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Full-genome characterization of G8P[8] rotavirus emerged among children with diarrhea in 1
Croatia, in 2006 2
3
Roberto Delogu1‡, Alessandra Lo Presti2‡, Franco Maria Ruggeri3#, Eleonora Cella2, Marta Giovanetti2, 4
Massimo Ciccozzi2, Suncanica Ljubin Sternak4, Suzana Bukovski-Simonoski5, Amarela Lukic-Grlic6, 5
Giovanni Ianiro1, Lucia Fiore1. 6
7
1National Center for Immunobiologicals Research and Evaluation, 2Deptartment of Infectious, 8
Parasitic and Immune-Mediated Diseases, and 3Department of Veterinary Public Health and Food 9
Safety, Istituto Superiore di Sanità, Rome, Italy; 4Croatian National Institute of Public Health, and 10
School of Medicine University of Zagreb, Zagreb, Croatia; 5University Hospital for Infectious Diseases 11
“Dr Fran Mihaljevic”, Zagreb, Croatia; 6Zagreb University Children’s Hospital, and School of 12
Medicine University of Zagreb, Zagreb, Croatia. 13
14
‡RDL and ALP have equally contributed to the work 15
16
Running title: Full-genome sequencing of G8P[8] rotavirus in Croatia 17
18
#Corresponding author: 19
Franco M Ruggeri, PhD 20
Dept. Veterinary Public Health & Food Safety 21
Istituto Superiore di Sanità, 22
Viale Regina Elena, 299, 00161 Rome, Italy 23
Phone: +39 06 4990 2980 24
Copyright © 2013, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.00396-13 JCM Accepts, published online ahead of print on 20 February 2013
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Fax: +39 06 4938 7101 25
E-mail: [email protected] 26
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Abstract 28
A 2006 epidemic G8P[8] rotavirus from Croatia was whole-genome sequenced, showing a Wa-like 29
genotype constellation. Its VP7 clustered with DS1-like G8 African rotaviruses, and a G8P[4] German 30
strain. Remaining genes clustered with contemporary Belgian G1P[8] rotaviruses, suggesting 31
reassortment between human G8 and G1P[8] rotaviruses, in Croatia or other European countries. 32
33
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Rotavirus A (RVA) causes acute gastroenteritis in man and animals globally, killing 450,000 children 34
per year, mainly in developing countries (1). 35
RVA genome includes 11 double-stranded RNA segments, encoding six viral (VP1-4,6,7) and six non-36
structural proteins (NSP1-6) (2). Rotavirus outer layer protein VP7 and spike protein VP4 are 37
neutralization antigens, determining virus genotypes G (glycoprotein, VP7) and P (protease-sensitive, 38
VP4). At least 27 G and 37 P genotypes have been identified among human, animal and avian RVAs 39
(3-5). 40
Five genotypes (G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]) represent over 90% of global human 41
RVAs (6), that may explain the efficacy of both monovalent (P[8], G1) Rotarix and pentavalent (P[8], 42
G1-4) RotaTeq vaccines against rotavirus diarrhea worldwide (7). 43
Serotypes other than G1-4, and strains evolving by mutation or genomic reassortment between human 44
and/or animal RVAs represent an emerging threat for children (8-10). Millions of people are exposed to 45
animal rotaviruses worldwide, and several uncommon human genotypes (G6, G8, G12, P3[9], P5A[3]) 46
closely resemble animal RVA strains (11). 47
Molecular epidemiology and viral typing can help monitor emergence of novel RVAs, confirming 48
current vaccine efficacy against unconventional genotypes. 49
Reassortment events can involve any dsRNA segments, and G and P typing may be insufficient to 50
investigate RVA origin and inter-species transmission. Genotyping based on sequencing all 11 genome 51
segments (3, 4) is very helpful, distinguishing 9 VP1 (R), 9 VP2 (C), 8 VP3 (M), 35 VP4 (P), 16 VP6 52
(I), 27 VP7 (G), 16 NSP1 (A), 9 NSP2 (N), 12 NSP3 (T), 14 NSP4 (E), and 11 NSP5 (H) genotypes. 53
Bovine-like G8 rotavirus was first reported in an Indonesian child (12), and further G8 RVA human 54
cases occurred worldwide, including industrialized countries (13-21). Using full-genome sequencing, 55
G8P[8] African RVAs were shown to involve reassortment between at least four human, swine, and 56
bovine strains (22). 57
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This paper reports the whole genome characterization of a G8P[8] human rotavirus strain emerged in 58
Croatia in 2006 (23). 59
Cases showed severe gastroenteritis, and included <5 year-old children from Croatian hospitals in 60
2005-2006. Stools were screened by Rotalex, and RVA characterized by RT-PCR (23). Agarose gel 61
purified amplicons were sequenced using RT-PCR primers (8, 18, 24, 25), assembling consensus 62
sequences for all gene segments (18, 22, 26). 63
Twelve datasets were built, including all NCBI sequences (http://www.ncbi.nlm.nih.gov/pubmed), 64
showing >89% similarity with query sequences (between 116 for VP2 and 127 for NSP4). 65
Sequences were aligned using ClustalX software (27), and edited by Bioedit software. The ModelTest 66
v3.0 (28) with hierarchical likelihood ratio test was used to select the best-fit models for sequence data 67
analysis. 68
Bayesian phylogenetic trees were constructed for VP7, VP4 and NSP4 datasets (29), using GTR+I+G 69
nucleotide substitution model for VP7, and HKY+G model for VP4/NSP4. 70
Markov Chain Monte Carlo searches were made (50x106 generations, tree-sampling every 5000 71
generation, and 10% burn-in fraction. Clade statistical support followed >0.90 posterior probability). 72
Maximum-likelihood (ML) phylogenetic trees of NSP1-3, NSP5-6, VP1-3 and VP6 were generated 73
with PAUP* 4.0 package (30), using GTR+I+G for VP6, NSP1, VP1; HKY+G model for NSP6/NSP5; 74
TrN+I+G for NSP2, NSP3, VP2 and VP3 datasets. Statistical robustness and reliability of branching 75
order within trees was confirmed by bootstrap analysis (1000 replicates), considering bootstrap >70% 76
as clades support. 77
Sequences were deposited in GenBank database (accession numbers JQ988894-JQ988904). 78
79
Initial sequencing of genes encoding, VP4 and NSP4 for 10/31 G8P[8] RVAs showed nearly complete 80
nucleotide identity suggesting circulation of a single G8P[8] strain in Croatia in 2005-6. The consensus 81
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Croatia 2006 (CR2006) VP7 gene showed >98% nucleotide identity with other human G8 GenBank 82
strains, particularly from neighboring Slovenia, 2006, and Africa, 2000-2004. CR2006 VP4 and NSP4 83
recalled human G3P[8] strains reported in Italy, 2005 (31). To investigate Croatian G8P[8] origin, full-84
genome sequencing was performed, using RotaC (http://rotac.regatools.be) (32). CR2006 presented a 85
genotype G8-P[8]-I1-R1-C1-M1-A1-N1-T1-E1-H1. 86
Bayesian analysis identified CR2006 VP7 in a statistically supported cluster with African (Malawi, 87
Tunisia, Cameroon, Ethiopia), and European strains (Slovenia [Sl-885/06], Germany [GER1H-09]) 88
(Fig 1A). In VP4 and NSP4 trees (Fig 1B and 2), CR2006 also clustered with European and African 89
RVAs, including Hu/Slo/2006/G8P[8]/Sl-885/06, Hu/Italy/2005/G3P[8], 90
Hu/Ethiopia/2004/G8P[8]/ARN, Hu/Tunisia/2000-2004/G8P[8]/ARN. 91
CR2006 clustered with Wa-like genotype constellation Hu/Bel/2005-2009/G1P[8], 92
Hu/Ger/G3G12P[8]/GER126-08, and other global RVAs in NSP1-NSP6, VP1-VP3, and VP6 93
(Supplementary Fig S1-9). 94
G8P[8] rotavirus circulated abnormally in Croatia in 2006, reaching 15.8% of children hospitalized 95
with diarrhea (23). Full-genome sequencing of these viruses was performed to investigate links 96
between their emergence and genome characters. 97
The G8 rotavirus genotype, normally combined with P[1],[5],[11] or [21] types (11, 33, 34), is typical 98
of bovine strains, but has also been sporadically detected in humans since 1978 (35) associated with 99
different P-types (13, 16-18, 36-38). Human G8 strains normally exhibit P[4] or P[6] both in developed 100
countries and in Africa, where they are particularly prevalent (15-17, 26). Recently, full-genome 101
sequencing of a human G8P[8] rotavirus in Democratic Republic of Congo (DRC) in 2003 (18, 22) 102
suggested this strain to originate from reassortment between a bovine G8P[6] ancestor with three or 103
more different rotaviruses, including human Wa- and DS1-like strains. This strain exhibited a DS-1-104
like genotype constellation. 105
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Differently, Croatian CR2006 G8P[8] presented the Wa-like constellation (3) G8-P[8]-I1-R1-C1-M1-106
A1-N1-T1-E1-H1, all genes showing close evolutionary relationship to prototype G1P[8] human strain 107
Wa, except the VP7 gene, related to African and Slovenian human G8 strains. Rotaviruses are usually 108
species-specific, and gene reassortment with human strains appears necessary for zoonotic adaptation 109
(26, 39-41). Characterization of epidemic CR2006 further supports this view. As VP7 plays a major 110
role in the immune response (42, 43), CR2006 emergence and epidemic spread may have involved 111
reassortment with common rotaviruses pre-existing in Croatia. 112
CR2006 was more frequent in younger children (23) than any other rotavirus genotypes co-circulating 113
in Croatia, 2006. This could be fortuitous, or suggest that maternal antibodies protected younger 114
children from CR2006 less than from other serotypes. Maternal immunity fades quickly (44, 45), and it 115
is conceivable that older Croatian children in 2006 were similarly unprotected hence susceptible to G8 116
or any other common rotavirus genotypes. 117
Phylogenetic analyses revealed that, except for the VP7 gene, CR2006 was more closely related to 118
humans G1P[8] strains identified between 2005-2009 in Belgium. Therefore, CR2006 origin likely 119
involved reassortment of a Wa-like rotavirus circulating in one or more countries of Europe, including 120
Belgium and possibly Croatia, with a co-circulating G8 virus. In 2006, a similar G8P[8] strain was 121
reported and partially sequenced in Slovenia (20), whereas no G8P[8] rotaviruses were detected in 122
other European countries. It is possible that the reassortment event leading to CR2006 may have 123
occurred in or near Croatia. Low VP7 correlation between CR2006 and G8 DS1-like rotaviruses 124
characterized in African DRC (18) indicates independent evolution of these G8 reassortants. 125
Conversely, CR2006 shared a similar gene 9 with human G8 strains from Northern/Central Africa in 126
the early 2000s, suggesting that the G8 gene followed immigration routes from Africa. A closely 127
similar VP7 was found in a G8P[4] human strain identified in Germany two years later (21), suggesting 128
that the CR2006 G8 gene participated to additional reassortments with European DS1-like rotaviruses. 129
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The German strain was strictly correlated with the DRC G8 rotaviruses reported earlier, except for VP4 130
(18), and may represent a further evolution of G8P[8] rotaviruses imported from Africa. The VP7 of all 131
European G8 strains differed markedly from bovine G8 rotaviruses, contrary to a recent human-bovine 132
reassortment event within Europe. 133
Rotavirus genetic evolution is certainly influenced by immune pressure (46), that may favor G8 and 134
other uncommon rotavirus G-types spreading (19, 20, 47), as occurred for G9 strains originated from 135
swine (48, 49). International transmission of genotypes is facilitated by globalization and travelling, 136
urging worldwide rotavirus strain surveillance to anticipate emergence of novel strains and 137
animal/human reassortants, and monitor effectiveness of current human rotavirus vaccines. 138
139
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Acknowledgments 140
Grant sponsor: Ministry of Health, Italy (to LF CCM ‘‘Epidemiologia molecolare di rotavirus in eta` 141
pediatrica in Italia. Creazione di una rete di sorveglianza per monitorare la diffusione e l’evoluzione di 142
genotipi virali’’ and to FMR Strategic Program: ‘‘Preparedness and response to emerging zoonoses and 143
exotic viral infection through an integrated medical and veterinary approach’’); Grant sponsor: 144
ISS/NIH (to FMR ‘‘Molecular and antigenic evolution of rotavirus strains of human and animal 145
origin’’); Grant sponsor: EuroRotaNet (http://www.eurorota.net). 146
147
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Legends to the Figures 299
300
FIG. 1. Bayesian phylogenetic analysis of Human Rotavirus VP7 and VP4 nucleotide sequences. The 301
trees were rooted by using the midpoint rooting method. Branch lengths were estimated with the best 302
fitting nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant 303
statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle 304
for simplicity, and its size is an indication of the number of sequences it represents. The number of the 305
strain for each place of isolation is indicated in parenthes. RV strain isolated in Croatia is in boldface 306
type. For each strain, the following data are given: species of origin/place of isolation/year/strain name. 307
The number of substitutions per site is indicated by the scale bar. 308
(A) Phylogenetic tree based on VP7 gene nucleotide sequence. (B) Detail of the phylogenetic 309
relationship between G8 genotype RV strains. 310
(C) Phylogenetic tree based on VP4 gene nucleotide sequence. (D) Detail of the phylogenetic 311
relationship between CR2006 and other RV strains. 312
313
FIG. 2. Bayesian phylogenetic analysis of Human Rotavirus NSP4 nucleotide sequences. The tree was 314
rooted by using the midpoint rooting method. Branch lengths were estimated with the best fitting 315
nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant 316
statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle 317
for simplicity, and its size is an indication of the number of sequences it represents. The number of the 318
strain for each place of isolation is indicated in parenthes. Phylogenetic clusters containing CR2006 are 319
boxed and the strain is in boldface type. For each strain, the following data are given: species of 320
origin/place of isolation/year/strain name. The number of substitutions per site is indicated by the scale 321
bar. 322
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VP7
A.
G8
G1
G12G3
G10
G2
G4
0.2
Hu / Jap / 1982 / G3P9 / AU-1 La / Italy / 1996 / G3P14 / 30/96
Bo / India / 2007 / G8P14 (3)
Hu / Philip / G12P4 / L26
Hu / Africa / 2004 / G8P6 / SA4948JHB/04
Hu / Australia / 2002 / G8P14 (2)
Hu / G1P8 / KU
Bo / USA / 1996 / G8 / Cody I801
Hu / India / G9 / RMC321
Hu / China / 1996 / G2P4 / TB-ChenBo / G6 / UK - NCDV
Bo / Kor / G8 (28)
Bo / USA / 2003 / G8 (4)
Hu / Malawi / 2000 / G8P8 / MW4103
Hu / India / G8P1 / MP409
Bo / USA / 2007 / G8
Hu / Finland / HAL 1166Hu / Australia / G8 (3)
Hu / Congo / 2003 / G8P8 / DRC88
Po / Kor / 2006 (15)
Bo / Thailand / G10
Ov / Spain / G8P14 / OVR762
Hu / USA / 1976 / G2P4 / DS-1
Lama / Arg / 1998 / G8P14 (4)
Hu / Cameroon / 2000 / G8 / ARN (4)
Hu / Nigerian / 2003 / G8 (2)
Po / USA / 1976 / G4P6 / Gottfried
Po / G5P7 / OSU
Bo / Jap / G8 (5)
Hu / UK / 1985 / G8 (2)
Hu / USA / 1974 / G1P8 / Wa
Si / Kenia / 1999 / G8P6 / Si/KY1646
Hu / Egypt / G8P14 / EGY1850
Bo / Nigeria / 2003 / G8 / NGRBg8
Hu / Tunisia / 2002-04 / G8 (3)
Hu / Switz / G8 / U25.96
Hu / Croatia / 2006 / G8P8 / CR2006
Hu / Congo / 2003 / G8P6 / DRC86
Hu / Malawi / 2000-01 / G8 (7)
Si / Sa11 / G3Hu / Bangl / 2000-03 / G12P6 (2)
Hu / Africa / G8 / QEH14262
Bo / Thailand / G8 / A5Hu / Jap / 1994-05 / G8 (2)
Hu / Egypt / G8P14 / EGY2295
Hu / 69M / G8P10
Hu / India / G10P11 / I321
Hu / Ger / 2009 / G8P4 / GER1H-09
Hu / Ethiopia / 2004 / G8 / ARN6810
Si / USA / PTRV
Hu / Kenia / 2002 / G8 (3)
Hu / Brazil / G8P4 / R291
Hu / UK / 678
Hu / Slo / 2006 / G8P8 / SI-885/06
1
0,97
1
1
1
1
1
1
0,951
1
0,991
1
1
0,94
0,91
1
0,91
1
1
1
1
Hu / Cameroon / 2000 / G8 / ARN6780
Hu / Ethiopia / 2004 / G8 / ARN6810
Hu / Tunisia / 2000 / G8 / ARN6862
Hu / Malawi / 2000 / G8P8 / MW4103
Hu / Tunisia / 2004 / G8 / ARN6736
Hu / Ger / 2009 / G8P4 / GER1H-09
Hu / Cameroon / 2000 / G8 / ARN6809
Hu / Cameroon / 2000 / G8 / ARN6782
Hu / Croatia / 2006 / G8P8 / CR2006
Hu / Tunisia / 2002 / G8 / ARN6854
Hu / Slo / 2006 / G8P8 / SI-885/06
Hu / Cameroon / 2000 / G8 / ARN67871
0,94
B.
FIG. 1. Bayesian phylogenetic trees of Human Rotavirus VP7 and VP4 nucleotide sequences. The trees were rooted by using the midpoint rooting method. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle for simplicity, and its size is an indication of the number of sequences it represents. The number of the strain for each place of isolation is indicated in parenthes. RV strain isolated in Croatia is in boldface type. For each strain, the following data are given: species of origin/place of isolation/year/strain name. The number of substitutions per site is indicated by the scale bar. (A) Phylogenetic tree based on VP7 gene nucleotide sequence. (B) Detail of the phylogenetic relationship between G8 genotype RV strains. (C) Phylogenetic tree based on VP4 gene nucleotide sequence.
0.3
Hu / Ger / 2009 / G8P4 / GER1H-09
Hu / India / G9 / RMC321
Hu / Bel / 2003 / G12P8 / B4633-03
Hu / China / 2007 / G3P8 (4)
Hu / Croatia / 2006 / G8P8 / CR2006Hu / Slo / 2006 / G8P8 / SI-885/06
Hu / Bangl / / G12P8 / Dhaka25-02
Hu / India / 2006 / G1P8 (3)
Hu / Bel / 2009 / G1P8 (8)
Hu / USA / 1974 / G1P8 / Wa
Hu / Jap / P8 (2)
Hu / USA / 2010 / G9P11 / 116E/AG
Hu / Italy / 2005 / G9P8 / BIA2
Hu / Bel / 2008 / G1P8 (6)
Hu / Italy / 2006 / G9P8 (2)
Hu / Russia / 2004-09 / P8 (16)
Hu / Russia / 2004-07 / P8 (2)
Hu / Russia / 2008 / G4P8 (3)
Hu / Bangl / 2003 / G12P6 (2)
Hu / China / 2007 / G3P8 (4)
Hu / Congo / 2003 / G8P8 / DRC88
Hu / Africa / 2000-04 / G8 / ARN (5)
Hu / Russia / 2009 / G4P8 (4)
Hu / Thailand / 2009 / P8 (7)
La / Italy / 1996 / G3P14 / 30/96
Hu / China / 2007 / P8 (6)
Hu / USA / 2007 / G1P8
Hu / Bangl / 2005 / P8 (9)
Hu / China / 1996 / G2P4 / TB-ChenHu / USA / 1976 / G2P4 / DS-1
Hu / India / 2006 / P8 / mani-133/06
Hu / Congo / 2003 / G8P6 / DRC86
Hu / GBR / 1975 / G4P6 / ST3
Hu / Bel / 2006 / G1P8 (7)Hu / Russia / 2006 / G9P8 / RUS-Nov06-1289
Po / USA / 1976 / G4P6 / Gottfried
Hu / USA / G1P8 / US6668
Hu / Ger / 2008 / G3G12P8 / GER126-08
Hu / Malawi / 1997 / P8 (4)
Hu / Italy / 2005 / G9P8 (2)
0,93
0,99
1
0,95
1
1
0,93
1
1
0,98
1
1
1
0,99
VP4
C.
P[8]
P[4]
P[11]
P[6]
P[14]
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0.3
Hu / Jap / G3 (2)
Hu / Tunisia / 2000-04 / G8 / ARN (3)
Hu / G3P2 / RV3
Hu / Jap / G3 / ITO
Hu / Bangl / 2000-03 / G12P6 (4)
Hu / Bel / 2009 / G1P8 (2)
Hu / G1P8 / KU
La / Italy / 1996 / G3P14 / 30/96
Hu / GBR / 1975 / G4 / ST3
Hu / Italy / 2006 / G3P6 / 128/07-34
Hu / South Africa / 1986 / G4P6 (4)
Hu / Australia / G4 (2)
Hu / Italy / 2005 / G3P8 / PA116
Hu / Russia / G1P8 / N.N.12813
Hu / USA / 2008 / G1P8
Hu / China / G3 (4)
Hu / Ger / 2008 / G3G12P8 / GER126-08
Hu / Russia / 2008-09 / G9P8 (2)
Hu / China / G1 (2)
Hu / Russia / 2004-09 / P8 (6)
Hu / USA / 2005-08 / G1P8 (3)
Hu / USA / G1P8 (3)
Hu / Slo / 2006 / G8P8 / SI-885/06
Hu / Ethiopia / 2004 / G8 / ARN6810
Hu / Bel / 2006 / G1P8 (2)
Hu / China / 1996 / G2P4 / TB-ChenHu / Congo / 2003 / G8 / DRC (2)
Hu / Russia / G1P8 / N.N.131
Hu / Russia / 2007 / G4P8 / Omsk07-78
Hu / USA / 1976 / G2P4 / DS-1
Hu / Italy / 2005 / G3P8 / PA106
Hu / Italy / 2005 / G3P8 / PA110
Hu / Jap / G3 (2)
Hu / USA / G1P8 (7)
Hu / China / 1994 / G4 (2)Hu / Jap / 1990 / G4 (2)
Hu / Jap / G3 (4)
Hu / Italy / 2005 / G3P8 / PA49
Hu / Italy / 2005 / G3P8 / PA57
Hu / Ger / 2009 / G8P4 / GER1H-09
Hu / India / G9/ RMC321
Hu / Bel / 2004-08 / G1P8 (4)
Po / OSU / G5P7
Hu / Bel / 2006-08 / G1P8 (3)
Hu / Russia / 2009 / G1P8 / D83Hu / India / 2006 / G1P8 / AM06-I
Hu / Russia / G4P8 / N.N.13518
Hu / Bel / 2007-09 / G1P8 (4)
Hu / Cameroon / 2000 / G8 / ARN6780
Hu / Jap /1982 / G3P9 / AU-1
Hu / Bel / 2005-06 / G1P8 (2)
Hu / Croatia / 2006 / G8P8 / CR2006
Hu / Italy / 2005 / G3P8 / PA105
Hu / Bel / 2007 / G1P8
Hu / India / G10 / I321Bo / Kor / CBN-1
Hu / USA / G1P8 / Wa (6)
Hu / Jap / G3-G4 (2)
1
1
11
1
1
1
1
1
0,96
1
1
1
1 1
1
1
1
1
1
1
1NSP4
E1
E2
E3E5
FIG. 2. Bayesian phylogenetic tree of Human Rotavirus NSP4 nucleotide sequence. The tree was rooted by using the midpoint rooting method. Branch lengths were estimated with the best fitting nucleotide substitution model according to a hierarchical likelihood ratio test. For the significant statistical support, posterior probabilities > 0.90 are reported. Several clusters are replaced by a triangle for simplicity, and its size is an indication of the number of sequences it represents. The number of the strain for each place of isolation is indicated in parenthes. Phylogenetic clusters containing CR2006 are boxed and the strain is in boldface type. For each strain, the following data are given: species of origin/place of isolation/year/strain name. The number of substitutions per site is indicated by the scale bar.
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