A Novel Genetic Group of Bovine Hepacivirus in Archival ...downloads.hindawi.com/journals/bmri/2017/4732520.pdf · Archival Serum Samples from Brazilian Cattle CláudioW.Canal,1 MatheusN.Weber,1

Research ArticleA Novel Genetic Group of Bovine Hepacivirus inArchival Serum Samples from Brazilian Cattle

ClaacuteudioW Canal1 Matheus N Weber1 Samuel P Cibulski1 Mariana S Silva1

Daniela E Puhl1 Hanspeter Stalder2 and Ernst Peterhans2

1Laboratorio de Virologia Faculdade de Veterinaria Universidade Federal do Rio Grande do Sul (UFRGS) Porto Alegre RS Brazil2Institute of Veterinary Virology University of Bern Bern Switzerland

Correspondence should be addressed to Claudio W Canal claudiocanalufrgsbr

Received 26 April 2017 Revised 3 July 2017 Accepted 20 July 2017 Published 20 August 2017

Academic Editor Christen Rune Stensvold

Copyright copy 2017 Claudio W Canal et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Hepatitis C virus (HCV) (genus Hepacivirus family Flaviviridae) is a major human pathogen causing persistent infection andhepatic injury Recently emerging HCV-like viruses were described infecting wild animals such as bats and rodents and domesticanimals including dogs horses and cattle Using degenerate primers for detecting bovine pestiviruses in a 1996 survey three bovineserum samples showed a low identity with the genus Pestivirus of the Flaviviridae family A virus could not be isolated in cell cultureThe description of bovine hepaciviruses (BovHepV) in 2015 allowed us to retrospectively identify the sequences as BovHepV witha 889 nucleotide identity In a reconstructed phylogenetic tree the Brazilian BovHepV samples grouped within the bovine HCV-like cluster in a separated terminal node that was more closely related to the putative bovineHepacivirus common ancestor than tobovine hepaciviruses detected in Europe and Africa

1 Introduction

Hepatitis C virus (HCV) is an enveloped single strandedRNA virus that represents the type species of theHepacivirusgenus within the Flaviviridae family Its genomewith a lengthof about 96 kb contains two untranslated regions (UTR)at the 51015840 and 31015840 ends Hepaciviruses differ from the otherFlaviviridae genera Flavivirus and Pestivirus by their limitedmultiplication in cultured cells [1]

HCV represent one of the most significant threats tohuman health leading to hepatitis liver cirrhosis and hep-atocellular carcinoma [2 3] Currently about 160 millionindividuals are persistently infected with HCV [4] AcuteHCV infection is asymptomatic in many cases but 50ndash80of infected individuals are unable to clear the virus leading toa state of persistent viral replication andhepatic inflammationtakes place [2 3]

Recently novel HCV-related hepaciviruses were detectedin nonprimate hosts In 2011 HCV-like viruses were reported

in dogs displaying signs of hepatic injury [5] and respiratoryillness [6] Further investigations revealed that the naturalreservoirs of this HCV-like virus are not dogs but horses[5 7] and that it was not associated with liver disease in dogs[8] These strains were designated nonprimate hepaciviruses(NPHV) [7 9] Moreover a great diversity of Hepacivirussequences was detected in rodents and bats worldwide [10ndash12]

These previous reports highlight the importance of thesearch for possible newHepacivirus reservoirs and of investi-gating the risks arising for public health In 2015 a study per-formed in Germany reported HCV-like viruses that showedliver tropism and chronic infection in domestic cattle withoutany signs of clinical disease [13] Another study performed inAfrica also found closely related viruses [14] In the presentstudy we describe and characterize a bovine Hepacivirus(BovHepV) detected in cattle from Southern Brazil during aBVDV survey that emphasizes a possible worldwide spreadof this emerging group of viruses

HindawiBioMed Research InternationalVolume 2017 Article ID 4732520 4 pageshttpsdoiorg10115520174732520

2 BioMed Research International

Table 1 Nucleotide sequence of the primers used for RT-PCR

Primer Sequence (51015840 to 31015840)230a TAG CCA TGC CCT TAG TAG GAC TAG C230b TAG CCA TGC CCT TAG TAG GAC AAG C230c TAG CCA TGC CCT TAG TAG GAG TAG C230d TAA CCA TAC CCT TAG TAG GAC TAG C230e TAG CCA TAC CCG TAG TAG GAC TAG C230f TAG CCA TAC ACG TAG TAG GAC TAG C230g TAG CCA TGC CCA TAG TAG GAC TAG C230h TAG CCA TGC CCA CAG TAG GAC TAG C

2 Methods

Six bovine serum samples from a farm located in CarazinhoRio Grande do Sul State Southern Brazil were collectedin 1996 when screening for bovine viral diarrhea virus(BVDV) The RNA was isolated using TRIzol LS Reagent(Invitrogen Carlsbad CA USA) in a total volume of 250120583Laccording to the manufacturerrsquos instructions The cDNA wassynthetized using SuperScript II (Invitrogen) according tothe manufacturerrsquos recommendation

For PCR we used the reverse primer 326 previouslydescribed by Vilcek et al with a final concentration of06 120583M [15] As forward primers we used a mixture of theeight different primers (Table 1) which were mixed at equalamounts to give a final concentration of 06120583M [16] Theforward primers bind approximately at position 52ndash76 andthe reverse primer at position 284ndash304 in the BovHepV-B1Ger213 strain (GenBank accession number KP641123)(Table 1)

PCR amplification products were purified using IllustraGFX PCR DNA and Gel Band Purification Kit (GE Health-care Life Sciences Uppsala Sweden) and both strands weresequenced three times with an ABI PRISM 3100 GeneticAnalyzer (Applied Biosystems) using a BigDye Termina-tor v31 cycle sequencing kit (Applied Biosystems) Thesequences were assembled using Geneious software version814 (Biomatters Auckland New Zealand) The sequencesdetected in the present study were deposited in GenBankunder accession numbers KY439906ndashKY439908

Sequences of 24 hepaciviruses including reference andrepresentative strains were retrieved fromGenBank (httpswwwncbinlmnihgovgenbank) and aligned using MUS-CLE software [17] Phylogenetic trees were reconstructedwith MrBayes v321 [18] using Bayesian analysis coupledwith Markov Chain Monte Carlo methods of phylogeneticinference For Bayesian analysis the Jukes-Cantor modelwas chosen using jModeltest and used as the substitutionmodel (rates variation across sites invariable + gamma)Markov Chain Monte Carlo chains were run for 1100000generations sampling every 100 generations and the first100000 sampled trees were discarded as burn-in Treesobtained before convergent and stable likelihood values werediscarded (ie the 100000 first generations were burn-in)

RT-PCR-positive serum samples were submitted to virusisolation in cell culture using the cell lines Madin Darby

bovine kidney (MDBK) (ATCC CCL-22) baby hamsterkidney 21 (BHK-21) (ATCCCCL-10) rabbit kidney 13 (RK-13) (ATCC CCL-37) mouse fibroblast NCTC clone 929(L929) (ATCC CCL-1) and bovine testicle and bovineturbinate primary cell cultures The cells were grown inminimal essential medium (MEM) supplemented with L-glutamine (14mM) gentamicin (50mgliter) and 10 fetalbovine serum (FBS) For virus isolation 25 cm2 flasks con-taining 70 confluent cell monolayers were inoculated withserum samples and incubated at 37∘C for 72 to 96 h Followingone freeze-thaw cycle the suspension was centrifuged for10min at 1000timesg Supernatants were collected aliquotedand submitted to two more passages in the cell lines followedby RT-PCR as above described to verify the virus presence

3 Results and Discussion

During a 1996 survey of bovine pestiviruses using degenerateprimers aiming to amplify bovine pestiviruses three bovineserum samples were positive by RT-PCR The sequencedamplification product showed a low identity with the knownFlaviviridaemembers Importantly the sequences were unre-lated to known pestiviruses However a retrospective com-parison revealed that the three 217 bp amplicons were 889identical with the recently discovered bovine hepaciviruses(BovHepV) [13 14]

Remarkably the RT-PCR protocol developed for detect-ing bovine pestiviruses in our study successfully amplified51015840UTR of BovHepV It is important to point out that theprimers used by us were modified [16] from those commonlyused to amplify 51015840UTR of pestiviruses [15] The 51015840UTR of theFlaviviridae members contains an internal ribosome entrysite (IRES) and is conserved [1] providing an opportunity foradaptation of protocols to detect novel viruses

The Bayesian phylogenetic tree (Figure 1) reconstructedwith the Brazilian BovHepV and representative strains withinthe genus presented two well separated branches supportedby posterior probability values of 1 where bovine and rodenthepaciviruses grouped in the same branch and HCV batcanine and equine hepaciviruses in the other BR75 BR78and BR79 grouped in the bovine BovHepV cluster supportedby a posterior probability value of 1 but were located in aseparated terminal node that was more closely related toa putative BovHepV common ancestor than to the virusesdetected in Europe and Africa [13 14] The data presentedhere showed that Brazilian BovHepV diverge from Europeanand African strains and suggest that variants of BovHepVmay circulate in cattle worldwide None of the cell culturesused in our study for the isolation of BR75 BR78 and BR79supported the multiplication of BovHepV which supportsearlier observations of a limited capacity of hepacivirusesto grow in cultured cells [1 19 20] Only one single HCVstrain JFH1 has been found to efficiently infect culturedcells and this was the case only in a human hepatoma cellline (Huh7) [19] Although the nucleotide sequences reportedhere do not comprise full genomes the data suggest thatBovHepV may have circulated in Brazilian cattle at least in1996 some 20 years before those described in Germany andAfrica [13 14]The sequences of the Brazilian BovHepV differ

BioMed Research International 3

KY439906 BR75 KY439907 BR78KY439908 BR79

KP265946 GHC52NC 026797 GHC25

KP641124 BovHepV_209Ger2014KP641125 BovHepV_379Ger2014KP641127 BovHepV_463Ger2014KP641126 BovHepV_438Ger2014KP641123 BovHepV_B1Ger2013

KC796074 PDB-829 KC815312 RHV-089NC 021153 RHV-339

KC796090 PDB-452 KR349930 20000546KP325401 NZP1

NC_024889 JPN3JAPAN2013KU747002 LG-1HailunChinaJX948116 NPHV EF369 11JKF177391 horseDH1HUN2013

JX948117 NPHV EF317JX948120 NPHV EF330

NC 004102 HCV genotype 1NC 009827 HCV genotype 6



NC 009825 HCV genotype 4

01

Brazil

Ghana

Germany

091

096

055

1

1

098

07

053

052

061

05

1

095

089

057

1

098

1

1

1

098

Figure 1 Phylogenetic tree of 51015840UTR of hepaciviruses Sequences from Brazil (BR75 BR78 and BR79) and representative hepaciviruses strainswere analyzed by Bayesian analysis coupled with Markov Chain Monte Carlo methods of phylogenetic inference The length of each pairof branches represents the distance between sequence pairs in the rectangular tree The scale bar represents the percentage of nucleotidedifferences in the rectangular tree

significantly from those of the previously described viruseswhich suggests that hepaciviruses of cattle may be morediverse than originally assumed It is important to highlightthat it is the first report of BovHepV in Brazilian cattle

Finally it is important to note that the Brazilian BovHepVdescribed in this work were detected when screening cattlesera for BVDV using primers designed to detect a broaderspectrum of bovine pestiviruses [16] This highlights thegenetic relationship between the Flaviviridae members in51015840UTR FBS is used in media for growing viruses in culturedcells and is known to be a risk factor for spreading BVDVespecially in live vaccines [21ndash23] Keeping in mind thediversemechanisms of high genetic plasticity of RNA virusesincluding recombination it may be of interest to extendthe screening protocols for adventitious agents in FBS toBovHepV

Conflicts of Interest

The authors declare that they have no conflicts of interest

Acknowledgments

Conselho Nacional de Desenvolvimento Cientıfico e Tec-nologico (CNPq) Fundacao de Amparo a Pesquisa do

Estado do Rio Grande do Sul (FAPERGS) Coordenacaode Aperfeicoamento de Pessoal de Nıvel Superior (CAPES)PropesqUFRGS and a grant by Robert and Dorotee Wylersupported this work

References

[1] P Simmonds P Becher M S Collet et al ldquoFlaviviridaerdquoin Proceeding of the Virus Taxonomy Ninth Report of theInternational Committee on Taxonomy of Viruses pp 1003ndash1020 Academic Press San Diego Calif USA 2011

[2] T Poynard M-F Yuen V Ratziu and C L Lai ldquoViral hepatitisCrdquoThe Lancet vol 362 no 9401 pp 2095ndash2100 2003

[3] J F Perz G L Armstrong L A Farrington Y J F Hutinand B P Bell ldquoThe contributions of hepatitis B virus andhepatitis C virus infections to cirrhosis and primary liver cancerworldwiderdquo Journal of Hepatology vol 45 no 4 pp 529ndash5382006

[4] D Lavanchy ldquoEvolving epidemiology of hepatitis C virusrdquoClinical Microbiology and Infection vol 17 no 2 pp 107ndash1152011

[5] A Kapoor P Simmonds G Gerold et al ldquoCharacterization of acanine homolog of hepatitis C virusrdquo Proceedings of theNationalAcademy of Sciences of the United States of America vol 108 no28 pp 11608ndash11613 2011


[6] J Bukh ldquoHepatitis C homolog in dogs with respiratory illnessrdquoProceedings of the National Academy of Sciences of the UnitedStates of America vol 108 no 31 pp 12563-12564 2011

[7] P D Burbelo E J Dubovi P Simmonds et al ldquoSerology-enabled discovery of genetically diverse hepaciviruses in a newhostrdquo Journal of Virology vol 86 no 11 pp 6171ndash6178 2012

[8] L J W Van Der Laan P E De Ruiter I M Van Gils et alldquoCanine hepacivirus and idiopathic hepatitis in dogs from aDutch cohortrdquo Journal of Viral Hepatitis vol 21 no 12 pp 894ndash896 2014

[9] S Lyons A Kapoor C Sharp et al ldquoNonprimate hepacivirusesin domestic horses United Kingdomrdquo Emerging InfectiousDiseases vol 18 no 12 pp 1976ndash1982 2012

[10] J F Drexler V M Corman M A Muller et al ldquoEvidence fornovel hepaciviruses in rodentsrdquo PLoS Pathogens vol 9 no 6Article ID e1003438 2013

[11] P Quan C Firth J M Conte et al ldquoBats are a major naturalreservoir for hepaciviruses and pegivirusesrdquo Proceedings of theNational Academy of Sciences vol 110 no 20 pp 8194ndash81992013

[12] C Firth M Bhat M A Firth et al ldquoDetection of zoonoticpathogens and characterization of novel viruses carried bycommensal rattus norvegicus in New York cityrdquo mBio vol 5no 5 Article ID e01933-14 2014

[13] C Baechlein N Fischer A Grundhoff et al ldquoIdentification ofa novel hepacivirus in domestic cattle from Germanyrdquo Journalof Virology vol 89 no 14 pp 7007ndash7015 2015

[14] V M Corman A Grundhoff C Baechlein et al ldquoHighly diver-gent hepaciviruses from African cattlerdquo Journal of Virology vol89 no 11 pp 5876ndash5882 2015

[15] S Vilcek A J Herring J A Herring P F Nettleton J PLowings and D J Paton ldquoPestiviruses isolated from pigscattle and sheep can be allocated into at least three genogroupsusing polymerase chain reaction and restriction endonucleaseanalysisrdquo Archives of Virology vol 136 no 3-4 pp 309ndash3231994

[16] H Stalder C Hug R Zanoni et al ldquoA nationwide databaselinking information on the hosts with sequence data of theirvirus strains A useful tool for the eradication of bovine viraldiarrhea (BVD) in SwitzerlandrdquoVirus Research vol 218 pp 49ndash56 2015

[17] R C Edgar ldquoMUSCLE multiple sequence alignment with highaccuracy and high throughputrdquo Nucleic Acids Research vol 32no 5 pp 1792ndash1797 2004

[18] J P Huelsenbeck and F Ronquist ldquoMrBayes Bayesian inferenceof phylogenetic treesrdquo Bioinformatics vol 17 no 8 pp 754-7552001

[19] T K H Scheel J M Gottwein T B Jensen et al ldquoDevelopmentof JFH1-based cell culture systems for hepatitis C virus genotype4a and evidence for cross-genotype neutralizationrdquo Proceedingsof the National Academy of Sciences of the United States ofAmerica vol 105 no 3 pp 997ndash1002 2008

[20] A Kaul I Worz and R Bartenschlager ldquoAdaptation of thehepatitis C virus to cell culturerdquo in Hepatitis C Methods andProtocols vol 510 ofMethods in Molecular Biology pp 361ndash372Humana Press Totowa NJ 2009

[21] H Xia B Vijayaraghavan S Belak and L Liu ldquoDetection andidentification of the atypical bovine pestiviruses in commercialfoetal bovine serum batchesrdquo PLoS ONE vol 6 no 12 ArticleID e28553 2011

[22] T Kozasa H Aoki N Nakajima A Fukusho M Ishimaru andS Nakamura ldquoMethods to select suitable fetal bovine serum foruse in quality control assays for the detection of adventitiousviruses from biological productsrdquo Biologicals vol 39 no 4 pp242ndash248 2011

[23] S Silveira M N Weber A C S Mosena et al ldquoGenetic diver-sity of brazilian bovine pestiviruses detected between 1995 and2014rdquo Transboundary and Emerging Diseases vol 64 no 2 pp613ndash623 2017

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Molecular Biology International

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


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Evolutionary BiologyInternational Journal of



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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


Table 1 Nucleotide sequence of the primers used for RT-PCR

Primer Sequence (51015840 to 31015840)230a TAG CCA TGC CCT TAG TAG GAC TAG C230b TAG CCA TGC CCT TAG TAG GAC AAG C230c TAG CCA TGC CCT TAG TAG GAG TAG C230d TAA CCA TAC CCT TAG TAG GAC TAG C230e TAG CCA TAC CCG TAG TAG GAC TAG C230f TAG CCA TAC ACG TAG TAG GAC TAG C230g TAG CCA TGC CCA TAG TAG GAC TAG C230h TAG CCA TGC CCA CAG TAG GAC TAG C

2 Methods

Six bovine serum samples from a farm located in CarazinhoRio Grande do Sul State Southern Brazil were collectedin 1996 when screening for bovine viral diarrhea virus(BVDV) The RNA was isolated using TRIzol LS Reagent(Invitrogen Carlsbad CA USA) in a total volume of 250120583Laccording to the manufacturerrsquos instructions The cDNA wassynthetized using SuperScript II (Invitrogen) according tothe manufacturerrsquos recommendation

For PCR we used the reverse primer 326 previouslydescribed by Vilcek et al with a final concentration of06 120583M [15] As forward primers we used a mixture of theeight different primers (Table 1) which were mixed at equalamounts to give a final concentration of 06120583M [16] Theforward primers bind approximately at position 52ndash76 andthe reverse primer at position 284ndash304 in the BovHepV-B1Ger213 strain (GenBank accession number KP641123)(Table 1)

PCR amplification products were purified using IllustraGFX PCR DNA and Gel Band Purification Kit (GE Health-care Life Sciences Uppsala Sweden) and both strands weresequenced three times with an ABI PRISM 3100 GeneticAnalyzer (Applied Biosystems) using a BigDye Termina-tor v31 cycle sequencing kit (Applied Biosystems) Thesequences were assembled using Geneious software version814 (Biomatters Auckland New Zealand) The sequencesdetected in the present study were deposited in GenBankunder accession numbers KY439906ndashKY439908

Sequences of 24 hepaciviruses including reference andrepresentative strains were retrieved fromGenBank (httpswwwncbinlmnihgovgenbank) and aligned using MUS-CLE software [17] Phylogenetic trees were reconstructedwith MrBayes v321 [18] using Bayesian analysis coupledwith Markov Chain Monte Carlo methods of phylogeneticinference For Bayesian analysis the Jukes-Cantor modelwas chosen using jModeltest and used as the substitutionmodel (rates variation across sites invariable + gamma)Markov Chain Monte Carlo chains were run for 1100000generations sampling every 100 generations and the first100000 sampled trees were discarded as burn-in Treesobtained before convergent and stable likelihood values werediscarded (ie the 100000 first generations were burn-in)

RT-PCR-positive serum samples were submitted to virusisolation in cell culture using the cell lines Madin Darby

bovine kidney (MDBK) (ATCC CCL-22) baby hamsterkidney 21 (BHK-21) (ATCCCCL-10) rabbit kidney 13 (RK-13) (ATCC CCL-37) mouse fibroblast NCTC clone 929(L929) (ATCC CCL-1) and bovine testicle and bovineturbinate primary cell cultures The cells were grown inminimal essential medium (MEM) supplemented with L-glutamine (14mM) gentamicin (50mgliter) and 10 fetalbovine serum (FBS) For virus isolation 25 cm2 flasks con-taining 70 confluent cell monolayers were inoculated withserum samples and incubated at 37∘C for 72 to 96 h Followingone freeze-thaw cycle the suspension was centrifuged for10min at 1000timesg Supernatants were collected aliquotedand submitted to two more passages in the cell lines followedby RT-PCR as above described to verify the virus presence

3 Results and Discussion

During a 1996 survey of bovine pestiviruses using degenerateprimers aiming to amplify bovine pestiviruses three bovineserum samples were positive by RT-PCR The sequencedamplification product showed a low identity with the knownFlaviviridaemembers Importantly the sequences were unre-lated to known pestiviruses However a retrospective com-parison revealed that the three 217 bp amplicons were 889identical with the recently discovered bovine hepaciviruses(BovHepV) [13 14]

Remarkably the RT-PCR protocol developed for detect-ing bovine pestiviruses in our study successfully amplified51015840UTR of BovHepV It is important to point out that theprimers used by us were modified [16] from those commonlyused to amplify 51015840UTR of pestiviruses [15] The 51015840UTR of theFlaviviridae members contains an internal ribosome entrysite (IRES) and is conserved [1] providing an opportunity foradaptation of protocols to detect novel viruses

The Bayesian phylogenetic tree (Figure 1) reconstructedwith the Brazilian BovHepV and representative strains withinthe genus presented two well separated branches supportedby posterior probability values of 1 where bovine and rodenthepaciviruses grouped in the same branch and HCV batcanine and equine hepaciviruses in the other BR75 BR78and BR79 grouped in the bovine BovHepV cluster supportedby a posterior probability value of 1 but were located in aseparated terminal node that was more closely related toa putative BovHepV common ancestor than to the virusesdetected in Europe and Africa [13 14] The data presentedhere showed that Brazilian BovHepV diverge from Europeanand African strains and suggest that variants of BovHepVmay circulate in cattle worldwide None of the cell culturesused in our study for the isolation of BR75 BR78 and BR79supported the multiplication of BovHepV which supportsearlier observations of a limited capacity of hepacivirusesto grow in cultured cells [1 19 20] Only one single HCVstrain JFH1 has been found to efficiently infect culturedcells and this was the case only in a human hepatoma cellline (Huh7) [19] Although the nucleotide sequences reportedhere do not comprise full genomes the data suggest thatBovHepV may have circulated in Brazilian cattle at least in1996 some 20 years before those described in Germany andAfrica [13 14]The sequences of the Brazilian BovHepV differ


KY439906 BR75 KY439907 BR78KY439908 BR79

KP265946 GHC52NC 026797 GHC25



KC796090 PDB-452 KR349930 20000546KP325401 NZP1







01

Brazil

Ghana

Germany

091

096

055

1

1

098

07

053

052

061

05

1

095

089

057

1

098

1

1

1

098






Acknowledgments



References
































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


KY439906 BR75 KY439907 BR78KY439908 BR79

KP265946 GHC52NC 026797 GHC25



KC796090 PDB-452 KR349930 20000546KP325401 NZP1







01

Brazil

Ghana

Germany

091

096

055

1

1

098

07

053

052

061

05

1

095

089

057

1

098

1

1

1

098






Acknowledgments



References
































Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



























Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 201






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

A Novel Genetic Group of Bovine Hepacivirus in Archival ...downloads.hindawi.com/journals/bmri/2017/4732520.pdf · Archival Serum Samples from Brazilian Cattle CláudioW.Canal,1 MatheusN.Weber,1