from 917 to 994 as well as 666to 695 compared with VR-2332 Majority of the sequenceswithout deletion shared high homology with HB-1(sh)2002showing the amino acid similarity of 981ndash992 Mean-while the JL1101 and GZ1101 displayed the highest homologywith VR-2332 with amino acid similarities of 992 and967 respectively

To further gain a better understanding of the geneticrelationship the phylogenetic analysis based on deduced

4 BioMed Research International

Table2Geographico

rigin

andam

plified

sequ

ence

sizefrom

clinicalsam

ples

inthisstu

dy

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

1AHQS0901

200908

Anh

ui1014

765

603

65HB0

908

200912

Hebei

1014

765

NA

2AHQS0902

200908

Anh

uiNA

765

603

66HB0

909

200912

Hebei

1014

765

603

3AHQS0903

200908

Anh

ui1014

765

603

67HB1001

201004

Hebei

1014

765

603

4AHQS0905

200908

Anh

ui1014

765

603

68HB1002

201004

Hebei

1014

765

603

5AHQS0906

200908

Anh

uiNA

765

603

69HB1101

201101

Hebei

1104

765

603

6AHQS0907

200908

Anh

ui1014

765

603

70HB1102

201101

Hebei

1014

765

603

7AHQS0908

200908

Anh

uiNA

765

603

71HB1103

201111

Hebei

1104

765

603

8AHQS0909

200908

Anh

uiNA

765

603

72HB1104

201111

Hebei

1014

765

603

9AHQS0910

200908

Anh

uiNA

765

603

73HB1105

201103

Hebei

1014

765

603

10AHSS0901

200908

Anh

ui1014

765

603

74HB1106

201104

Hebei

1014

765

NA

11AHSS0902

200908

Anh

ui1014

765

603

75HB1201

201205

Hebei

1014

765

603

12AHSS0903

200908

Anh

ui1014

765

603

76HEN

1001

201003

Henan

NA

NA

603

13AHSS0904

200908

Anh

uiNA

765

603

77HEN

1002

201011

Henan

1014

765

603

14AHSS0905

200908

Anh

uiNA

765

603

78HEN

1003

201011

Henan

1014

765

603

15BJ0803

200804

Beijing

1014

765

603

79HEN

1101

201101

Henan

1014

765

603

16BJ0804

200809

Beijing

1014

765

603

80HEN

1102

201109

Henan

1104

765

603

17BJ0805

200809

Beijing

1014

765

603

81HEN

1103

201109

Henan

1014

765

603

18BJ0806

200801

Beijing

1014

765

603

82HLJ1101

201109

Heilong

jiang

1014

765

603

19BJ0807

200801

Beijing

1014

765

603

83HLJ1102

201109

Heilong

jiang

1014

765

603

20BJ0808

200801

Beijing

NA

765

603

84HLJ110

3201109

Heilong

jiang

1014

765

603

21BJ0809

200801

Beijing

NA

765

603

85HLJ1104

201109

Heilong

jiang

1014

765

603

22BJ0810

200811

Beijing

1104

765

603

86HN0901

200903

Henan

1014

765

NA

23BJ0901

200902

Beijing

1014

765

603

87HN0902

200905

Henan

1014

765

603

24BJ0902

200902

Beijing

1014

765

603

88HN0903

200909

Henan

1104

765

603

25BJ0903

200904

Beijing

NA

765

603

89HuB

1001

201008

Hub

ei1014

765

603

26BJ0904

200905

Beijing

1014

NA

NA

90HuN

1001

201008

Hun

an110

4765

NA

27BJ0905

200905

Beijing

1104

765

603

91HuN

1002

201012

Hun

an1014

765

603

28BJ0906

200905

Beijing

NA

765

603

92HuN

1003

201012

Hun

an1014

765

603

29BJ0907

200905

Beijing

NA

NA

603

93HuN

1101

201111

Hun

an1014

765

603

30BJ0908

200906

Beijing

1014

765

603

94HuN

1102

201101

Hun

an1014

NA

603

31BJ0909

200909

Beijing

1104

765

603

95JL110

1201101

Jilin

1104

765

603

32BJ0910

200911

Beijing

NA

765

603

96JL110

2201103

Jilin

1014

765

603

33BJ0911

200912

Beijing

NA

765

603

97JX1001

201008

Jiang

xi1014

765

603

BioMed Research International 5

Table2Con

tinued

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

34BJ0912

200912

Beijing

NA

765

603

98JX1002

201009

Jiang

xi1014

765

603

35BJ1001

201003

Beijing

1014

765

603

99LN

0801

200801

Liaoning

1014

765

603

36BJ1002

201003

Beijing

1104

765

603

100

LN0901

200906

Liaoning

1014

765

603

37BJ1003

201005

Beijing

1014

765

603

101

LN0902

200911

Liaoning

1014

765

603

38BJ1004

201007

Beijing

1014

765

603

102

LN1001

201009

Liaoning

1014

765

603

39BJ1005

201007

Beijing

1014

765

603

103

LN110

1201101

Liaoning

1050

765

603

40BJ1006

201008

Beijing

1104

765

603

104

NMG0801

200809

Inner

Mon

golia

NA

765

NA

41BJ1007

201009

Beijing

1014

765

603

105

NMG0802

200809

Inner

Mon

golia

NA

765

NA

42BJ1008

201009

Beijing

1014

765

603

106

SC0801

200801

Sichuan

1014

765

603

43BJ1009

201011

Beijing

NA

765

NA

107

SC110

1201103

Sichuan

1014

765

NA

44BJ1010

201011

Beijing

1014

765

603

108

SC1201

201204

Sichuan

1014

765

603

45BJ110

1201101

Beijing

1014

765

603

109

SD0901

200904

Shando

ng1014

765

603

46BJ110

2201101

Beijing

1014

765

603

110SD

0902

200912

Shando

ng1014

765

603

47GX1001

201006

Guang

xi1014

765

603

111SD

0903

200912

Shando

ng1014

765

603

48GX1002

201006

Guang

xi1014

765

603

112

SD0904

200912

Shando

ng1014

765

603

49GZ1001

201009

Guizhou

1014

765

603

113SD

0905

200912

Shando

ng1014

765

603

50GZ1002

201011

Guizhou

1014

765

603

114SD

0906

200912

Shando

ng1014

765

603

51GZ1101

201103

Guizhou

1095

765

600

115SD

1001

201003

Shando

ng1014

765

603

52HB0

801

200807

Hebei

NA

765

603

116

SD1002

201003

Shando

ngNA

765

603

53HB0

802

200801

Hebei

1014

765

603

117SD

1003

201011

Shando

ng1014

765

603

54HB0

803

200801

Hebei

1014

765

603

118SD

1004

201011

Shando

ng1014

765

603

55HB0

804

200801

Hebei

NA

765

603

119SD

1101

201103

Shando

ng1014

765

603

56HB0

805

200801

Hebei

NA

NA

603

120

SX1101

201105

Shanxi

1014

765

603

57HB0

806

200811

Hebei

1014

765

603

121

TJ0901

200911

Tianjin

NA

765

603

58HB0

901

200903

Hebei

NA

765

NA

122

TJ0902

200911

Tianjin

NA

765

603

59HB0

902

200903

Hebei

1014

765

603

123

TJ0903

200911

Tianjin

NA

765

603

60HB0

903

200903

Hebei

1014

765

603

124

TJ110

1201104

Tianjin

1014

765

603

61HB0

904

200904

Hebei

1014

765

603

125

ZJ1001

201001

Zhejiang

1014

765

603

62HB0

905

200911

Hebei

1104

765

603

126

ZJ1101

201102

Zhejiang

1014

765

603

63HB0

906

200911

Hebei

1104

765

603

127

ZJ110

2201104

Zhejiang

1014

765

603

64HB0

907

200911

Hebei

1014

765

603

128

ZJ1103

201104

Zhejiang

1014

765

603

lowast

Each

samplew

asnamed

accordingto

ther

egionandcollectionyearNAnot

amplified

6 BioMed Research International

amino acid sequence of NSP2 HV region was conducted byusing the 101 NSP2 sequences obtained in this study togetherwith downloaded representative sequences (SupplementaryTable S1) The phylogenetic tree revealed that all 101 NSP2sequences belonged to genotype 2 of PRRSV and all ChinesePRRSV strains could be classified into three main subgroups(Figure 1) The JL1101 and GZ1101 were located in subgroup 1with the representative strains VR-2332 BJ-4 and RespPRRSMLV the other 99 were clustered into the subgroup 3 withmultiple branches together with the representative strainsHB-1(sh)2002 JXwn06 JXA1 and JXA1 P80 No strains inthis study were clustered into subgroup 2 with representativestrain CH-1a the earliest Chinese strain This means that thegenetic diversity of NSP2 still existed and the strains with 30-aa deletion inNSP2-coding region remain to be the dominantviruses in the field Compared with the data from 2006 to2007 the percentage of NSP2-deleted strains increased [25]However these subgroups did not appear to be associatedwith epidemiological features based on geography or date

Interestingly a minor branch with JXA1 P80 the HP-PRRSV JXA1 derived vaccine strain was observed in theNSP2 phylogenetic tree Four strainsHB1105 HB1201 SC1101and BJ1101 collected later than the year 2011 when theJXA1-derived vaccine was launched commercially were alsoclustered in this branch whereas the parental strain JXA1was out of this branch suggesting that there is the possibilitythat the four strains directly derived from the vaccine strainJAX1 P80However few earlier strains were also clustered intothis minor branch Even though the analysis from this studydoes not fully reflect that a great number of emergence ofPRRSV were due to the use of HP-PRRSV-derived MLV thepotential risk of the reversion of MLV to virulent strains andthe recombination between the vaccine virus and field virusesare worthy to pay more attention to in the future [35]

33 Sequence Alignment and Phylogenetic Analysis of ORF3Gene All the obtainedORF3 genes in this study had the samesize of 725 nt The sequences alignments indicated that theyshared 925ndash100 amino acid similarity with each otherand 894ndash953 amino acid similarity with JXwn06 as wellas 807ndash850 with VR-2332 The regions residues 33ndash46120ndash133 and 162ndash198 were conserved among these strainsotherwise majority of amino acid substitutions were locatedin two hypervariable regions the residues 58ndash71 and 216ndash226 Especially 63 out of 123 contained the I66-T66mutationcomparing with those in JXwn06 and VR-2332

The phylogenetic analysis of deduced amino acidsequences of ORF3 indicated that all Chinese genotype 2strains were distributed into three subgroups (Figure 2)Three genes JL1101 HB1103 and GZ1101 were clustered intosubgroup 1 with the representative strains VR-2332 and BJ-4and no strains in this study were clustered into subgroup 2with the representative strains CH-1a HB-1(sh)2002 andHB-2(sh)2002 All the other strains were clustered intosubgroup 3 which contained most Chinese strains collectedlater than 2004

34 Sequence Alignment and Phylogenetic Analysis of ORF5Gene Except for the GZ1101 which had one amino acid

deletion at the position aa 34 in ORF5-coding region theother 117 genes had the same size of 603 nt as that of VR-2332Sequences alignments showed that the amino acid similarityamong the 117 ORF5 genes ranged from 770 to 100 andthey shared 78ndash99 amino acid similarity with VR-2332 aswell as 865ndash99 with JXwn06 Similar as previous reportthe residue 3ndash39 the putative signal sequence was the mostvariable region whereas the regions 40ndash57 67ndash90 107ndash120138ndash160 and 165ndash184were relatively conserved [25]Howevera novel substitution E170-G170 which was conserved in theChinese strains collected during the period from 2006 to2007 was observed in recent strains

The phylogenetic tree conducted by using the deducedamino acid sequences of ORF5 showed that the Chinesestrains of genotype 2 PRRSV could be divided into 3 differ-ent subgroups (Figure 3) Three strains JL1101 HB1103 andGZ1101 were in subgroup 1 with the representative strainsVR-2332 BJ-4 and CH-1a and the SD1003 was the onlystrain clustered in subgroup 2 with the representative strainMN184A all other 115 strains were clustered into subgroup3 with multiple branches which were completely composedof Chinese strains with the representative JXwn06 and HB-1(sh)2002 Similar to the NSP2 phylogenetic tree a minorbranchwith the JXA1 P80 contained the strains collected bothearlier and later than 2011

35 Full-Length Genomic Analysis of 4 New PRRSV IsolatesThree strains SD0901 LN1101 and GZ1101 with charac-teristic deletion or insertion in NSP2 or ORF5 genes andanother strain BJ1102 were successfully isolated from theclinical samples using MARC-145 cells or PAMs The fourstrains were subjected to full-length genomic sequencingafter plaque purification of three rounds The SD0901 (Gen-Bank Accession number NJ256115) and BJ1102 (GenBankAccession number KF751237) shared same size of completegenome with 15320 nt in length excluding the ploy (A) tailsThe genome sizes of LN1101 (GenBank Accession numberKF751238) and GZ1101 were 15356 nt and 15404 nt respec-tively The BJ1102 was isolated from clinical samples of Tibetmini-pig with acute PRRS symptom in a pig farm whereHP-PRRSV-derived vaccine was used before importing Tibetmini-pig

Sequence alignments indicated that the 51015840UTRof the fourstrains shared nucleotide identities of 910ndash100 with therepresentative genotype 2 PRRSV strains A nucleotide ldquoArdquoinsertion at the position nt 75 of GZ1101 51015840UTR was firstobserved in this study It was shown that major variationswere located in NSP2-coding region including 3-aa deletionat the position aa 593ndash595 in GZ1101 18-aa deletion at theposition aa 482ndash499 in LN1101 30-aa deletion at the positionsaa 482 and aa 533ndash561 in BJ1102 and 31-aa deletion at thepositions aa 468 aa 482 and aa 533ndash561 and an amino acidldquoPrdquo insertion between aa 585 and aa 586 in SD0901 (Figure 4)In addition a new deletion at the position aa 34 of GP5 wasfound inGZ1101 (Figure 5)The individual homology analysisof the other genes was also summarized in SupplementaryTables S2ndashS5

To further classify the evolutionary relationship of these4 isolates the phylogenetic tree was conducted based on their

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 2: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

2 BioMed Research International

genetic diversities [25 26]The genetically extensive variationwith geneticantigenic diverse strains in the field is regardedas an important reason for vaccination failure and occasionaloutbreaks of more severe forms of PRRS [21 26]

Since the first outbreak of PRRS in China was docu-mented at the end of 1995 [27] this disease has been accom-panying the Chinese swine industry [28] Considering Chinahas the largest number of pig farms with diversity of size anddifferent levels of biosecurity control and management theeconomical cost caused by PRRS in China should be higherthan that in theUnited States whichwas estimated to be $664million per year [4 29] Especially in 2006 a large-scale out-break caused by the highly pathogenic PRRSV (HP-PRRSV)was characterized by prolonged high fiver rubefaction on theskin and increased morbidity and mortality in all ages ofpigs resulting in unprecedented damage to theChinese swineindustry [25 30 31]Thephylogenetic analyses have indicatedthat the causative pathogen HP-PRRSV was evolved by agradual variation and accumulation progress of genomechanges from the early Chinese domestic strain [25 26] Inthe following years the HP-PRRSV has been becoming thedominant strains in the field [25] In the year 2011 the Chi-nese HP-PRRSV-derived commercial vaccines which wereattenuated by serial passaging on the MARC-145 cells wereapproved to put on the domesticmarket In the same year theEuropean PRRSV isolates were first reported in China [32]Considering the risk of potential reversion to virulence andrecombination the two events increased the complexity ofPRRSV epidemic situation in China which will attract moreattention on the molecular epidemiology analysis

In this study we phylogenetically analyzed the PRRSVNSP2 hypervariable (HV) region andORF3 and ORF5 geneswhich were directly amplified from the clinical samples col-lected from various pig farms around the pig-producing areasof China during the period from 2008 to early 2012 Simul-taneously we described the complete genomic sequences offour new Chinese PRRSV isolates including one strain fromTibet mini-pig and three sharing novel characteristic geneticvariations and compared their genetic characterization withprevious strains Finally a phylogenetic tree based on the full-length genomic sequence is conducted in order to analyze theevolutionary relationship of these strains

2 Materials and Methods

21 Sample Collection and Geographic Distribution Duringthe period from 2008 to early 2012 128 clinical samplesincluding lung brain spleen lymph node and sera whichwere positive for PRRSV by conventional laboratory detec-tion and diagnosis were collected from pig farms distributedin 18 regions of China These samples were further used forPRRSV isolation or NSP2 HV region and ORF3 and ORF5genes amplification and sequencing

22 RNA Extraction and RT-PCR Amplification andSequencing Total RNA was extracted from 250 120583L oftissue homogenates or serum by using TRIzol LS reagent(Invitrogen Corporation Auckland NY USA) Then reversetranscription was performed by using M-MLV reverse

transcriptase (Promega Madison WI USA) and specificantisense primers (Table 1) Resulting cDNA was amplifiedby using PrimeSTARHS DNA polymerase (TaKaRa Biotech-nology Co Dalian China) in the following process 34 cyclesof denaturation at 98∘C for 12 s annealing at 56∘C for 10 s andextension at 72∘C for 1minkbThe PCR products were exam-ined by gel electrophoresis and purified by using AgaroseGel DNA Extraction Kit (BioDev Co Beijing China) andthen subjected to BGI (Beijing China) for sequencing

23 Cells and Virus MARC-145 cells were grown at 37∘Cin Dulbeccorsquos minimum essential medium (DMEM) supple-mented with 10 fetal bovine serum (FBS) and antibioticsThe pulmonary alveolar macrophage (PAM) cells were pre-pared as described previously [33] andmaintained in 10FBS1640 medium Serum or supernatant of tissue homogenatesfrom PRRSV-positive samples were used to inoculate theMARC-145 cells or PAM for PRRSV isolation

24 Full-Length Genomic Sequencing of PRRSV IsolatesFourteen pairs of primers for genotype 2 PRRSV (Table 1)covering the full-length genomes were designed based onJXwn06 (Accession number EF641008) Each fragment of theisolates was amplified and cloned into pEASY-Blunt vector(Transgen Tech Co Beijing China) as described previously[34] The 51015840 and 31015840 ends region was amplified using 51015840 and31015840 full RACE kit (TaKaRa Dalian China) according to themanufacturerrsquos instructions The PCR products or plasmidwith cloned PRRSV fragments was subjected to BGI (BeijingChina) for sequencing

25 Sequence Alignment and Phylogenetic Analysis Thenucleotide and deduced amino acid sequences were alignedby ClustalW in software Lasergene (DNASTAR Inc Madi-son WI USA) to determine sequence homology And phy-logenetic and molecular evolutionary analyses were con-ducted using MEGA version 5 (Tamura Peterson Peter-son Stecher Nei and Kumar 2011) along with multiplesequences of representative PRRSV available in GenBankfrom various countries and areas (Supplementary Table S1see Table S1 in Supplementary Material available online athttpdxdoiorg1011552014748068)

3 Results

31 Number of NSP2 HV Fragment and ORF3 and ORF5Genes Amplified from PRRSV-Positive Clinical Samples Thefragments of NSP2 HV region and ORF3 and ORF5 genesamplified fromPRRSV-positive samples were sequencedTheresults showed that totally 101 NSP2 123 ORF3 and 118ORF5 sequenceswere successfully obtained from 128 PRRSV-positive samples collected during the period from 2008 toearly 2012 (Table 2)

32 Sequence Alignment and Phylogenetic Analysis of NSP2HVRegion TheamplifiedNSP2HV region exhibited varioussizes in length (Table 2) Nucleotide and deduced amino acidsequences analysis revealed that 86 out of 101 NSP2 HV

BioMed Research International 3

Table 1 Primers used for amplification and sequencing of PRRSV genome and NSP2 HV region and ORF3 and ORF5 genes

Name Primer sequence Locationlowast Length (bp)51015840-Outer-R TTTCACTTCATCCCCACCAC 800 81951015840-Inner-R CCCCGTTCATAAACTTGTAGAG 542 563W1F ATGACGTATAGGTGTTGGCTCT 1W1R TACTCTTTCAGGAAGGGTGG 1575 1575W2F ACGCTCTGGTGCGACTACTA 1362W2R AGGTTGTTCGGTTGTCTGATT 2253 892W3F CCTCCGTGGCGCAACAAGTCTTG 2115W3R CGATGATGGCTTGAGCTGAGTAT 3178 1064W4F TGAGCCTCTGGATTTGTCTGC 2949W4R GGCGATCTCATTAGGAGCAGTT 4329 1381W5F TGCTTAGGCTTGGCATTGTTG 4214W5R ACGGTGTTCAGTGAGGGCTTT 5564 1351W6F ACTAACATTGCTGGTCTCGTCA 5350W6R AAGGAAATCCAAGTCCTCGTC 6750 1401W7F TTGTGACCTCGCCAGTCCCAGTG 6500W7R CCAAAGCGTGCCATCAATCCC 7922 1423W8F GGTTGATGGTGGTGTTGTGCT 7787W8R GTCTTCTTTGGGTCCGTCTGG 9226 1440W9F TGGTCACCCTCATGGCCTTCT 9038W9R CAAATACATAGCAATGGGAGTCAAA 10323 1286W10F TTCCTGGATGAAGCGGCGTAT 10194W10R AACTCGGATGTATGAGGCGTAG 11573 1380W11F GGTGCTGGAAAGTGATGTTGG 11440W11R AAAGCGGGCATACCGTGTAAT 12774 1335W12F AGTGGTTTGGATGTGGTGGCT 12400W12R TGTTGTTGTTGCTGGCGTTGA 13803 1404W13F ATGTGCGACTGCTTCATTTCA 13599W13R TTTGCTGCTTGCCGTTGTTAT 14826 1228W14F TCCACTACGGTCAACGGCACAT 14702W14R GGATCCGGTACCTCTAGATCAGA 63731015840race adaptor-T GGATCCGGTACCTCTAGATCAGATTTTTTTTTTTTTTTTTTNsp2-F CCTCCGTGGTGCAACAAATCTTG 2115Nsp2-R CGATGATGGCTTGAGCTGAGTAT 3178 10641154ORF3-F CAGGGTCAAATGTAACCATAGTG 12506ORF3-R GGCAAGAAGAAAGCATGAGGAG 13457 952ORF5-F AGCCTGTCTTTTTGCCATTCT 13654ORF5-R CTTTTGTGGAGCCGTGCTATC 14335 682ORF7F TGATAACCACGCATTTGTCG 14668ORF7R GCCATTCACCACACATTCTTC 15228 561lowastThe location is according to the genomic sequences of JXwn06 (GenBank Accession number EF641008)

region sequences had the same length of 1014 nucleotide (nt)containing the same 30-aa deletion at aa 482 and aa 533ndash561as JXwn06 and other HP-PRRSV strains compared with thetype 2 prototype VR-2332 and the Chinese early strains TheLN1101 and GZ1101 showed two novel deletion patterns intheir NSP2 regions whose nucleotide sequences length was1050 nt and 1095 nt respectivelyThe other 13NSP2 sequenceswere 1104 nt in length same as those of VR-2332

Pairwise comparisons showed that those 86 sequenceswith 30-aa deletions in NSP2 shared 876ndash100 amino acid

similarity with each other And their amino acid similaritieswith JXwn06 ranged from 917 to 994 as well as 666to 695 compared with VR-2332 Majority of the sequenceswithout deletion shared high homology with HB-1(sh)2002showing the amino acid similarity of 981ndash992 Mean-while the JL1101 and GZ1101 displayed the highest homologywith VR-2332 with amino acid similarities of 992 and967 respectively

To further gain a better understanding of the geneticrelationship the phylogenetic analysis based on deduced

4 BioMed Research International

Table2Geographico

rigin

andam

plified

sequ

ence

sizefrom

clinicalsam

ples

inthisstu

dy

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

1AHQS0901

200908

Anh

ui1014

765

603

65HB0

908

200912

Hebei

1014

765

NA

2AHQS0902

200908

Anh

uiNA

765

603

66HB0

909

200912

Hebei

1014

765

603

3AHQS0903

200908

Anh

ui1014

765

603

67HB1001

201004

Hebei

1014

765

603

4AHQS0905

200908

Anh

ui1014

765

603

68HB1002

201004

Hebei

1014

765

603

5AHQS0906

200908

Anh

uiNA

765

603

69HB1101

201101

Hebei

1104

765

603

6AHQS0907

200908

Anh

ui1014

765

603

70HB1102

201101

Hebei

1014

765

603

7AHQS0908

200908

Anh

uiNA

765

603

71HB1103

201111

Hebei

1104

765

603

8AHQS0909

200908

Anh

uiNA

765

603

72HB1104

201111

Hebei

1014

765

603

9AHQS0910

200908

Anh

uiNA

765

603

73HB1105

201103

Hebei

1014

765

603

10AHSS0901

200908

Anh

ui1014

765

603

74HB1106

201104

Hebei

1014

765

NA

11AHSS0902

200908

Anh

ui1014

765

603

75HB1201

201205

Hebei

1014

765

603

12AHSS0903

200908

Anh

ui1014

765

603

76HEN

1001

201003

Henan

NA

NA

603

13AHSS0904

200908

Anh

uiNA

765

603

77HEN

1002

201011

Henan

1014

765

603

14AHSS0905

200908

Anh

uiNA

765

603

78HEN

1003

201011

Henan

1014

765

603

15BJ0803

200804

Beijing

1014

765

603

79HEN

1101

201101

Henan

1014

765

603

16BJ0804

200809

Beijing

1014

765

603

80HEN

1102

201109

Henan

1104

765

603

17BJ0805

200809

Beijing

1014

765

603

81HEN

1103

201109

Henan

1014

765

603

18BJ0806

200801

Beijing

1014

765

603

82HLJ1101

201109

Heilong

jiang

1014

765

603

19BJ0807

200801

Beijing

1014

765

603

83HLJ1102

201109

Heilong

jiang

1014

765

603

20BJ0808

200801

Beijing

NA

765

603

84HLJ110

3201109

Heilong

jiang

1014

765

603

21BJ0809

200801

Beijing

NA

765

603

85HLJ1104

201109

Heilong

jiang

1014

765

603

22BJ0810

200811

Beijing

1104

765

603

86HN0901

200903

Henan

1014

765

NA

23BJ0901

200902

Beijing

1014

765

603

87HN0902

200905

Henan

1014

765

603

24BJ0902

200902

Beijing

1014

765

603

88HN0903

200909

Henan

1104

765

603

25BJ0903

200904

Beijing

NA

765

603

89HuB

1001

201008

Hub

ei1014

765

603

26BJ0904

200905

Beijing

1014

NA

NA

90HuN

1001

201008

Hun

an110

4765

NA

27BJ0905

200905

Beijing

1104

765

603

91HuN

1002

201012

Hun

an1014

765

603

28BJ0906

200905

Beijing

NA

765

603

92HuN

1003

201012

Hun

an1014

765

603

29BJ0907

200905

Beijing

NA

NA

603

93HuN

1101

201111

Hun

an1014

765

603

30BJ0908

200906

Beijing

1014

765

603

94HuN

1102

201101

Hun

an1014

NA

603

31BJ0909

200909

Beijing

1104

765

603

95JL110

1201101

Jilin

1104

765

603

32BJ0910

200911

Beijing

NA

765

603

96JL110

2201103

Jilin

1014

765

603

33BJ0911

200912

Beijing

NA

765

603

97JX1001

201008

Jiang

xi1014

765

603

BioMed Research International 5

Table2Con

tinued

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

34BJ0912

200912

Beijing

NA

765

603

98JX1002

201009

Jiang

xi1014

765

603

35BJ1001

201003

Beijing

1014

765

603

99LN

0801

200801

Liaoning

1014

765

603

36BJ1002

201003

Beijing

1104

765

603

100

LN0901

200906

Liaoning

1014

765

603

37BJ1003

201005

Beijing

1014

765

603

101

LN0902

200911

Liaoning

1014

765

603

38BJ1004

201007

Beijing

1014

765

603

102

LN1001

201009

Liaoning

1014

765

603

39BJ1005

201007

Beijing

1014

765

603

103

LN110

1201101

Liaoning

1050

765

603

40BJ1006

201008

Beijing

1104

765

603

104

NMG0801

200809

Inner

Mon

golia

NA

765

NA

41BJ1007

201009

Beijing

1014

765

603

105

NMG0802

200809

Inner

Mon

golia

NA

765

NA

42BJ1008

201009

Beijing

1014

765

603

106

SC0801

200801

Sichuan

1014

765

603

43BJ1009

201011

Beijing

NA

765

NA

107

SC110

1201103

Sichuan

1014

765

NA

44BJ1010

201011

Beijing

1014

765

603

108

SC1201

201204

Sichuan

1014

765

603

45BJ110

1201101

Beijing

1014

765

603

109

SD0901

200904

Shando

ng1014

765

603

46BJ110

2201101

Beijing

1014

765

603

110SD

0902

200912

Shando

ng1014

765

603

47GX1001

201006

Guang

xi1014

765

603

111SD

0903

200912

Shando

ng1014

765

603

48GX1002

201006

Guang

xi1014

765

603

112

SD0904

200912

Shando

ng1014

765

603

49GZ1001

201009

Guizhou

1014

765

603

113SD

0905

200912

Shando

ng1014

765

603

50GZ1002

201011

Guizhou

1014

765

603

114SD

0906

200912

Shando

ng1014

765

603

51GZ1101

201103

Guizhou

1095

765

600

115SD

1001

201003

Shando

ng1014

765

603

52HB0

801

200807

Hebei

NA

765

603

116

SD1002

201003

Shando

ngNA

765

603

53HB0

802

200801

Hebei

1014

765

603

117SD

1003

201011

Shando

ng1014

765

603

54HB0

803

200801

Hebei

1014

765

603

118SD

1004

201011

Shando

ng1014

765

603

55HB0

804

200801

Hebei

NA

765

603

119SD

1101

201103

Shando

ng1014

765

603

56HB0

805

200801

Hebei

NA

NA

603

120

SX1101

201105

Shanxi

1014

765

603

57HB0

806

200811

Hebei

1014

765

603

121

TJ0901

200911

Tianjin

NA

765

603

58HB0

901

200903

Hebei

NA

765

NA

122

TJ0902

200911

Tianjin

NA

765

603

59HB0

902

200903

Hebei

1014

765

603

123

TJ0903

200911

Tianjin

NA

765

603

60HB0

903

200903

Hebei

1014

765

603

124

TJ110

1201104

Tianjin

1014

765

603

61HB0

904

200904

Hebei

1014

765

603

125

ZJ1001

201001

Zhejiang

1014

765

603

62HB0

905

200911

Hebei

1104

765

603

126

ZJ1101

201102

Zhejiang

1014

765

603

63HB0

906

200911

Hebei

1104

765

603

127

ZJ110

2201104

Zhejiang

1014

765

603

64HB0

907

200911

Hebei

1014

765

603

128

ZJ1103

201104

Zhejiang

1014

765

603

lowast

Each

samplew

asnamed

accordingto

ther

egionandcollectionyearNAnot

amplified

6 BioMed Research International

amino acid sequence of NSP2 HV region was conducted byusing the 101 NSP2 sequences obtained in this study togetherwith downloaded representative sequences (SupplementaryTable S1) The phylogenetic tree revealed that all 101 NSP2sequences belonged to genotype 2 of PRRSV and all ChinesePRRSV strains could be classified into three main subgroups(Figure 1) The JL1101 and GZ1101 were located in subgroup 1with the representative strains VR-2332 BJ-4 and RespPRRSMLV the other 99 were clustered into the subgroup 3 withmultiple branches together with the representative strainsHB-1(sh)2002 JXwn06 JXA1 and JXA1 P80 No strains inthis study were clustered into subgroup 2 with representativestrain CH-1a the earliest Chinese strain This means that thegenetic diversity of NSP2 still existed and the strains with 30-aa deletion inNSP2-coding region remain to be the dominantviruses in the field Compared with the data from 2006 to2007 the percentage of NSP2-deleted strains increased [25]However these subgroups did not appear to be associatedwith epidemiological features based on geography or date

Interestingly a minor branch with JXA1 P80 the HP-PRRSV JXA1 derived vaccine strain was observed in theNSP2 phylogenetic tree Four strainsHB1105 HB1201 SC1101and BJ1101 collected later than the year 2011 when theJXA1-derived vaccine was launched commercially were alsoclustered in this branch whereas the parental strain JXA1was out of this branch suggesting that there is the possibilitythat the four strains directly derived from the vaccine strainJAX1 P80However few earlier strains were also clustered intothis minor branch Even though the analysis from this studydoes not fully reflect that a great number of emergence ofPRRSV were due to the use of HP-PRRSV-derived MLV thepotential risk of the reversion of MLV to virulent strains andthe recombination between the vaccine virus and field virusesare worthy to pay more attention to in the future [35]

33 Sequence Alignment and Phylogenetic Analysis of ORF3Gene All the obtainedORF3 genes in this study had the samesize of 725 nt The sequences alignments indicated that theyshared 925ndash100 amino acid similarity with each otherand 894ndash953 amino acid similarity with JXwn06 as wellas 807ndash850 with VR-2332 The regions residues 33ndash46120ndash133 and 162ndash198 were conserved among these strainsotherwise majority of amino acid substitutions were locatedin two hypervariable regions the residues 58ndash71 and 216ndash226 Especially 63 out of 123 contained the I66-T66mutationcomparing with those in JXwn06 and VR-2332

The phylogenetic analysis of deduced amino acidsequences of ORF3 indicated that all Chinese genotype 2strains were distributed into three subgroups (Figure 2)Three genes JL1101 HB1103 and GZ1101 were clustered intosubgroup 1 with the representative strains VR-2332 and BJ-4and no strains in this study were clustered into subgroup 2with the representative strains CH-1a HB-1(sh)2002 andHB-2(sh)2002 All the other strains were clustered intosubgroup 3 which contained most Chinese strains collectedlater than 2004

34 Sequence Alignment and Phylogenetic Analysis of ORF5Gene Except for the GZ1101 which had one amino acid

deletion at the position aa 34 in ORF5-coding region theother 117 genes had the same size of 603 nt as that of VR-2332Sequences alignments showed that the amino acid similarityamong the 117 ORF5 genes ranged from 770 to 100 andthey shared 78ndash99 amino acid similarity with VR-2332 aswell as 865ndash99 with JXwn06 Similar as previous reportthe residue 3ndash39 the putative signal sequence was the mostvariable region whereas the regions 40ndash57 67ndash90 107ndash120138ndash160 and 165ndash184were relatively conserved [25]Howevera novel substitution E170-G170 which was conserved in theChinese strains collected during the period from 2006 to2007 was observed in recent strains

The phylogenetic tree conducted by using the deducedamino acid sequences of ORF5 showed that the Chinesestrains of genotype 2 PRRSV could be divided into 3 differ-ent subgroups (Figure 3) Three strains JL1101 HB1103 andGZ1101 were in subgroup 1 with the representative strainsVR-2332 BJ-4 and CH-1a and the SD1003 was the onlystrain clustered in subgroup 2 with the representative strainMN184A all other 115 strains were clustered into subgroup3 with multiple branches which were completely composedof Chinese strains with the representative JXwn06 and HB-1(sh)2002 Similar to the NSP2 phylogenetic tree a minorbranchwith the JXA1 P80 contained the strains collected bothearlier and later than 2011

35 Full-Length Genomic Analysis of 4 New PRRSV IsolatesThree strains SD0901 LN1101 and GZ1101 with charac-teristic deletion or insertion in NSP2 or ORF5 genes andanother strain BJ1102 were successfully isolated from theclinical samples using MARC-145 cells or PAMs The fourstrains were subjected to full-length genomic sequencingafter plaque purification of three rounds The SD0901 (Gen-Bank Accession number NJ256115) and BJ1102 (GenBankAccession number KF751237) shared same size of completegenome with 15320 nt in length excluding the ploy (A) tailsThe genome sizes of LN1101 (GenBank Accession numberKF751238) and GZ1101 were 15356 nt and 15404 nt respec-tively The BJ1102 was isolated from clinical samples of Tibetmini-pig with acute PRRS symptom in a pig farm whereHP-PRRSV-derived vaccine was used before importing Tibetmini-pig

Sequence alignments indicated that the 51015840UTRof the fourstrains shared nucleotide identities of 910ndash100 with therepresentative genotype 2 PRRSV strains A nucleotide ldquoArdquoinsertion at the position nt 75 of GZ1101 51015840UTR was firstobserved in this study It was shown that major variationswere located in NSP2-coding region including 3-aa deletionat the position aa 593ndash595 in GZ1101 18-aa deletion at theposition aa 482ndash499 in LN1101 30-aa deletion at the positionsaa 482 and aa 533ndash561 in BJ1102 and 31-aa deletion at thepositions aa 468 aa 482 and aa 533ndash561 and an amino acidldquoPrdquo insertion between aa 585 and aa 586 in SD0901 (Figure 4)In addition a new deletion at the position aa 34 of GP5 wasfound inGZ1101 (Figure 5)The individual homology analysisof the other genes was also summarized in SupplementaryTables S2ndashS5

To further classify the evolutionary relationship of these4 isolates the phylogenetic tree was conducted based on their

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 3: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

BioMed Research International 3

Table 1 Primers used for amplification and sequencing of PRRSV genome and NSP2 HV region and ORF3 and ORF5 genes

Name Primer sequence Locationlowast Length (bp)51015840-Outer-R TTTCACTTCATCCCCACCAC 800 81951015840-Inner-R CCCCGTTCATAAACTTGTAGAG 542 563W1F ATGACGTATAGGTGTTGGCTCT 1W1R TACTCTTTCAGGAAGGGTGG 1575 1575W2F ACGCTCTGGTGCGACTACTA 1362W2R AGGTTGTTCGGTTGTCTGATT 2253 892W3F CCTCCGTGGCGCAACAAGTCTTG 2115W3R CGATGATGGCTTGAGCTGAGTAT 3178 1064W4F TGAGCCTCTGGATTTGTCTGC 2949W4R GGCGATCTCATTAGGAGCAGTT 4329 1381W5F TGCTTAGGCTTGGCATTGTTG 4214W5R ACGGTGTTCAGTGAGGGCTTT 5564 1351W6F ACTAACATTGCTGGTCTCGTCA 5350W6R AAGGAAATCCAAGTCCTCGTC 6750 1401W7F TTGTGACCTCGCCAGTCCCAGTG 6500W7R CCAAAGCGTGCCATCAATCCC 7922 1423W8F GGTTGATGGTGGTGTTGTGCT 7787W8R GTCTTCTTTGGGTCCGTCTGG 9226 1440W9F TGGTCACCCTCATGGCCTTCT 9038W9R CAAATACATAGCAATGGGAGTCAAA 10323 1286W10F TTCCTGGATGAAGCGGCGTAT 10194W10R AACTCGGATGTATGAGGCGTAG 11573 1380W11F GGTGCTGGAAAGTGATGTTGG 11440W11R AAAGCGGGCATACCGTGTAAT 12774 1335W12F AGTGGTTTGGATGTGGTGGCT 12400W12R TGTTGTTGTTGCTGGCGTTGA 13803 1404W13F ATGTGCGACTGCTTCATTTCA 13599W13R TTTGCTGCTTGCCGTTGTTAT 14826 1228W14F TCCACTACGGTCAACGGCACAT 14702W14R GGATCCGGTACCTCTAGATCAGA 63731015840race adaptor-T GGATCCGGTACCTCTAGATCAGATTTTTTTTTTTTTTTTTTNsp2-F CCTCCGTGGTGCAACAAATCTTG 2115Nsp2-R CGATGATGGCTTGAGCTGAGTAT 3178 10641154ORF3-F CAGGGTCAAATGTAACCATAGTG 12506ORF3-R GGCAAGAAGAAAGCATGAGGAG 13457 952ORF5-F AGCCTGTCTTTTTGCCATTCT 13654ORF5-R CTTTTGTGGAGCCGTGCTATC 14335 682ORF7F TGATAACCACGCATTTGTCG 14668ORF7R GCCATTCACCACACATTCTTC 15228 561lowastThe location is according to the genomic sequences of JXwn06 (GenBank Accession number EF641008)

region sequences had the same length of 1014 nucleotide (nt)containing the same 30-aa deletion at aa 482 and aa 533ndash561as JXwn06 and other HP-PRRSV strains compared with thetype 2 prototype VR-2332 and the Chinese early strains TheLN1101 and GZ1101 showed two novel deletion patterns intheir NSP2 regions whose nucleotide sequences length was1050 nt and 1095 nt respectivelyThe other 13NSP2 sequenceswere 1104 nt in length same as those of VR-2332

Pairwise comparisons showed that those 86 sequenceswith 30-aa deletions in NSP2 shared 876ndash100 amino acid

similarity with each other And their amino acid similaritieswith JXwn06 ranged from 917 to 994 as well as 666to 695 compared with VR-2332 Majority of the sequenceswithout deletion shared high homology with HB-1(sh)2002showing the amino acid similarity of 981ndash992 Mean-while the JL1101 and GZ1101 displayed the highest homologywith VR-2332 with amino acid similarities of 992 and967 respectively

To further gain a better understanding of the geneticrelationship the phylogenetic analysis based on deduced

4 BioMed Research International

Table2Geographico

rigin

andam

plified

sequ

ence

sizefrom

clinicalsam

ples

inthisstu

dy

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

1AHQS0901

200908

Anh

ui1014

765

603

65HB0

908

200912

Hebei

1014

765

NA

2AHQS0902

200908

Anh

uiNA

765

603

66HB0

909

200912

Hebei

1014

765

603

3AHQS0903

200908

Anh

ui1014

765

603

67HB1001

201004

Hebei

1014

765

603

4AHQS0905

200908

Anh

ui1014

765

603

68HB1002

201004

Hebei

1014

765

603

5AHQS0906

200908

Anh

uiNA

765

603

69HB1101

201101

Hebei

1104

765

603

6AHQS0907

200908

Anh

ui1014

765

603

70HB1102

201101

Hebei

1014

765

603

7AHQS0908

200908

Anh

uiNA

765

603

71HB1103

201111

Hebei

1104

765

603

8AHQS0909

200908

Anh

uiNA

765

603

72HB1104

201111

Hebei

1014

765

603

9AHQS0910

200908

Anh

uiNA

765

603

73HB1105

201103

Hebei

1014

765

603

10AHSS0901

200908

Anh

ui1014

765

603

74HB1106

201104

Hebei

1014

765

NA

11AHSS0902

200908

Anh

ui1014

765

603

75HB1201

201205

Hebei

1014

765

603

12AHSS0903

200908

Anh

ui1014

765

603

76HEN

1001

201003

Henan

NA

NA

603

13AHSS0904

200908

Anh

uiNA

765

603

77HEN

1002

201011

Henan

1014

765

603

14AHSS0905

200908

Anh

uiNA

765

603

78HEN

1003

201011

Henan

1014

765

603

15BJ0803

200804

Beijing

1014

765

603

79HEN

1101

201101

Henan

1014

765

603

16BJ0804

200809

Beijing

1014

765

603

80HEN

1102

201109

Henan

1104

765

603

17BJ0805

200809

Beijing

1014

765

603

81HEN

1103

201109

Henan

1014

765

603

18BJ0806

200801

Beijing

1014

765

603

82HLJ1101

201109

Heilong

jiang

1014

765

603

19BJ0807

200801

Beijing

1014

765

603

83HLJ1102

201109

Heilong

jiang

1014

765

603

20BJ0808

200801

Beijing

NA

765

603

84HLJ110

3201109

Heilong

jiang

1014

765

603

21BJ0809

200801

Beijing

NA

765

603

85HLJ1104

201109

Heilong

jiang

1014

765

603

22BJ0810

200811

Beijing

1104

765

603

86HN0901

200903

Henan

1014

765

NA

23BJ0901

200902

Beijing

1014

765

603

87HN0902

200905

Henan

1014

765

603

24BJ0902

200902

Beijing

1014

765

603

88HN0903

200909

Henan

1104

765

603

25BJ0903

200904

Beijing

NA

765

603

89HuB

1001

201008

Hub

ei1014

765

603

26BJ0904

200905

Beijing

1014

NA

NA

90HuN

1001

201008

Hun

an110

4765

NA

27BJ0905

200905

Beijing

1104

765

603

91HuN

1002

201012

Hun

an1014

765

603

28BJ0906

200905

Beijing

NA

765

603

92HuN

1003

201012

Hun

an1014

765

603

29BJ0907

200905

Beijing

NA

NA

603

93HuN

1101

201111

Hun

an1014

765

603

30BJ0908

200906

Beijing

1014

765

603

94HuN

1102

201101

Hun

an1014

NA

603

31BJ0909

200909

Beijing

1104

765

603

95JL110

1201101

Jilin

1104

765

603

32BJ0910

200911

Beijing

NA

765

603

96JL110

2201103

Jilin

1014

765

603

33BJ0911

200912

Beijing

NA

765

603

97JX1001

201008

Jiang

xi1014

765

603

BioMed Research International 5

Table2Con

tinued

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

34BJ0912

200912

Beijing

NA

765

603

98JX1002

201009

Jiang

xi1014

765

603

35BJ1001

201003

Beijing

1014

765

603

99LN

0801

200801

Liaoning

1014

765

603

36BJ1002

201003

Beijing

1104

765

603

100

LN0901

200906

Liaoning

1014

765

603

37BJ1003

201005

Beijing

1014

765

603

101

LN0902

200911

Liaoning

1014

765

603

38BJ1004

201007

Beijing

1014

765

603

102

LN1001

201009

Liaoning

1014

765

603

39BJ1005

201007

Beijing

1014

765

603

103

LN110

1201101

Liaoning

1050

765

603

40BJ1006

201008

Beijing

1104

765

603

104

NMG0801

200809

Inner

Mon

golia

NA

765

NA

41BJ1007

201009

Beijing

1014

765

603

105

NMG0802

200809

Inner

Mon

golia

NA

765

NA

42BJ1008

201009

Beijing

1014

765

603

106

SC0801

200801

Sichuan

1014

765

603

43BJ1009

201011

Beijing

NA

765

NA

107

SC110

1201103

Sichuan

1014

765

NA

44BJ1010

201011

Beijing

1014

765

603

108

SC1201

201204

Sichuan

1014

765

603

45BJ110

1201101

Beijing

1014

765

603

109

SD0901

200904

Shando

ng1014

765

603

46BJ110

2201101

Beijing

1014

765

603

110SD

0902

200912

Shando

ng1014

765

603

47GX1001

201006

Guang

xi1014

765

603

111SD

0903

200912

Shando

ng1014

765

603

48GX1002

201006

Guang

xi1014

765

603

112

SD0904

200912

Shando

ng1014

765

603

49GZ1001

201009

Guizhou

1014

765

603

113SD

0905

200912

Shando

ng1014

765

603

50GZ1002

201011

Guizhou

1014

765

603

114SD

0906

200912

Shando

ng1014

765

603

51GZ1101

201103

Guizhou

1095

765

600

115SD

1001

201003

Shando

ng1014

765

603

52HB0

801

200807

Hebei

NA

765

603

116

SD1002

201003

Shando

ngNA

765

603

53HB0

802

200801

Hebei

1014

765

603

117SD

1003

201011

Shando

ng1014

765

603

54HB0

803

200801

Hebei

1014

765

603

118SD

1004

201011

Shando

ng1014

765

603

55HB0

804

200801

Hebei

NA

765

603

119SD

1101

201103

Shando

ng1014

765

603

56HB0

805

200801

Hebei

NA

NA

603

120

SX1101

201105

Shanxi

1014

765

603

57HB0

806

200811

Hebei

1014

765

603

121

TJ0901

200911

Tianjin

NA

765

603

58HB0

901

200903

Hebei

NA

765

NA

122

TJ0902

200911

Tianjin

NA

765

603

59HB0

902

200903

Hebei

1014

765

603

123

TJ0903

200911

Tianjin

NA

765

603

60HB0

903

200903

Hebei

1014

765

603

124

TJ110

1201104

Tianjin

1014

765

603

61HB0

904

200904

Hebei

1014

765

603

125

ZJ1001

201001

Zhejiang

1014

765

603

62HB0

905

200911

Hebei

1104

765

603

126

ZJ1101

201102

Zhejiang

1014

765

603

63HB0

906

200911

Hebei

1104

765

603

127

ZJ110

2201104

Zhejiang

1014

765

603

64HB0

907

200911

Hebei

1014

765

603

128

ZJ1103

201104

Zhejiang

1014

765

603

lowast

Each

samplew

asnamed

accordingto

ther

egionandcollectionyearNAnot

amplified

6 BioMed Research International

amino acid sequence of NSP2 HV region was conducted byusing the 101 NSP2 sequences obtained in this study togetherwith downloaded representative sequences (SupplementaryTable S1) The phylogenetic tree revealed that all 101 NSP2sequences belonged to genotype 2 of PRRSV and all ChinesePRRSV strains could be classified into three main subgroups(Figure 1) The JL1101 and GZ1101 were located in subgroup 1with the representative strains VR-2332 BJ-4 and RespPRRSMLV the other 99 were clustered into the subgroup 3 withmultiple branches together with the representative strainsHB-1(sh)2002 JXwn06 JXA1 and JXA1 P80 No strains inthis study were clustered into subgroup 2 with representativestrain CH-1a the earliest Chinese strain This means that thegenetic diversity of NSP2 still existed and the strains with 30-aa deletion inNSP2-coding region remain to be the dominantviruses in the field Compared with the data from 2006 to2007 the percentage of NSP2-deleted strains increased [25]However these subgroups did not appear to be associatedwith epidemiological features based on geography or date

Interestingly a minor branch with JXA1 P80 the HP-PRRSV JXA1 derived vaccine strain was observed in theNSP2 phylogenetic tree Four strainsHB1105 HB1201 SC1101and BJ1101 collected later than the year 2011 when theJXA1-derived vaccine was launched commercially were alsoclustered in this branch whereas the parental strain JXA1was out of this branch suggesting that there is the possibilitythat the four strains directly derived from the vaccine strainJAX1 P80However few earlier strains were also clustered intothis minor branch Even though the analysis from this studydoes not fully reflect that a great number of emergence ofPRRSV were due to the use of HP-PRRSV-derived MLV thepotential risk of the reversion of MLV to virulent strains andthe recombination between the vaccine virus and field virusesare worthy to pay more attention to in the future [35]

33 Sequence Alignment and Phylogenetic Analysis of ORF3Gene All the obtainedORF3 genes in this study had the samesize of 725 nt The sequences alignments indicated that theyshared 925ndash100 amino acid similarity with each otherand 894ndash953 amino acid similarity with JXwn06 as wellas 807ndash850 with VR-2332 The regions residues 33ndash46120ndash133 and 162ndash198 were conserved among these strainsotherwise majority of amino acid substitutions were locatedin two hypervariable regions the residues 58ndash71 and 216ndash226 Especially 63 out of 123 contained the I66-T66mutationcomparing with those in JXwn06 and VR-2332

The phylogenetic analysis of deduced amino acidsequences of ORF3 indicated that all Chinese genotype 2strains were distributed into three subgroups (Figure 2)Three genes JL1101 HB1103 and GZ1101 were clustered intosubgroup 1 with the representative strains VR-2332 and BJ-4and no strains in this study were clustered into subgroup 2with the representative strains CH-1a HB-1(sh)2002 andHB-2(sh)2002 All the other strains were clustered intosubgroup 3 which contained most Chinese strains collectedlater than 2004

34 Sequence Alignment and Phylogenetic Analysis of ORF5Gene Except for the GZ1101 which had one amino acid

deletion at the position aa 34 in ORF5-coding region theother 117 genes had the same size of 603 nt as that of VR-2332Sequences alignments showed that the amino acid similarityamong the 117 ORF5 genes ranged from 770 to 100 andthey shared 78ndash99 amino acid similarity with VR-2332 aswell as 865ndash99 with JXwn06 Similar as previous reportthe residue 3ndash39 the putative signal sequence was the mostvariable region whereas the regions 40ndash57 67ndash90 107ndash120138ndash160 and 165ndash184were relatively conserved [25]Howevera novel substitution E170-G170 which was conserved in theChinese strains collected during the period from 2006 to2007 was observed in recent strains

The phylogenetic tree conducted by using the deducedamino acid sequences of ORF5 showed that the Chinesestrains of genotype 2 PRRSV could be divided into 3 differ-ent subgroups (Figure 3) Three strains JL1101 HB1103 andGZ1101 were in subgroup 1 with the representative strainsVR-2332 BJ-4 and CH-1a and the SD1003 was the onlystrain clustered in subgroup 2 with the representative strainMN184A all other 115 strains were clustered into subgroup3 with multiple branches which were completely composedof Chinese strains with the representative JXwn06 and HB-1(sh)2002 Similar to the NSP2 phylogenetic tree a minorbranchwith the JXA1 P80 contained the strains collected bothearlier and later than 2011

35 Full-Length Genomic Analysis of 4 New PRRSV IsolatesThree strains SD0901 LN1101 and GZ1101 with charac-teristic deletion or insertion in NSP2 or ORF5 genes andanother strain BJ1102 were successfully isolated from theclinical samples using MARC-145 cells or PAMs The fourstrains were subjected to full-length genomic sequencingafter plaque purification of three rounds The SD0901 (Gen-Bank Accession number NJ256115) and BJ1102 (GenBankAccession number KF751237) shared same size of completegenome with 15320 nt in length excluding the ploy (A) tailsThe genome sizes of LN1101 (GenBank Accession numberKF751238) and GZ1101 were 15356 nt and 15404 nt respec-tively The BJ1102 was isolated from clinical samples of Tibetmini-pig with acute PRRS symptom in a pig farm whereHP-PRRSV-derived vaccine was used before importing Tibetmini-pig

Sequence alignments indicated that the 51015840UTRof the fourstrains shared nucleotide identities of 910ndash100 with therepresentative genotype 2 PRRSV strains A nucleotide ldquoArdquoinsertion at the position nt 75 of GZ1101 51015840UTR was firstobserved in this study It was shown that major variationswere located in NSP2-coding region including 3-aa deletionat the position aa 593ndash595 in GZ1101 18-aa deletion at theposition aa 482ndash499 in LN1101 30-aa deletion at the positionsaa 482 and aa 533ndash561 in BJ1102 and 31-aa deletion at thepositions aa 468 aa 482 and aa 533ndash561 and an amino acidldquoPrdquo insertion between aa 585 and aa 586 in SD0901 (Figure 4)In addition a new deletion at the position aa 34 of GP5 wasfound inGZ1101 (Figure 5)The individual homology analysisof the other genes was also summarized in SupplementaryTables S2ndashS5

To further classify the evolutionary relationship of these4 isolates the phylogenetic tree was conducted based on their

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 4: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

4 BioMed Research International

Table2Geographico

rigin

andam

plified

sequ

ence

sizefrom

clinicalsam

ples

inthisstu

dy

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

1AHQS0901

200908

Anh

ui1014

765

603

65HB0

908

200912

Hebei

1014

765

NA

2AHQS0902

200908

Anh

uiNA

765

603

66HB0

909

200912

Hebei

1014

765

603

3AHQS0903

200908

Anh

ui1014

765

603

67HB1001

201004

Hebei

1014

765

603

4AHQS0905

200908

Anh

ui1014

765

603

68HB1002

201004

Hebei

1014

765

603

5AHQS0906

200908

Anh

uiNA

765

603

69HB1101

201101

Hebei

1104

765

603

6AHQS0907

200908

Anh

ui1014

765

603

70HB1102

201101

Hebei

1014

765

603

7AHQS0908

200908

Anh

uiNA

765

603

71HB1103

201111

Hebei

1104

765

603

8AHQS0909

200908

Anh

uiNA

765

603

72HB1104

201111

Hebei

1014

765

603

9AHQS0910

200908

Anh

uiNA

765

603

73HB1105

201103

Hebei

1014

765

603

10AHSS0901

200908

Anh

ui1014

765

603

74HB1106

201104

Hebei

1014

765

NA

11AHSS0902

200908

Anh

ui1014

765

603

75HB1201

201205

Hebei

1014

765

603

12AHSS0903

200908

Anh

ui1014

765

603

76HEN

1001

201003

Henan

NA

NA

603

13AHSS0904

200908

Anh

uiNA

765

603

77HEN

1002

201011

Henan

1014

765

603

14AHSS0905

200908

Anh

uiNA

765

603

78HEN

1003

201011

Henan

1014

765

603

15BJ0803

200804

Beijing

1014

765

603

79HEN

1101

201101

Henan

1014

765

603

16BJ0804

200809

Beijing

1014

765

603

80HEN

1102

201109

Henan

1104

765

603

17BJ0805

200809

Beijing

1014

765

603

81HEN

1103

201109

Henan

1014

765

603

18BJ0806

200801

Beijing

1014

765

603

82HLJ1101

201109

Heilong

jiang

1014

765

603

19BJ0807

200801

Beijing

1014

765

603

83HLJ1102

201109

Heilong

jiang

1014

765

603

20BJ0808

200801

Beijing

NA

765

603

84HLJ110

3201109

Heilong

jiang

1014

765

603

21BJ0809

200801

Beijing

NA

765

603

85HLJ1104

201109

Heilong

jiang

1014

765

603

22BJ0810

200811

Beijing

1104

765

603

86HN0901

200903

Henan

1014

765

NA

23BJ0901

200902

Beijing

1014

765

603

87HN0902

200905

Henan

1014

765

603

24BJ0902

200902

Beijing

1014

765

603

88HN0903

200909

Henan

1104

765

603

25BJ0903

200904

Beijing

NA

765

603

89HuB

1001

201008

Hub

ei1014

765

603

26BJ0904

200905

Beijing

1014

NA

NA

90HuN

1001

201008

Hun

an110

4765

NA

27BJ0905

200905

Beijing

1104

765

603

91HuN

1002

201012

Hun

an1014

765

603

28BJ0906

200905

Beijing

NA

765

603

92HuN

1003

201012

Hun

an1014

765

603

29BJ0907

200905

Beijing

NA

NA

603

93HuN

1101

201111

Hun

an1014

765

603

30BJ0908

200906

Beijing

1014

765

603

94HuN

1102

201101

Hun

an1014

NA

603

31BJ0909

200909

Beijing

1104

765

603

95JL110

1201101

Jilin

1104

765

603

32BJ0910

200911

Beijing

NA

765

603

96JL110

2201103

Jilin

1014

765

603

33BJ0911

200912

Beijing

NA

765

603

97JX1001

201008

Jiang

xi1014

765

603

BioMed Research International 5

Table2Con

tinued

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

34BJ0912

200912

Beijing

NA

765

603

98JX1002

201009

Jiang

xi1014

765

603

35BJ1001

201003

Beijing

1014

765

603

99LN

0801

200801

Liaoning

1014

765

603

36BJ1002

201003

Beijing

1104

765

603

100

LN0901

200906

Liaoning

1014

765

603

37BJ1003

201005

Beijing

1014

765

603

101

LN0902

200911

Liaoning

1014

765

603

38BJ1004

201007

Beijing

1014

765

603

102

LN1001

201009

Liaoning

1014

765

603

39BJ1005

201007

Beijing

1014

765

603

103

LN110

1201101

Liaoning

1050

765

603

40BJ1006

201008

Beijing

1104

765

603

104

NMG0801

200809

Inner

Mon

golia

NA

765

NA

41BJ1007

201009

Beijing

1014

765

603

105

NMG0802

200809

Inner

Mon

golia

NA

765

NA

42BJ1008

201009

Beijing

1014

765

603

106

SC0801

200801

Sichuan

1014

765

603

43BJ1009

201011

Beijing

NA

765

NA

107

SC110

1201103

Sichuan

1014

765

NA

44BJ1010

201011

Beijing

1014

765

603

108

SC1201

201204

Sichuan

1014

765

603

45BJ110

1201101

Beijing

1014

765

603

109

SD0901

200904

Shando

ng1014

765

603

46BJ110

2201101

Beijing

1014

765

603

110SD

0902

200912

Shando

ng1014

765

603

47GX1001

201006

Guang

xi1014

765

603

111SD

0903

200912

Shando

ng1014

765

603

48GX1002

201006

Guang

xi1014

765

603

112

SD0904

200912

Shando

ng1014

765

603

49GZ1001

201009

Guizhou

1014

765

603

113SD

0905

200912

Shando

ng1014

765

603

50GZ1002

201011

Guizhou

1014

765

603

114SD

0906

200912

Shando

ng1014

765

603

51GZ1101

201103

Guizhou

1095

765

600

115SD

1001

201003

Shando

ng1014

765

603

52HB0

801

200807

Hebei

NA

765

603

116

SD1002

201003

Shando

ngNA

765

603

53HB0

802

200801

Hebei

1014

765

603

117SD

1003

201011

Shando

ng1014

765

603

54HB0

803

200801

Hebei

1014

765

603

118SD

1004

201011

Shando

ng1014

765

603

55HB0

804

200801

Hebei

NA

765

603

119SD

1101

201103

Shando

ng1014

765

603

56HB0

805

200801

Hebei

NA

NA

603

120

SX1101

201105

Shanxi

1014

765

603

57HB0

806

200811

Hebei

1014

765

603

121

TJ0901

200911

Tianjin

NA

765

603

58HB0

901

200903

Hebei

NA

765

NA

122

TJ0902

200911

Tianjin

NA

765

603

59HB0

902

200903

Hebei

1014

765

603

123

TJ0903

200911

Tianjin

NA

765

603

60HB0

903

200903

Hebei

1014

765

603

124

TJ110

1201104

Tianjin

1014

765

603

61HB0

904

200904

Hebei

1014

765

603

125

ZJ1001

201001

Zhejiang

1014

765

603

62HB0

905

200911

Hebei

1104

765

603

126

ZJ1101

201102

Zhejiang

1014

765

603

63HB0

906

200911

Hebei

1104

765

603

127

ZJ110

2201104

Zhejiang

1014

765

603

64HB0

907

200911

Hebei

1014

765

603

128

ZJ1103

201104

Zhejiang

1014

765

603

lowast

Each

samplew

asnamed

accordingto

ther

egionandcollectionyearNAnot

amplified

6 BioMed Research International

amino acid sequence of NSP2 HV region was conducted byusing the 101 NSP2 sequences obtained in this study togetherwith downloaded representative sequences (SupplementaryTable S1) The phylogenetic tree revealed that all 101 NSP2sequences belonged to genotype 2 of PRRSV and all ChinesePRRSV strains could be classified into three main subgroups(Figure 1) The JL1101 and GZ1101 were located in subgroup 1with the representative strains VR-2332 BJ-4 and RespPRRSMLV the other 99 were clustered into the subgroup 3 withmultiple branches together with the representative strainsHB-1(sh)2002 JXwn06 JXA1 and JXA1 P80 No strains inthis study were clustered into subgroup 2 with representativestrain CH-1a the earliest Chinese strain This means that thegenetic diversity of NSP2 still existed and the strains with 30-aa deletion inNSP2-coding region remain to be the dominantviruses in the field Compared with the data from 2006 to2007 the percentage of NSP2-deleted strains increased [25]However these subgroups did not appear to be associatedwith epidemiological features based on geography or date

Interestingly a minor branch with JXA1 P80 the HP-PRRSV JXA1 derived vaccine strain was observed in theNSP2 phylogenetic tree Four strainsHB1105 HB1201 SC1101and BJ1101 collected later than the year 2011 when theJXA1-derived vaccine was launched commercially were alsoclustered in this branch whereas the parental strain JXA1was out of this branch suggesting that there is the possibilitythat the four strains directly derived from the vaccine strainJAX1 P80However few earlier strains were also clustered intothis minor branch Even though the analysis from this studydoes not fully reflect that a great number of emergence ofPRRSV were due to the use of HP-PRRSV-derived MLV thepotential risk of the reversion of MLV to virulent strains andthe recombination between the vaccine virus and field virusesare worthy to pay more attention to in the future [35]

33 Sequence Alignment and Phylogenetic Analysis of ORF3Gene All the obtainedORF3 genes in this study had the samesize of 725 nt The sequences alignments indicated that theyshared 925ndash100 amino acid similarity with each otherand 894ndash953 amino acid similarity with JXwn06 as wellas 807ndash850 with VR-2332 The regions residues 33ndash46120ndash133 and 162ndash198 were conserved among these strainsotherwise majority of amino acid substitutions were locatedin two hypervariable regions the residues 58ndash71 and 216ndash226 Especially 63 out of 123 contained the I66-T66mutationcomparing with those in JXwn06 and VR-2332

The phylogenetic analysis of deduced amino acidsequences of ORF3 indicated that all Chinese genotype 2strains were distributed into three subgroups (Figure 2)Three genes JL1101 HB1103 and GZ1101 were clustered intosubgroup 1 with the representative strains VR-2332 and BJ-4and no strains in this study were clustered into subgroup 2with the representative strains CH-1a HB-1(sh)2002 andHB-2(sh)2002 All the other strains were clustered intosubgroup 3 which contained most Chinese strains collectedlater than 2004

34 Sequence Alignment and Phylogenetic Analysis of ORF5Gene Except for the GZ1101 which had one amino acid

deletion at the position aa 34 in ORF5-coding region theother 117 genes had the same size of 603 nt as that of VR-2332Sequences alignments showed that the amino acid similarityamong the 117 ORF5 genes ranged from 770 to 100 andthey shared 78ndash99 amino acid similarity with VR-2332 aswell as 865ndash99 with JXwn06 Similar as previous reportthe residue 3ndash39 the putative signal sequence was the mostvariable region whereas the regions 40ndash57 67ndash90 107ndash120138ndash160 and 165ndash184were relatively conserved [25]Howevera novel substitution E170-G170 which was conserved in theChinese strains collected during the period from 2006 to2007 was observed in recent strains

The phylogenetic tree conducted by using the deducedamino acid sequences of ORF5 showed that the Chinesestrains of genotype 2 PRRSV could be divided into 3 differ-ent subgroups (Figure 3) Three strains JL1101 HB1103 andGZ1101 were in subgroup 1 with the representative strainsVR-2332 BJ-4 and CH-1a and the SD1003 was the onlystrain clustered in subgroup 2 with the representative strainMN184A all other 115 strains were clustered into subgroup3 with multiple branches which were completely composedof Chinese strains with the representative JXwn06 and HB-1(sh)2002 Similar to the NSP2 phylogenetic tree a minorbranchwith the JXA1 P80 contained the strains collected bothearlier and later than 2011

35 Full-Length Genomic Analysis of 4 New PRRSV IsolatesThree strains SD0901 LN1101 and GZ1101 with charac-teristic deletion or insertion in NSP2 or ORF5 genes andanother strain BJ1102 were successfully isolated from theclinical samples using MARC-145 cells or PAMs The fourstrains were subjected to full-length genomic sequencingafter plaque purification of three rounds The SD0901 (Gen-Bank Accession number NJ256115) and BJ1102 (GenBankAccession number KF751237) shared same size of completegenome with 15320 nt in length excluding the ploy (A) tailsThe genome sizes of LN1101 (GenBank Accession numberKF751238) and GZ1101 were 15356 nt and 15404 nt respec-tively The BJ1102 was isolated from clinical samples of Tibetmini-pig with acute PRRS symptom in a pig farm whereHP-PRRSV-derived vaccine was used before importing Tibetmini-pig

Sequence alignments indicated that the 51015840UTRof the fourstrains shared nucleotide identities of 910ndash100 with therepresentative genotype 2 PRRSV strains A nucleotide ldquoArdquoinsertion at the position nt 75 of GZ1101 51015840UTR was firstobserved in this study It was shown that major variationswere located in NSP2-coding region including 3-aa deletionat the position aa 593ndash595 in GZ1101 18-aa deletion at theposition aa 482ndash499 in LN1101 30-aa deletion at the positionsaa 482 and aa 533ndash561 in BJ1102 and 31-aa deletion at thepositions aa 468 aa 482 and aa 533ndash561 and an amino acidldquoPrdquo insertion between aa 585 and aa 586 in SD0901 (Figure 4)In addition a new deletion at the position aa 34 of GP5 wasfound inGZ1101 (Figure 5)The individual homology analysisof the other genes was also summarized in SupplementaryTables S2ndashS5

To further classify the evolutionary relationship of these4 isolates the phylogenetic tree was conducted based on their

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 5: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

BioMed Research International 5

Table2Con

tinued

Seria

lnu

mber

Sample

desig

natio

nlowastCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

Seria

lnu

mber

Sample

desig

natio

nCollection

date

Area

NSP

2(b

p)

ORF

3(b

p)

ORF

5(b

p)

34BJ0912

200912

Beijing

NA

765

603

98JX1002

201009

Jiang

xi1014

765

603

35BJ1001

201003

Beijing

1014

765

603

99LN

0801

200801

Liaoning

1014

765

603

36BJ1002

201003

Beijing

1104

765

603

100

LN0901

200906

Liaoning

1014

765

603

37BJ1003

201005

Beijing

1014

765

603

101

LN0902

200911

Liaoning

1014

765

603

38BJ1004

201007

Beijing

1014

765

603

102

LN1001

201009

Liaoning

1014

765

603

39BJ1005

201007

Beijing

1014

765

603

103

LN110

1201101

Liaoning

1050

765

603

40BJ1006

201008

Beijing

1104

765

603

104

NMG0801

200809

Inner

Mon

golia

NA

765

NA

41BJ1007

201009

Beijing

1014

765

603

105

NMG0802

200809

Inner

Mon

golia

NA

765

NA

42BJ1008

201009

Beijing

1014

765

603

106

SC0801

200801

Sichuan

1014

765

603

43BJ1009

201011

Beijing

NA

765

NA

107

SC110

1201103

Sichuan

1014

765

NA

44BJ1010

201011

Beijing

1014

765

603

108

SC1201

201204

Sichuan

1014

765

603

45BJ110

1201101

Beijing

1014

765

603

109

SD0901

200904

Shando

ng1014

765

603

46BJ110

2201101

Beijing

1014

765

603

110SD

0902

200912

Shando

ng1014

765

603

47GX1001

201006

Guang

xi1014

765

603

111SD

0903

200912

Shando

ng1014

765

603

48GX1002

201006

Guang

xi1014

765

603

112

SD0904

200912

Shando

ng1014

765

603

49GZ1001

201009

Guizhou

1014

765

603

113SD

0905

200912

Shando

ng1014

765

603

50GZ1002

201011

Guizhou

1014

765

603

114SD

0906

200912

Shando

ng1014

765

603

51GZ1101

201103

Guizhou

1095

765

600

115SD

1001

201003

Shando

ng1014

765

603

52HB0

801

200807

Hebei

NA

765

603

116

SD1002

201003

Shando

ngNA

765

603

53HB0

802

200801

Hebei

1014

765

603

117SD

1003

201011

Shando

ng1014

765

603

54HB0

803

200801

Hebei

1014

765

603

118SD

1004

201011

Shando

ng1014

765

603

55HB0

804

200801

Hebei

NA

765

603

119SD

1101

201103

Shando

ng1014

765

603

56HB0

805

200801

Hebei

NA

NA

603

120

SX1101

201105

Shanxi

1014

765

603

57HB0

806

200811

Hebei

1014

765

603

121

TJ0901

200911

Tianjin

NA

765

603

58HB0

901

200903

Hebei

NA

765

NA

122

TJ0902

200911

Tianjin

NA

765

603

59HB0

902

200903

Hebei

1014

765

603

123

TJ0903

200911

Tianjin

NA

765

603

60HB0

903

200903

Hebei

1014

765

603

124

TJ110

1201104

Tianjin

1014

765

603

61HB0

904

200904

Hebei

1014

765

603

125

ZJ1001

201001

Zhejiang

1014

765

603

62HB0

905

200911

Hebei

1104

765

603

126

ZJ1101

201102

Zhejiang

1014

765

603

63HB0

906

200911

Hebei

1104

765

603

127

ZJ110

2201104

Zhejiang

1014

765

603

64HB0

907

200911

Hebei

1014

765

603

128

ZJ1103

201104

Zhejiang

1014

765

603

lowast

Each

samplew

asnamed

accordingto

ther

egionandcollectionyearNAnot

amplified

6 BioMed Research International

amino acid sequence of NSP2 HV region was conducted byusing the 101 NSP2 sequences obtained in this study togetherwith downloaded representative sequences (SupplementaryTable S1) The phylogenetic tree revealed that all 101 NSP2sequences belonged to genotype 2 of PRRSV and all ChinesePRRSV strains could be classified into three main subgroups(Figure 1) The JL1101 and GZ1101 were located in subgroup 1with the representative strains VR-2332 BJ-4 and RespPRRSMLV the other 99 were clustered into the subgroup 3 withmultiple branches together with the representative strainsHB-1(sh)2002 JXwn06 JXA1 and JXA1 P80 No strains inthis study were clustered into subgroup 2 with representativestrain CH-1a the earliest Chinese strain This means that thegenetic diversity of NSP2 still existed and the strains with 30-aa deletion inNSP2-coding region remain to be the dominantviruses in the field Compared with the data from 2006 to2007 the percentage of NSP2-deleted strains increased [25]However these subgroups did not appear to be associatedwith epidemiological features based on geography or date

Interestingly a minor branch with JXA1 P80 the HP-PRRSV JXA1 derived vaccine strain was observed in theNSP2 phylogenetic tree Four strainsHB1105 HB1201 SC1101and BJ1101 collected later than the year 2011 when theJXA1-derived vaccine was launched commercially were alsoclustered in this branch whereas the parental strain JXA1was out of this branch suggesting that there is the possibilitythat the four strains directly derived from the vaccine strainJAX1 P80However few earlier strains were also clustered intothis minor branch Even though the analysis from this studydoes not fully reflect that a great number of emergence ofPRRSV were due to the use of HP-PRRSV-derived MLV thepotential risk of the reversion of MLV to virulent strains andthe recombination between the vaccine virus and field virusesare worthy to pay more attention to in the future [35]

33 Sequence Alignment and Phylogenetic Analysis of ORF3Gene All the obtainedORF3 genes in this study had the samesize of 725 nt The sequences alignments indicated that theyshared 925ndash100 amino acid similarity with each otherand 894ndash953 amino acid similarity with JXwn06 as wellas 807ndash850 with VR-2332 The regions residues 33ndash46120ndash133 and 162ndash198 were conserved among these strainsotherwise majority of amino acid substitutions were locatedin two hypervariable regions the residues 58ndash71 and 216ndash226 Especially 63 out of 123 contained the I66-T66mutationcomparing with those in JXwn06 and VR-2332

The phylogenetic analysis of deduced amino acidsequences of ORF3 indicated that all Chinese genotype 2strains were distributed into three subgroups (Figure 2)Three genes JL1101 HB1103 and GZ1101 were clustered intosubgroup 1 with the representative strains VR-2332 and BJ-4and no strains in this study were clustered into subgroup 2with the representative strains CH-1a HB-1(sh)2002 andHB-2(sh)2002 All the other strains were clustered intosubgroup 3 which contained most Chinese strains collectedlater than 2004

34 Sequence Alignment and Phylogenetic Analysis of ORF5Gene Except for the GZ1101 which had one amino acid

deletion at the position aa 34 in ORF5-coding region theother 117 genes had the same size of 603 nt as that of VR-2332Sequences alignments showed that the amino acid similarityamong the 117 ORF5 genes ranged from 770 to 100 andthey shared 78ndash99 amino acid similarity with VR-2332 aswell as 865ndash99 with JXwn06 Similar as previous reportthe residue 3ndash39 the putative signal sequence was the mostvariable region whereas the regions 40ndash57 67ndash90 107ndash120138ndash160 and 165ndash184were relatively conserved [25]Howevera novel substitution E170-G170 which was conserved in theChinese strains collected during the period from 2006 to2007 was observed in recent strains

The phylogenetic tree conducted by using the deducedamino acid sequences of ORF5 showed that the Chinesestrains of genotype 2 PRRSV could be divided into 3 differ-ent subgroups (Figure 3) Three strains JL1101 HB1103 andGZ1101 were in subgroup 1 with the representative strainsVR-2332 BJ-4 and CH-1a and the SD1003 was the onlystrain clustered in subgroup 2 with the representative strainMN184A all other 115 strains were clustered into subgroup3 with multiple branches which were completely composedof Chinese strains with the representative JXwn06 and HB-1(sh)2002 Similar to the NSP2 phylogenetic tree a minorbranchwith the JXA1 P80 contained the strains collected bothearlier and later than 2011

35 Full-Length Genomic Analysis of 4 New PRRSV IsolatesThree strains SD0901 LN1101 and GZ1101 with charac-teristic deletion or insertion in NSP2 or ORF5 genes andanother strain BJ1102 were successfully isolated from theclinical samples using MARC-145 cells or PAMs The fourstrains were subjected to full-length genomic sequencingafter plaque purification of three rounds The SD0901 (Gen-Bank Accession number NJ256115) and BJ1102 (GenBankAccession number KF751237) shared same size of completegenome with 15320 nt in length excluding the ploy (A) tailsThe genome sizes of LN1101 (GenBank Accession numberKF751238) and GZ1101 were 15356 nt and 15404 nt respec-tively The BJ1102 was isolated from clinical samples of Tibetmini-pig with acute PRRS symptom in a pig farm whereHP-PRRSV-derived vaccine was used before importing Tibetmini-pig

Sequence alignments indicated that the 51015840UTRof the fourstrains shared nucleotide identities of 910ndash100 with therepresentative genotype 2 PRRSV strains A nucleotide ldquoArdquoinsertion at the position nt 75 of GZ1101 51015840UTR was firstobserved in this study It was shown that major variationswere located in NSP2-coding region including 3-aa deletionat the position aa 593ndash595 in GZ1101 18-aa deletion at theposition aa 482ndash499 in LN1101 30-aa deletion at the positionsaa 482 and aa 533ndash561 in BJ1102 and 31-aa deletion at thepositions aa 468 aa 482 and aa 533ndash561 and an amino acidldquoPrdquo insertion between aa 585 and aa 586 in SD0901 (Figure 4)In addition a new deletion at the position aa 34 of GP5 wasfound inGZ1101 (Figure 5)The individual homology analysisof the other genes was also summarized in SupplementaryTables S2ndashS5

To further classify the evolutionary relationship of these4 isolates the phylogenetic tree was conducted based on their

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 6: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

6 BioMed Research International

amino acid sequence of NSP2 HV region was conducted byusing the 101 NSP2 sequences obtained in this study togetherwith downloaded representative sequences (SupplementaryTable S1) The phylogenetic tree revealed that all 101 NSP2sequences belonged to genotype 2 of PRRSV and all ChinesePRRSV strains could be classified into three main subgroups(Figure 1) The JL1101 and GZ1101 were located in subgroup 1with the representative strains VR-2332 BJ-4 and RespPRRSMLV the other 99 were clustered into the subgroup 3 withmultiple branches together with the representative strainsHB-1(sh)2002 JXwn06 JXA1 and JXA1 P80 No strains inthis study were clustered into subgroup 2 with representativestrain CH-1a the earliest Chinese strain This means that thegenetic diversity of NSP2 still existed and the strains with 30-aa deletion inNSP2-coding region remain to be the dominantviruses in the field Compared with the data from 2006 to2007 the percentage of NSP2-deleted strains increased [25]However these subgroups did not appear to be associatedwith epidemiological features based on geography or date

Interestingly a minor branch with JXA1 P80 the HP-PRRSV JXA1 derived vaccine strain was observed in theNSP2 phylogenetic tree Four strainsHB1105 HB1201 SC1101and BJ1101 collected later than the year 2011 when theJXA1-derived vaccine was launched commercially were alsoclustered in this branch whereas the parental strain JXA1was out of this branch suggesting that there is the possibilitythat the four strains directly derived from the vaccine strainJAX1 P80However few earlier strains were also clustered intothis minor branch Even though the analysis from this studydoes not fully reflect that a great number of emergence ofPRRSV were due to the use of HP-PRRSV-derived MLV thepotential risk of the reversion of MLV to virulent strains andthe recombination between the vaccine virus and field virusesare worthy to pay more attention to in the future [35]

33 Sequence Alignment and Phylogenetic Analysis of ORF3Gene All the obtainedORF3 genes in this study had the samesize of 725 nt The sequences alignments indicated that theyshared 925ndash100 amino acid similarity with each otherand 894ndash953 amino acid similarity with JXwn06 as wellas 807ndash850 with VR-2332 The regions residues 33ndash46120ndash133 and 162ndash198 were conserved among these strainsotherwise majority of amino acid substitutions were locatedin two hypervariable regions the residues 58ndash71 and 216ndash226 Especially 63 out of 123 contained the I66-T66mutationcomparing with those in JXwn06 and VR-2332

The phylogenetic analysis of deduced amino acidsequences of ORF3 indicated that all Chinese genotype 2strains were distributed into three subgroups (Figure 2)Three genes JL1101 HB1103 and GZ1101 were clustered intosubgroup 1 with the representative strains VR-2332 and BJ-4and no strains in this study were clustered into subgroup 2with the representative strains CH-1a HB-1(sh)2002 andHB-2(sh)2002 All the other strains were clustered intosubgroup 3 which contained most Chinese strains collectedlater than 2004

34 Sequence Alignment and Phylogenetic Analysis of ORF5Gene Except for the GZ1101 which had one amino acid

deletion at the position aa 34 in ORF5-coding region theother 117 genes had the same size of 603 nt as that of VR-2332Sequences alignments showed that the amino acid similarityamong the 117 ORF5 genes ranged from 770 to 100 andthey shared 78ndash99 amino acid similarity with VR-2332 aswell as 865ndash99 with JXwn06 Similar as previous reportthe residue 3ndash39 the putative signal sequence was the mostvariable region whereas the regions 40ndash57 67ndash90 107ndash120138ndash160 and 165ndash184were relatively conserved [25]Howevera novel substitution E170-G170 which was conserved in theChinese strains collected during the period from 2006 to2007 was observed in recent strains

The phylogenetic tree conducted by using the deducedamino acid sequences of ORF5 showed that the Chinesestrains of genotype 2 PRRSV could be divided into 3 differ-ent subgroups (Figure 3) Three strains JL1101 HB1103 andGZ1101 were in subgroup 1 with the representative strainsVR-2332 BJ-4 and CH-1a and the SD1003 was the onlystrain clustered in subgroup 2 with the representative strainMN184A all other 115 strains were clustered into subgroup3 with multiple branches which were completely composedof Chinese strains with the representative JXwn06 and HB-1(sh)2002 Similar to the NSP2 phylogenetic tree a minorbranchwith the JXA1 P80 contained the strains collected bothearlier and later than 2011

35 Full-Length Genomic Analysis of 4 New PRRSV IsolatesThree strains SD0901 LN1101 and GZ1101 with charac-teristic deletion or insertion in NSP2 or ORF5 genes andanother strain BJ1102 were successfully isolated from theclinical samples using MARC-145 cells or PAMs The fourstrains were subjected to full-length genomic sequencingafter plaque purification of three rounds The SD0901 (Gen-Bank Accession number NJ256115) and BJ1102 (GenBankAccession number KF751237) shared same size of completegenome with 15320 nt in length excluding the ploy (A) tailsThe genome sizes of LN1101 (GenBank Accession numberKF751238) and GZ1101 were 15356 nt and 15404 nt respec-tively The BJ1102 was isolated from clinical samples of Tibetmini-pig with acute PRRS symptom in a pig farm whereHP-PRRSV-derived vaccine was used before importing Tibetmini-pig

Sequence alignments indicated that the 51015840UTRof the fourstrains shared nucleotide identities of 910ndash100 with therepresentative genotype 2 PRRSV strains A nucleotide ldquoArdquoinsertion at the position nt 75 of GZ1101 51015840UTR was firstobserved in this study It was shown that major variationswere located in NSP2-coding region including 3-aa deletionat the position aa 593ndash595 in GZ1101 18-aa deletion at theposition aa 482ndash499 in LN1101 30-aa deletion at the positionsaa 482 and aa 533ndash561 in BJ1102 and 31-aa deletion at thepositions aa 468 aa 482 and aa 533ndash561 and an amino acidldquoPrdquo insertion between aa 585 and aa 586 in SD0901 (Figure 4)In addition a new deletion at the position aa 34 of GP5 wasfound inGZ1101 (Figure 5)The individual homology analysisof the other genes was also summarized in SupplementaryTables S2ndashS5

To further classify the evolutionary relationship of these4 isolates the phylogenetic tree was conducted based on their

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

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Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 7: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

BioMed Research International 7

99

99 99 99

99

99

99

62

79

68

65

55

50

95

72

71

70

74

85

92

81

59

91

63

65

65

75

56

50

988

8

74

58

5755

6261

51

Lelystad virus

NSP2

EuroPRRS

V NSP2

MN184A NSP

2

QYYZ NSP

2

QY2010NSP

2

JL1101

BJ-4NSP2

PL97-1

NSP2

RespPRRS M

LVNSP2

DYNSP2

GS2003NSP2

CC-1NSP2

VR-2332NSP2

HN1NSP2

YN-2011NSP2

GZ1101

16244BNSP2

SPNSP2

Prime P

acNS

P2

HB-1(sh)

2002nsp

2Em

2007NSP

2SH

BNSP2

HB-2(sh)

2002NSP2

CH-1R

NSP2

HH08NSP2

CH-1a

NSP2

P129NSP

2NVSL

97-7

985IA

1-4-2NSP

2

IngelvacATP

NSP2

JA142NSP

2

GM2NSP

2

NB04NSP2

BJ1010

HLJ1

104

HLJ1

101

HLJ1

102

HB0906

BJ1006

HuN

1001

HB0905

HN0903

BJ1002

HEN

1102

BJ0905

BJ0810

BJ0909

BJ0806BJ0807HLJ1103LN0801

08HuNNSP

2

09HEB NSP

2

LN0902

SD0903

SD0905

HB1104

NVDC-GD2-2

011 NSP

2

BJ1102

GZ1002

SD0902

09HEN

1NSP

2

SD0906

SD0904

HB1001

HB1002

HEN1103SX1101LN090110-10JL NSP2LN1101

HEN1101SD110109HUB1 NSP2SD1003SX2009 NSP2BJ0804BJ0805JSyx NSP2WUH1 NSP2

SD0901aSD

0901 NSP2

HB1106

HuN1101

HuN1102

AHSS0

902

SD1001

GX1002

AHSS090

1

GX1001

AHQS090

5

AHQS090

1

ZJ1102

SD1004

TJ1101

10-LW3-7 NSP2

AHQS0903

AHQS0907

10-10QN NSP2

HEN1003HB0806HB0903

FSNSP

2

HN0901

HN0902

TJNSP

2TP

NSP

2CW

Z-1-F3NSP2

SC0801

BJ0908

GDQJ

NSP2

HB1103

JL1102

HB0902

BJ0901

BJ0902

BJ0706NSP2

HB0907

BJ0803

BJ0904

ZJ1101

ZJ1103

AHSS0

903

ZJ1001

JXA1

P80NSP2

JL-04

12NSP2

SC1101

BJ1101

GX1003NS

P2

HB1101

LN1001

BJ1005

GZ1001

JX1001

BJ1007

BJ1008

HB0

908

JX1002

HEN

1002

BJ1004

HuN

1003

BJ1003

BJsy06NSP

2

DC

NSP

2

NT0

801NSP

2

BJ1001

HB0

909

HB0802HB0803HuN1002HUB2NSP2GDBY1NSP2HEB1NSP2SY0608

NSP2 HB1102

JX143NSP2 SD16

NSP2 Henan-1NSP2 GDQ

Y1NSP2 HuB1001 YN9

NSP2 JXw

n06NSP

2

JXA1NSP

2

HUN4

NSP2

SC1201 YN

2008

NSP2

CGNSP2

HB0904 GD

NSP2

HB1105

YDNSP2

H

Shaanxi-2

NSP2

HB1201

Subgroup 3

Subgroup 1

Subgroup 2

Figure 1 Phylogenetic tree based on the deduced amino acid sequence of NSP2 HV region The bootstrap consensus tree is shown Thesequence downloaded from GenBank had a suffix ldquoNSP2rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccinestrains were labeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

full-length genomic sequence together with both genotype 1and genotype 2 representative strains It was shown that theSD0901 and BJ1102 were clustered in the subgroup of ChineseHP-PRRSV and HP-PRRSV-derived vaccine virus sharinghigh identity 987 and 984 with JXwn06 respectivelyin addition the LN1101 was the neighbor of HB-1(sh)2002in the same minor branch which share 988 identity witheach other The GZ1101 was close to the minor branch withprototype VR-2332 and BJ-4 (Figure 6) The four strainsexhibited 883ndash978 nucleotide identity with each other

The findings suggest that various PRRSV strains from differ-ent clusters simultaneously circulate and spread in pig farmsin China

4 Discussion

PRRSV is characterized of its extensive geneticantigenicvariation in the field [36] Low replication fidelity of RNApolymerase abundance of quasispecies RNA recombina-tion and immune pressure selection are regarded as the

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 8: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

8 BioMed Research International

99

99

51

65

99

50

74

65

63

98 53 91

99

85

53

84

56

79

99

58

81

8087

62

50

8270

83

59

90

Lelystad virus

ORF3

EuroPRRS

V ORF3

SP ORF3

Prime Pa

c ORF3

MN184A

ORF3

YN-201

1OR

F3

GS2003ORF

3

BJ-4OR

F3

PL97-1

ORF3

VR-23

32OR

F3

RespPRRS M

LVOR

F3

CC-1OR

F3

JL1101

HN1ORF3

HB1103

DYORF3

GZ1101

16244BOR

F3

NVSL

97-7985IA

1-4-2ORF

3

P129ORF

3Ingelvac

ATPORF

3

JA142ORF

3CH-1R

ORF3

HH08ORF

3

HB-2(sh)2002ORF

3

CH-1aORF3

HB-1(sh)2002ORF

3

QYYZ

ORF

3

QY2010ORF

3GM

2ORF

3SD

1003 BJ0

905

NMG0801

BJ1006

HEN

1102

BJ0910

BJ1002

HB1101

BJ0909

HB1102

BJ0810

SHBORF

3HN0903

HB0906

SD1002

BJ0908

Em2007ORF

3

BJ1009

ZJ1001

HLJ1101

HLJ1103

HLJ1102

BJ1007

BJ1008

HLJ1104

JX1002

BJ1004

BJ1005

SC0801

BJ0809

SD16ORF

3

HB0905

HB0907

LN1101

BJ0706ORF

3

NB04ORF

3

BJ0912GZ1001AHQS0905SD1004AHQS0903AHQS090210-LW3-7 ORF3SD1001AHQS090110-10QN ORF3GDQY1 ORF3GDQJ ORF3 210-10JL ORF3HEN1103HuN110109HEN1 ORF3

Henan-1 ORF3NVDC-GD2-2011 ORF3

08HuN ORF3JL1102GD ORF3

SX2009 ORF3BJ1001DC ORF3

HB110409HUB

1 ORF3

HB0909

BJ1010JX1001

09HEB ORF3

HB0904

BJ1101

TJ1101

SD0901

AHSS0

903

AHSS0

905

SD0906

BJ0804

BJ0805

AHSS0

902

AHSS09

01

AHQS0

910

AHQS090

9

AHQS090

8

AHQS0907

AHQS0906

AHSS090

4

HEN1101

FS ORF3

HuN1003

HUN4ORF

3BJ0906

JSyx

ORF

3HN0901

HN0902

HB0

903

BJ0901

BJ0902

GX1002

HEN

1002

HEN

1003

BJ0807

BJ0808

BJ0903

HB0806

SX1101

HB0901

SC1201

SD1101

BJ0806

HB0804

HB0802

HB0803

BJ0803

JXA1OR

F3

GZ1002

JX143

ORF3

HB1106

YDORF3

HB1105

GX1003OR

F3

HB1201

GDBY1OR

F3

SD0903

SD0905

CWZ-1-F3OR

F3

YN9OR

F3

ZJ1102

CGOR

F3

HB1002

GX1001

HB1001

BJsy06ORF

3

TPORF

3

Shaanxi-2

ORF

3

HB0

902

SC1101

BJ1102

LN0801

HuN

1001

BJ1003

HuB

1001

SY0608ORF

3

BJ0911

HB0

908

NMG0802

SD0901ORF3YN2008ORF3TJORF3LN0902LN1001SD0902SD0904WUH1ORF3 JL-0412ORF3 JXA1

P80ORF3TJ090

3 HUB2ORF3 TJ0902

JXwn06

ORF3

HEB1ORF

3

TJ0901 HB0

801 LN0

901 HuN

1002

NT0801

ORF3

ZJ1101 ZJ1

103

Subgroup 3

Subgroup 2

Subgroup 1

Figure 2 Phylogenetic tree based on the deduced amino acid sequence of ORF3 The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF3rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

mechanisms of generating viral heterogeneity and diversitywhich promotes the evolution of PRRSV [37ndash39] Theemergence and reemergence of acute form PRRS is ofteninfluenced by the genetics of PRRSV [36] Since the PRRSoutbreak in China was first documented in 1995 this virusis always accompanied with the Chinese pig industry [27]In 2006 an unparalleled large-scale atypical PRRS outbreakwas reported in China [25 30 31] In the following 1-2 yearsthe HP-PRRSV with 30-aa deletion in NSP2-coding region

rapidly became the dominant in the field meanwhile theclassical and low-pathogenic strains could also be isolatedfrom pig farms [25] In 2011 the HP-PRRSV-derived MLVwas licensed and widely used afterward in the fieldThis situ-ationmight greatly increase the immune selective pressure inpig herds to accelerate the variation and evolution of PRRSV[39] Meanwhile the European genotype 1 PRRSV strainsalso emerged in China [32] resulting in the complexity ofPRRS Therefore it is meaningful to continually survey the

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 9: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

BioMed Research International 9

100

98

93

90

79

72

57 100

53100

8956

9770

52

52

66

52

83

71

87

8157

56

54

64

67

70

59

Lelystad virus

ORF5

EuroPRR

SV ORF5

BJ0907

NVSL 97

-7985IA 1-4-

2ORF

5

Em2007ORF

5

HB-2(sh

)2002ORF

5

JA142

ORF5

Ingelva

c ATP

ORF5

P129OR

F5

CH-1a

ORF5

CH-1R

ORF5

HH08

ORF5

CC-1OR

F5

RespP

PRS M

LVORF5

DYORF5

JL1101

HN1OR

F5

YN-20

11OR

F5

GS2003OR

F5

BJ-4OR

F5PL97-1

ORF5

HB1103

VR-2332OR

F516244BOR

F5GZ

1101

SPOR

F5Prim

ePacORF

5SD

1003

MN184AORF

5GM2ORF

5QYY

ZORF

5QY2

010ORF

5

HB0909

HEB1ORF

5

09H

UB1ORF

5

BJ1004

HEN

1003

SD1001

AHQS0910

AHQS0907

AHQS0908

SD0904

SD0902

GX1002

AHSS0905

AHSS0904

AHSS0902

AHSS0901

AHQS0909

AHQS0906

AHQS0905

AHQS0903

AHSS0903

SD0906

10-10JL

ORF

5AHQS0901

AHQS0902

NB04 ORF5SHB ORF5BJ0706 ORF5BJ1001TJ0901TJ0902TJ0903BJ0803BJ080910-LW3-7 ORF5

SY0608 ORF5

HLJ1101SD1101

BJ110210-10QNORF

5

ZJ1101ZJ1103HuN1002

GDQJ ORF5

09HEB

ORF5

08HuN

ORF5

HuB1001

HuN1102

09HEN

1ORF

5

HB1001

HB1002

HB-1(sh)2002ORF5

HB0906

HN0903

BJ1006

LN1101

HB0907

HEN11

02

BJ0810

HB0905

BJ0909

BJ0905

BJ0910

HEN1103

SX1101

JL1102

BJ0807

BJ0808HLJ1103

HB0904BJ0902BJ1010HB0903BJ0806HB0902BJ0908HB1104HuN1003BJ0805BJ0804ZJ1102

HLJ1102

HB1

201

BJ1007

BJ1008

HLJ1104

BJ1003

SD1004

ZJ1001

BJ1002

SD0901a

BJ1101

JX1002

HB0

806

YN9ORF

5HEN

1101

SD0905

BJ0912

GDBY

1ORF

5BJ0911

SD1002

BJ0906

BJ1005

HEN

1001

SC0801

BJ0903

GZ1002

NVDC

-GD2

-2011OR

F5

LN0801

HB0802

HB0803

HB0804

HB0805

BJ0901

LN0901

HB1101

HB1102

HUN4ORF5 HUB

2ORF5

Henan-1ORF

5

HB0801 SC1

201 YD

ORF5 GZ

1001 HN

0902 NT

0801

ORF5

JXA1OR

F5

JL-04

12OR

F5

GX1003

ORF5

HuN1101

HB1105 JX

A1P80ORF5

Shaan

xi-2ORF5

GX1001

GDQY

1OR

F5

JSyxOR

F5

TJOR

F5

GDOR

F5

YN2008OR

F5

CWZ-1-F3OR

F5

FSOR

F5

HEN

1002

CGOR

F5

DCOR

F5

BJsy06ORF

5

JXwn

06ORF

5

TPORF5WUH1ORF5TJ1101SX2009ORF5SD16ORF5SD0903SD0901ORF5 LN1001LN090

2 JX143ORF5 JX100

1

Subgroup 3

Subgroup 1

Subgroup 2

Figure 3 Phylogenetic tree based on the deduced amino acid sequence of ORF5The bootstrap consensus tree is shown The sequencedownloaded from GenBank had a suffix ldquoORF5rdquo The representative strains were labeled with ldquoblack trianglerdquo and the vaccine strains werelabeled with ldquoblack diamondrdquo The bootstrap values were shown close to the branches

diversity of PRRSV and analyze the phylogenetic relationshipand evolutionary process of field strains

In this studywe amplified and gained 101NSP2HV regionsequences from 128 PRRSV-positive clinical samples Out ofthem 86 had the same 30-aa deletion in NSP2-coding regionas that of JXwn06 and other early HP-PRRSV strains The 86new sequences shared 876ndash100amino acid similaritywitheach other as well as 666ndash695with VR-2332 which wereboth lower than previous corresponding data 934ndash998and 771ndash778 we obtained in 2006-2007 [25] Meanwhile3 novel patterns of deletion or insertion in NSP2-codingregion were first found in this study These results suggest

that the diversity of PRRSV NSP2 region has expanded from2006-2007 to 2008ndash2012The phylogenetic analysis on aminoacid sequence of NSP2 indicated that all new strains in thisstudy were clustered into 2 out of 3 subgroups 2 strains insubgroup 1 with the representative strains VR-2332 BJ-4 andRespPRRS MLV and the other 99 in the subgroup 3 withthe representative strains HB-1(sh)2002 JXwn06 JXA1 andJXA1 P80 suggesting that the strains with 30-aa deletion inNSP2-coding region are still prevailing in the field Amongthem the BJ1102 with low pathogenicity (data not shown)which was closely related with vaccine virus was clusteredtogether with HP-PRRSV-derived vaccine virus in the same

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 10: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

10 BioMed Research International

570 660650640630620610600590580

470 560550540530520510500490480

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

VR-2332 NSP2BJ-4 NSP2CH-1a NSP2NB04 NSP2HB-1(sh)2002 NSP2HB-2(sh)2002 NSP2HUN4 NSP2JXwn06 NSP2JXA1 NSP2JXA1 P80 NSP2BJ1102 NSP2GZ1101 NSP2LN1101 NSP2SD0901 NSP2

L

N-

FC-LLNMIEAGQVGILNDNPREAH-LSNMIEAGQVILNDTNPPGKKEAHN-LSSNMSIEAGQVILNDTSPKKEACY-LLENIEAGQVGILDTNPKKEARHPF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHF-LSNMIEAGQVVILNDTNPKKEAHVF-LSNMIEARQVGVILNDTNPKKEAHKF-LSNIIEAGRVLNDTNPIKAKEAHSVP----YPHNP-LSSNMSIEAGQVILNDTNPKKEAHPLTSNMEAGQVIPNDTNPKCKEA

ILMDNGFGGLFSMLPLMPASHPVGVRMFSHEEANPTTLTLMDNGSETGGLNFSMMPLTPALRVPKLMGSVRMFSQHEEANPTTTLTLMDNGSTGGLNFSMMPLTPALRVPKLMDGSVRMFLSHEEANPTTTLTILA GGLNFSSLVLGPLMPASHVSVVRIMTFFWSEEANPTLTLMDNGSET-GGLNFSMMPLMPASRRVPKLMGSVRT-----------------------------

--------------------------------------------------------------------------------------------------------------------

-----------------------------

TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTLMDNGSET-GGLNFSMMPLPASRRVPKLMGSVRT TTLTILMDNGSET-GGLNFSLMMPLPASFVPKLMIGSVKT TTLTVVLMDNGS------------------PLTPALRVPKLMNGSVRMFLSHEEANPTTTLT-VMDNGSET-GGSLNFSMMPLPASRRVPKLMSGSVRT TTLT

VSLGGDVPNSWEDLAVSSPFDLPTPPEPATPSSELVIVSSPQCIFRPATPLSEPAPIPAPRGTVSRPVTPLSEPIPVPAPRRKFQQVKRLSSAAAIPP

------------

YQDEPLDLSASSQTEYEASP-PAPPQSGGVLGVEGHEAEETLSEISDMSGNIKPASVSSSSSLSSVRITRPKYSAQAIIDSGGPCSGHLQEVKETCLS

Figure 4The alignment ofNSP2 amino acid sequence of PRRSVAmultiple alignment of PRRSVNSP2 amino acid sequences was performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted or inserted residues arelabeled with box

10 20 30 40 50 60 70 80 90 100 110

MLEKCLTAGCCSRLLSLWCIVPFCFAVLANASNDSSSHLQLIYNLTLCELNGTDWLANKFDWAVESFVIFPVLTHIVSYGALTTSHFLDTVALVTVSTAGFVHGRYVLSS Q ACGT VNSNFTGYG YLVNNIQRTGAYYRG YLVNIKNNRTGYYRGY Q VSNPFERTGIYRGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQTGAYYGC FYLVNNIQNTGGAYYGC FFLNNIQTGAYY Q QGFG FYVANITGYYRGC FYLVNNIQTGAYY

-

CH-1a ORF5

HB-1(sh)2002 ORFSHB-2(sh)2002 ORF5

JXwn06 ORF5JXA1 ORF5JXA1 P80 ORF5

VR-2332ORF5BJ-4 ORF5

NB 04 ORF5

HUN4 ORF5

BJ1102 ORF5GZ1101ORF5LN1101ORF5SD0901 ORF5

Figure 5The alignment of PRRSVORF5 amino acid sequence Multiple alignments of PRRSVORF5 amino acid sequences were performedby ClustalW The sequence of VR-2332 is shown on the top the residues conserved with it are hidden The deleted residues are labeled withbox

branch As more and more low pathogenic strains have beenfound to have 30-aa deletion in NSP2-coding region thisdeletion will no longer be defined as the molecular markerof HP-PRRSV

The ORF3 sequences alignment showed that the residueswithin 3 regions including 33ndash46 120ndash133 and 162ndash198 wererelatively conserved among the strains in this study whilethe nt 58ndash71 and 216ndash226 of ORF 3 gene were hypervariableregions More than 50 strains contained the I66-T66 muta-tion which was located at the identified epitope in GP3 [40ndash43] A previous clue suggested that the residue substitutionat this position may be related with inducing neutralizingantibody [40] Whether this mutation is associated with theimmune pressure selection or immune invasion still needsfurther investigation

Even if the ORF5 is the highest variable region of PRRSVstructural proteins the deletion in this region is little recog-nized In this study one amino acid deletion at the positionaa 34 of GP5 was first found in GZ1101 and the sequencealignment showed that this strain had higher homology withVR-2332 and RespPRRS MLV implying that the virus mightbe evolved from the vaccine virus The lowest amino acid

similarity of ORF5 among these strains was 770 whichwas lower than the data (841) in our previous researchsupporting that the diversity of strains has increased since2008 The phylogenetic tree based on deduced amino acidsequence of ORF5 showed that the Chinese PRRSV strainscould be clustered into 3 different subgroups Compared withShi Mangrsquos phylogenetic result based on more than 8000sequences subgroup 1was composed of representative strainslocated in lineage 8 (VR-2332 and CH-1a) lineage 51 (VR-2332 and BJ-4) and lineage 7 (SP and prime Pac) subgroup2 contained representative strains MN184a from lineage 1and the other HP-PRRSV in subgroup 3 was late clusteredinto lineage 8 in Yanyan Nirsquos modified phylogenetic treeeven if the information of Chinese HP-PRRSV had not beenincluded in Shirsquos analysis [44 45]

Because of having novel genetic characterization or beingisolated from special host Tibet mini-pigs four strainsSD0901 LN1101 GZ1101 and BJ1102 in our study weresubjected to full-length genomic sequencing in order tobetter understand their characterizations of whole genomeComparative analysis showed that their complete genomesequence homology ranged from 883 to 978 and they

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 11: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

BioMed Research International 11

005

HUN4

JXA1

JXA1 P80JXwn 06

SD0901

BJ1102HB-1(sh)-2002

LN1101

CH-1aHB-2(sh)-2002

Ingelvac ATPNVSL 97-7985 IA 1-4-2

Prime PacSP

GZ1101VR-2332BJ-4MLV Resp PRRSResp PRRS MLV

MN184ALelystad Virus

100

100

100

100

100

100

100

100

100

100

100

99

99

56

64

94

Figure 6 Phylogenetic tree based on full-length genomic sequence of PRRSV The strains isolated in this study were labeled with ldquoblacktrianglerdquo The bootstrap values were shown close to the branches The numbers below the scale bar indicate amino acid substitution (100x)

were clustered into different branches of genotype 2 PRRSVfurther indicating that PRRSV strains with genetic diversitysimultaneously exist in the field in China

In this study the molecular sequence data of PRRSV wasutilized to characterize the epidemiology and evolutionaryprocess in phylogenetic analysis expecting that it could pro-vide an important clue formodification of diagnosis methodsand design of novel vaccine Hopefully these analyses willbe useful for PRRS control strategy Considering that themodern transportation in pork supply chains can easilyspread the virus nationwide or even internationally andmeanwhile the wide use of attenuated PRRSV live vaccinewill raise the risk of reversion to virulence and increasethe possibility of recombination between vaccine strainsand field strains the RPRSV diversity will be continuallyexpanded and the epidemic situation in the field will be moreand more complicated So if we try to gain a deeper viewof the PRRSV epidemiology the long-term investigationlinked observation between genetic diversity and phenotypicdifference and effort of explaining the mechanism of howHP-PRRSV strains gain the dominance in field should be firstconcerned in future

5 Conclusion

Our analysis results indicated that the genetic diversity ofPRRSV in the field further increased in recent years in Chinadue to the dramatic variations of NSP2 and ORF5 genes ofPRRSV and the 30-aa deletion inNSP2-coding region should

be no longer defined as the only molecular marker of theChinese HP-PRRSV as the PRRSV strain with same deletionand low pathogenicity emerged in the field and the attenuatedlive vaccines derived from HP-PRRSV were widely used inpig farms

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgments

Thisworkwas supported byNationalKeyBasic ResearchPlanGrant (2014CB542700) from the ChineseMinistry of Scienceand Technology and the earmarked fund for Modern Agro-industry Technology Research System of China (CARS-36) from the Chinese Ministry of Agriculture We alsothank Yanhong Chen and Zhenlin Cha for their technicalassistance

References

[1] G Wensvoort E P de Kluyver J M A Pol et al ldquoLelystadvirus the cause of porcine epidemic abortion and respiratorysyndrome a review of mystery swine disease research atLelystadrdquo Veterinary Microbiology vol 33 no 1ndash4 pp 185ndash1931992

[2] M G Garner I F Whan G P Gard and D Phillips ldquoTheexpected economic impact of selected exotic diseases on the pig

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 12: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

12 BioMed Research International

industry of Australiardquo OIE Revue Scientifique et Technique vol20 no 3 pp 671ndash685 2001

[3] J K Lunney D A Benfield and R R R Rowland ldquoPorcinereproductive and respiratory syndrome virus an update onan emerging and re-emerging viral disease of swinerdquo VirusResearch vol 154 no 1-2 pp 1ndash6 2010

[4] E J Neumann J B Kliebenstein C D Johnson et al ldquoAssess-ment of the economic impact of porcine reproductive andrespiratory syndrome on swine production in the UnitedStatesrdquo Journal of the American Veterinary Medical Associationvol 227 no 3 pp 385ndash392 2005

[5] Z Pejsak and I Markowska-Daniel ldquoLosses due to porcinereproductive and respiratory syndrome in a large swine farmrdquoComparative Immunology Microbiology and Infectious Diseasesvol 20 no 4 pp 345ndash352 1997

[6] K K Keffaber ldquoReproductive failure of unknown etiologyrdquoTheAmerican Association of Swine Veterinarians vol 1 no 2 pp1ndash9 1989

[7] G Wensvoort C Terpstra J M Pol et al ldquoMystery swine dis-ease inThe Netherlands the isolation of Lelystad virusrdquo Veteri-nary Quarterly vol 13 no 3 pp 121ndash130 1991

[8] T Baron E Albina Y Leforban et al ldquoReport on the first out-breaks of the porcine reproductive and respiratory syndrome(PRRS) in France Diagnosis and viral isolationrdquo Annales deRecherches Veterinaires vol 23 no 2 pp 161ndash166 1992

[9] R Bilodeau S Dea R A Sauvageau and G P MartineauldquolsquoPorcine reproductive and respiratory syndromersquo in QuebecrdquoVeterinary Record vol 129 no 5 pp 102ndash103 1991

[10] A Boslashtner J Nielsen and V Bille-Hansen ldquoIsolation of porcinereproductive and respiratory syndrome (PRRS) virus in aDanish swine herd and experimental infection of pregnant giltswith the virusrdquoVeterinaryMicrobiology vol 40 no 3-4 pp 351ndash360 1994

[11] H Kuwahara T Nunoya M Tajima A Kato and T SamejimaldquoAn outbreak of porcine reproductive and respiratory syndromein JapanrdquoThe Journal of Veterinary Medical Science vol 56 no5 pp 901ndash909 1994

[12] K-K Conzelmann N Visser P Van Woensel and H-J ThielldquoMolecular characterization of porcine reproductive and res-piratory syndrome virus a member of the arterivirus grouprdquoVirology vol 193 no 1 pp 329ndash339 1993

[13] J J MMeulenberg MM Hulst E J DeMeijer et al ldquoLelystadvirus the causative agent of porcine epidemic abortion andrespiratory syndrome (PEARS) is related to LDV and EAVrdquoVirology vol 192 no 1 pp 62ndash72 1993

[14] R Forsberg ldquoDivergence time of porcine reproductive andrespiratory syndrome virus subtypesrdquo Molecular Biology andEvolution vol 22 no 11 pp 2131ndash2134 2005

[15] J J M Meulenberg and A P-D Besten ldquoIdentification andcharacterization of a sixth structural protein of Lelystad virusthe glycoprotein GP2 encoded byORF2 is incorporated in virusparticlesrdquo Virology vol 225 no 1 pp 44ndash51 1996

[16] J J M Meulenberg A P-D Besten E P De Kluyver R JM Moormann W M M Schaaper and G Wensvoort ldquoCha-racterization of proteins encoded by ORFs 2 to 7 of Lelystadvirusrdquo Virology vol 206 no 1 pp 155ndash163 1995

[17] A P van Nieuwstadt J J M Meulenberg A van Essen-Zandbergen et al ldquoProteins encoded by open reading frames3 and 4 of the genome of Lelystad virus (Arteriviridae) arestructural proteins of the virionrdquo Journal of Virology vol 70 no7 pp 4767ndash4772 1996

[18] W-H Wu Y Fang R Farwell et al ldquoA 10-kDa structuralprotein of porcine reproductive and respiratory syndrome virusencoded by ORF2brdquo Virology vol 287 no 1 pp 183ndash191 2001

[19] J A den Boon K S Faaberg J J M Meulenberg et al ldquoPro-cessing and evolution of theN-terminal region of the arterivirusreplicase ORF1a protein identification of two papainlike cys-teine proteasesrdquo Journal of Virology vol 69 no 7 pp 4500ndash4505 1995

[20] E J Snijder A LMWassenaar andW JM Spaan ldquoProteolyticprocessing of the replicase ORF1a protein of equine arteritisvirusrdquo Journal of Virology vol 68 no 9 pp 5755ndash5764 1994

[21] L C vanDinten A LMWassenaar A E GorbalenyaW J MSpaan and E J Snijder ldquoProcessing of the equine arteritis virusreplicase ORF1b protein identification of cleavage productscontaining the putative viral polymerase and helicase domainsrdquoJournal of Virology vol 70 no 10 pp 6625ndash6633 1996

[22] A L M Wassenaar W J M Spaan A E Gorbalenya and EJ Snijder ldquoAlternative proteolytic processing of the arterivirusreplicase ORF1a polyprotein evidence that NSP2 acts as acofactor for the NSP4 serine proteaserdquo Journal of Virology vol71 no 12 pp 9313ndash9322 1997

[23] EM Bautista J JMMeulenberg C S Choi andTWMolitorldquoStructural polypeptides of the american (VR-2332) strain ofporcine reproductive and respiratory syndrome virusrdquo Archivesof Virology vol 141 no 7 pp 1357ndash1365 1996

[24] H Mardassi S Mounir and S Dea ldquoStructural gene analysisof a Quebec reference strain of porcine reproductive and res-piratory syndrome virus (PRRSV)rdquo Advances in ExperimentalMedicine and Biology vol 380 pp 277ndash281 1995

[25] L Zhou S Chen J Zhang et al ldquoMolecular variation analysis ofporcine reproductive and respiratory syndrome virus in ChinardquoVirus Research vol 145 no 1 pp 97ndash105 2009

[26] M Shi T T Lam C C Hon et al ldquoMolecular epidemiologyof PRRSV a phylogenetic perspectiverdquo Virus Research vol 154no 1-2 pp 7ndash17 2010

[27] B Guo Z Chen W Liu and Y Cui ldquoPorcine reproductiveand respiratory syndrome virus was isolated from abortive fetusof suspected PRRSrdquo Chinese Journal of Animal and PoultryInfectious Disease vol 87 no 2 pp 1ndash5 1996

[28] L Zhou and H Yang ldquoPorcine reproductive and respiratorysyndrome in Chinardquo Virus Research vol 154 no 1-2 pp 31ndash372010

[29] L Zhou Y Y Ni P Pineyro et al ldquoBroadening the heterologouscross-neutralizing antibody inducing ability of porcine repro-ductive and respiratory syndrome virus by breeding the GP4 orM genesrdquo PLoS ONE vol 8 no 6 2013

[30] K Tian X Yu T Zhao et al ldquoEmergence of fatal PRRSVvariants unparalleled outbreaks of atypical PRRS in China andmolecular dissection of the unique hallmarkrdquo PLoS ONE vol 2no 6 Article ID e526 2007

[31] Y-J Zhou X-F Hao Z-J Tian et al ldquoHighly virulent por-cine reproductive and respiratory syndrome virus emerged inChinardquo Transboundary and Emerging Diseases vol 55 no 3-4pp 152ndash164 2008

[32] N Chen Z Cao X Yu et al ldquoEmergence of novel Europeangenotype porcine reproductive and respiratory syndrome virusin mainland Chinardquo Journal of General Virology vol 92 no 4pp 880ndash892 2011

[33] H Zhang X Guo X Ge Y Chen Q Sun and H YangldquoChanges in the cellular proteins of pulmonary alveolar macro-phage infected with porcine reproductive and respiratory

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 13: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

BioMed Research International 13

syndrome virus by proteomics analysisrdquo Journal of ProteomeResearch vol 8 no 6 pp 3091ndash3097 2009

[34] L Zhou J Zhang J Zeng et al ldquoThe 30-amino-acid deletion inthe Nsp2 of highly pathogenic porcine reproductive and respi-ratory syndrome virus emerging in China is not related to itsvirulencerdquo Journal of Virology vol 83 no 10 pp 5156ndash51672009

[35] D Liu R Zhou J Zhang et al ldquoRecombination analysesbetween two strains of porcine reproductive and respiratorysyndrome virus in vivordquoVirus Research vol 155 no 2 pp 473ndash486 2011

[36] K F Key G Haqshenas D K Guenette S L Swenson T EToth and X-J Meng ldquoGenetic variation and phylogenetic ana-lyses of the ORF5 gene of acute porcine reproductive and res-piratory syndrome virus isolatesrdquo Veterinary Microbiology vol83 no 3 pp 249ndash263 2001

[37] R R R Rowland M Steffen T Ackerman and D A BenfieldldquoThe evolution of porcine reproductive and respiratory syn-drome virus quasispecies and emergence of a virus subpopu-lation during infection of pigs with VR-2332rdquoVirology vol 259no 2 pp 262ndash266 1999

[38] S Yuan C J Nelsen M P Murtaugh B J Schmitt and KS Faaberg ldquoRecombination between North American strainsof porcine reproductive and respiratory syndrome virusrdquo VirusResearch vol 61 no 1 pp 87ndash98 1999

[39] S Costers D J Lefebvre J van Doorsselaere M Vanhee P LDelputte and H J Nauwynck ldquoGP4 of porcine reproductiveand respiratory syndrome virus contains a neutralizing epitopethat is susceptible to immunoselection in vitrordquo Archives ofVirology vol 155 no 3 pp 371ndash378 2010

[40] L Zhou Y Y Ni P Pineyro et al ldquoDNA shuffling of theGP3 genes of porcine reproductive and respiratory syndromevirus (PRRSV) produces a chimeric virus with an improvedcross-neutralizing ability against a heterologous PRRSV strainrdquoVirology vol 434 no 1 pp 96ndash109 2012

[41] M de Lima A K Pattnaik E F Flores and F A Osorio ldquoSero-logic marker candidates identified among B-cell linear epitopesof Nsp2 and structural proteins of a North American strain ofporcine reproductive and respiratory syndrome virusrdquoVirologyvol 353 no 2 pp 410ndash421 2006

[42] M Vanhee W Van Breedam S Costers M Geldhof Y NoppeandH Nauwynck ldquoCharacterization of antigenic regions in theporcine reproductive and respiratory syndrome virus by the useof peptide-specific serum antibodiesrdquo Vaccine vol 29 no 29-30 pp 4794ndash4804 2011

[43] Y-J Zhou T-Q An Y-X He et al ldquoAntigenic structure ana-lysis of glycosylated protein 3 of porcine reproductive and respi-ratory syndrome virusrdquo Virus Research vol 118 no 1-2 pp 98ndash104 2006

[44] M Shi T T-Y Lam C-C Hon et al ldquoPhylogeny-basedevolutionary demographical and geographical dissection ofnorth american type 2 porcine reproductive and respiratorysyndrome virusesrdquo Journal of Virology vol 84 no 17 pp 8700ndash8711 2010

[45] Y Y Ni T Opriessnig L Zhou et al ldquoAttenuation of porcinereproductive and respiratory syndrome virus by molecularbreeding of virus envelope genes from genetically divergentstrainsrdquo Journal of Virology vol 87 no 1 pp 304ndash313 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Page 14: Research Article Genetic Diversity Analysis of Genotype 2 ...downloads.hindawi.com/journals/bmri/2014/748068.pdf · Gel DNA Extraction Kit (BioDev Co., Beijing, China) and then subjected

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology


Genotype, Fenotype en Chromosomen

Genotype, Fenotype en Chromosomen

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ND Lokal Genotype 7

ND Lokal Genotype 7

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BIODEV published pdf

BIODEV published pdf

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Estrogen Ariticle - 5 Genotype

Estrogen Ariticle - 5 Genotype

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Genotype Phenotype Power Point Presentation

Genotype Phenotype Power Point Presentation

Education
Genotype analysis and graph databases · Genotype analysis and graph databases AndrewStephenLaw ... Chapter2 ... E7-8870v3CPUsrunningat2.10GHz

Genotype analysis and graph databases · Genotype analysis and graph databases AndrewStephenLaw ... Chapter2 ... E7-8870v3CPUsrunningat2.10GHz

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Genotype-phenotype correlation In PKU

Genotype-phenotype correlation In PKU

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Ne&as4 - California Lutheran Universityearth.callutheran.edu/.../Departments/BioDev/marcey/biol_426/mess_… · BIOLOGY: MESELSON AND STAHL HOURS A iC....._ 0 2.1 4.3: 14.9 ~~~~~

Ne&as4 - California Lutheran Universityearth.callutheran.edu/.../Departments/BioDev/marcey/biol_426/mess_… · BIOLOGY: MESELSON AND STAHL HOURS A iC....._ 0 2.1 4.3: 14.9 ~~~~~

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Genotype distribution of

Genotype distribution of

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Clinical Study Genotype by Energy Expenditure Interaction ...downloads.hindawi.com/journals/bmri/2014/845207.pdfClinical Study Genotype by Energy Expenditure Interaction and Body Composition

Clinical Study Genotype by Energy Expenditure Interaction ...downloads.hindawi.com/journals/bmri/2014/845207.pdfClinical Study Genotype by Energy Expenditure Interaction and Body Composition

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Phenotype-Genotype Integrator (PheGenI) Updates

Phenotype-Genotype Integrator (PheGenI) Updates

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Genotype and Phenotype

Genotype and Phenotype

Documents
Relationship between Genotype and Phenotype

Relationship between Genotype and Phenotype

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Genotype B5 Lineage 1 (2008)

Genotype B5 Lineage 1 (2008)

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Genotype vs. Phenotype

Genotype vs. Phenotype

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Genotype Determination

Genotype Determination

Documents
GENETIC ALTERATIONS IN CRANIOSYNOSTOSIS, GENOTYPE

GENETIC ALTERATIONS IN CRANIOSYNOSTOSIS, GENOTYPE

Documents
Identification and Sequencing of Genotype VII of Newcastle ...ejvs.journals.ekb.eg/article_4021_1c0cad942e86e4f576cfd7bbdb93919… · Identification and Sequencing of Genotype VII

Identification and Sequencing of Genotype VII of Newcastle ...ejvs.journals.ekb.eg/article_4021_1c0cad942e86e4f576cfd7bbdb93919… · Identification and Sequencing of Genotype VII

Documents
Phenomics: Genotype to Phenotype

Phenomics: Genotype to Phenotype

Documents
HEREDITARY - Sinoe Medical Associationsinoemedicalassociation.org/AP4/HEREDITARY.pdf · Genotype and Phenotype • Genotype: ... Dominant-Recessive Inheritance ... Affected female

HEREDITARY - Sinoe Medical Associationsinoemedicalassociation.org/AP4/HEREDITARY.pdf · Genotype and Phenotype • Genotype: ... Dominant-Recessive Inheritance ... Affected female

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My Genotype - Bite Sci-zed

My Genotype - Bite Sci-zed

Documents
Evolutionary Robotics The Genotype-to-Phenotype Map The genotype to phenotype map: the algorithm that transforms the genotype into the phenotype. Direct

Evolutionary Robotics The Genotype-to-Phenotype Map The genotype to phenotype map: the algorithm that transforms the genotype into the phenotype. Direct

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Studies Genotype Phenotype Association

Studies Genotype Phenotype Association

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Punnet Squares, Linked Genes and Pedigrees. Predicting offspring genotype If we know the genotype of the parents, we can predict the genotype of the offspring,

Punnet Squares, Linked Genes and Pedigrees. Predicting offspring genotype If we know the genotype of the parents, we can predict the genotype of the offspring,

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Genotype Tissue Expression ( GTEx ) Project

Genotype Tissue Expression ( GTEx ) Project

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Two-locus systems. Scheme of genotypes genotype Two-locus genotypes Multilocus genotypes genotype

Two-locus systems. Scheme of genotypes genotype Two-locus genotypes Multilocus genotypes genotype

Documents
Genotype Calling from Population-Genomic Sequencing Data...inferred (called) genotype of the individual is the genotype that max-imizes Equation 3. To avoid calling genotypes at false

Genotype Calling from Population-Genomic Sequencing Data...inferred (called) genotype of the individual is the genotype that max-imizes Equation 3. To avoid calling genotypes at false

Documents
Dominant vs. Recessive and Genotype vs. Phenotype Dominant vs. Recessive and Genotype vs. Phenotype

Dominant vs. Recessive and Genotype vs. Phenotype Dominant vs. Recessive and Genotype vs. Phenotype

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Evaluation Standard Estimated Multilocus Genotype

Evaluation Standard Estimated Multilocus Genotype

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Phenotype vs Genotype

Phenotype vs Genotype

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