Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Manancial - Repositório Digital da UFSM |
Texto Completo: | http://repositorio.ufsm.br/handle/1/28123 |
Resumo: | Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea vírus 1 (BVDV) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped by the analysis of single genomic regions, mainly the 5' untranslated region (5'UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classifications. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for subtyping BVDV-1 and BVDV-2. Initially, genomic regions of BVDV-1 and BVDV-2, previously described as the most suitable targets for bovine pestivirus subtyping, were analyzed for the design of high-coverage primers. Then, the putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 (BVDV-1) and 88 (BVDV-2) complete/near complete genomes (CNCGs) available on GenBank. This analysis was also performed considering the region amplifiable by primers HCV90-368, largely used used for classification/subtyping of bovine pestiviruses. After confirming agreement between the analyses performed with the putative amplicons from our primers versus those from the CNCGs, we optimized the RT-PCR assays and evaluated their performance in the amplification of several BVDV isolates/strains from Brazil, Argentina and the United States. Among potential targets for bovine pestivirus subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526bp amplicon) and NS5B (BVDV-2) (728bp). The phylogenetic classification based on these regions fully reproduced the subtyping of all CNCGs analyzed in the study. On the other hand, subtyping based on the putative amplicon from primers HCV90-368 showed four (BVDV-1) and twelve (BVDV-2) disagreements in relation to the CNCG classification. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains analyzed here. Finally, considering the phylogenetic analyses from our putative amplicons, as well as the performance of RT-PCR assays, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDV-1 and BVDV-2. |
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Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2Genomic targets for subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2PestivírusFilogeniaNS3NS4ANS5BPhylogenyCNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIAWhole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea vírus 1 (BVDV) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped by the analysis of single genomic regions, mainly the 5' untranslated region (5'UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classifications. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for subtyping BVDV-1 and BVDV-2. Initially, genomic regions of BVDV-1 and BVDV-2, previously described as the most suitable targets for bovine pestivirus subtyping, were analyzed for the design of high-coverage primers. Then, the putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 (BVDV-1) and 88 (BVDV-2) complete/near complete genomes (CNCGs) available on GenBank. This analysis was also performed considering the region amplifiable by primers HCV90-368, largely used used for classification/subtyping of bovine pestiviruses. After confirming agreement between the analyses performed with the putative amplicons from our primers versus those from the CNCGs, we optimized the RT-PCR assays and evaluated their performance in the amplification of several BVDV isolates/strains from Brazil, Argentina and the United States. Among potential targets for bovine pestivirus subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526bp amplicon) and NS5B (BVDV-2) (728bp). The phylogenetic classification based on these regions fully reproduced the subtyping of all CNCGs analyzed in the study. On the other hand, subtyping based on the putative amplicon from primers HCV90-368 showed four (BVDV-1) and twelve (BVDV-2) disagreements in relation to the CNCG classification. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains analyzed here. Finally, considering the phylogenetic analyses from our putative amplicons, as well as the performance of RT-PCR assays, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDV-1 and BVDV-2.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPESA análise filogenética do genoma completo, estratégia mais adequada para a subtipagem dos vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2, não é viável para muitos laboratórios. Consequentemente, isolados e cepas de BVDV têm sido frequentemente subtipados pela análise de determinadas regiões genômicas, principalmente a região não traduzida 5' (5' untranslated region, 5'UTR). Essa abordagem, no entanto, pode levar a classificações equivocadas e/ou com baixo suporte estatístico. Nesse contexto, o presente estudo descreve o desenho e utilização de novos pares de primers cujos amplicons podem ser facilmente sequenciados e utilizados para subtipagem de BVDV-1 e BVDV-2. Inicialmente, regiões genômicas do BVDV-1 e BVDV-2, previamente descritas na literatura como os alvos mais adequados para subtipagem dos pestivírus bovinos, foram analisadas para o desenho de primers de alta cobertura. Em seguida, as regiões amplificáveis por esses primers foram analisadas in silico quanto à capacidade de reproduzir a classificação filogenética de 118 (BVDV-1) e 88 (BVDV-2) genomas completos/quase completos (GCQCs) disponíveis no GenBank. Essa análise também foi realizada considerando a região amplificável pelos primers HCV90-368, a qual tem sido comumente utilizada para a classificação/subtipagem de pestivírus bovinos. Após comprovar a conformidade entre as análises realizadas com a região amplificável pelos primers do presente estudo versus as análises de GCQC, seguiuse com a otimização dos ensaios de RT-PCR e com a avaliação de sua capacidade de amplificar isolados e cepas de BVDV do Brasil, Argentina e Estados Unidos. As regiões de BVDV-1 e BVDV-2 que permitiram o desenho de primers de alta cobertura foram a NS3-NS4A (amplicon de 526pb) e NS5B (728pb), respectivamente. A classificação filogenética com base nessas regiões reproduziu integralmente a subtipagem de todos os GCQCs analisados. Por outro lado, a subtipagem baseada na região amplificável pelos primers HCV90-368 apresentou quatro (BVDV-1) e doze (BVDV-2) discordâncias em relação à classificação dos GCQCs. Os primers de NS3-NS4A e NS5B também permitiram a amplificação de todos os isolados/cepas de BVDV analisados. Por fim, considerando as análises filogenéticas das regiões amplificáveis pelos primers descritos aqui, bem como o desempenho dos ensaios de RT-PCR, sugere-se o seu uso em futuros estudos filogenéticos e epidemiológicos de BVDV-1 e BVDV-2.Universidade Federal de Santa MariaBrasilMedicina VeterináriaUFSMPrograma de Pós-Graduação em Medicina VeterináriaCentro de Ciências RuraisWeiblen, Rudihttp://lattes.cnpq.br/7946350215388090Silva Júnior, José Valter JoaquimMonteiro, Francielle LizSilveira, SimoneMucellini, Carolina Isabela2023-03-09T10:55:58Z2023-03-09T10:55:58Z2023-02-07info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://repositorio.ufsm.br/handle/1/28123porAttribution-NonCommercial-NoDerivatives 4.0 Internationalhttp://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccessreponame:Manancial - Repositório Digital da UFSMinstname:Universidade Federal de Santa Maria (UFSM)instacron:UFSM2023-03-09T10:55:58Zoai:repositorio.ufsm.br:1/28123Biblioteca Digital de Teses e Dissertaçõeshttps://repositorio.ufsm.br/ONGhttps://repositorio.ufsm.br/oai/requestatendimento.sib@ufsm.br||tedebc@gmail.comopendoar:2023-03-09T10:55:58Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM)false |
dc.title.none.fl_str_mv |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 Genomic targets for subtyping of bovine viral diarrhea virus 1 (BVDV-1) and BVDV-2 |
title |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 |
spellingShingle |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 Mucellini, Carolina Isabela Pestivírus Filogenia NS3 NS4A NS5B Phylogeny CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
title_short |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 |
title_full |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 |
title_fullStr |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 |
title_full_unstemmed |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 |
title_sort |
Alvos genômicos para subtipagem de vírus da diarreia viral bovina 1 (BVDV-1) e BVDV-2 |
author |
Mucellini, Carolina Isabela |
author_facet |
Mucellini, Carolina Isabela |
author_role |
author |
dc.contributor.none.fl_str_mv |
Weiblen, Rudi http://lattes.cnpq.br/7946350215388090 Silva Júnior, José Valter Joaquim Monteiro, Francielle Liz Silveira, Simone |
dc.contributor.author.fl_str_mv |
Mucellini, Carolina Isabela |
dc.subject.por.fl_str_mv |
Pestivírus Filogenia NS3 NS4A NS5B Phylogeny CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
topic |
Pestivírus Filogenia NS3 NS4A NS5B Phylogeny CNPQ::CIENCIAS AGRARIAS::MEDICINA VETERINARIA |
description |
Whole-genome phylogenetic analysis, the most suitable strategy for subtyping bovine viral diarrhea vírus 1 (BVDV) and BVDV-2, is not feasible for many laboratories. Consequently, BVDV isolates/strains have been frequently subtyped by the analysis of single genomic regions, mainly the 5' untranslated region (5'UTR). This approach, however, may lead to inaccurate and/or poorly statistically supported viral classifications. Herein, we describe novel primer sets whose amplicons may be easily sequenced and used for subtyping BVDV-1 and BVDV-2. Initially, genomic regions of BVDV-1 and BVDV-2, previously described as the most suitable targets for bovine pestivirus subtyping, were analyzed for the design of high-coverage primers. Then, the putative amplicons were analyzed in silico for their suitability to reproduce the phylogenetic classification of 118 (BVDV-1) and 88 (BVDV-2) complete/near complete genomes (CNCGs) available on GenBank. This analysis was also performed considering the region amplifiable by primers HCV90-368, largely used used for classification/subtyping of bovine pestiviruses. After confirming agreement between the analyses performed with the putative amplicons from our primers versus those from the CNCGs, we optimized the RT-PCR assays and evaluated their performance in the amplification of several BVDV isolates/strains from Brazil, Argentina and the United States. Among potential targets for bovine pestivirus subtyping, we designed high-coverage primers for NS3-NS4A (BVDV-1) (526bp amplicon) and NS5B (BVDV-2) (728bp). The phylogenetic classification based on these regions fully reproduced the subtyping of all CNCGs analyzed in the study. On the other hand, subtyping based on the putative amplicon from primers HCV90-368 showed four (BVDV-1) and twelve (BVDV-2) disagreements in relation to the CNCG classification. The NS3-NS4A and NS5B primers also allowed the amplification of all BVDV isolates/strains analyzed here. Finally, considering the phylogenetic analyses from our putative amplicons, as well as the performance of RT-PCR assays, we suggest the use of these primers in future phylogenetic and epidemiological studies of BVDV-1 and BVDV-2. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-03-09T10:55:58Z 2023-03-09T10:55:58Z 2023-02-07 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://repositorio.ufsm.br/handle/1/28123 |
url |
http://repositorio.ufsm.br/handle/1/28123 |
dc.language.iso.fl_str_mv |
por |
language |
por |
dc.rights.driver.fl_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Attribution-NonCommercial-NoDerivatives 4.0 International http://creativecommons.org/licenses/by-nc-nd/4.0/ |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Medicina Veterinária UFSM Programa de Pós-Graduação em Medicina Veterinária Centro de Ciências Rurais |
publisher.none.fl_str_mv |
Universidade Federal de Santa Maria Brasil Medicina Veterinária UFSM Programa de Pós-Graduação em Medicina Veterinária Centro de Ciências Rurais |
dc.source.none.fl_str_mv |
reponame:Manancial - Repositório Digital da UFSM instname:Universidade Federal de Santa Maria (UFSM) instacron:UFSM |
instname_str |
Universidade Federal de Santa Maria (UFSM) |
instacron_str |
UFSM |
institution |
UFSM |
reponame_str |
Manancial - Repositório Digital da UFSM |
collection |
Manancial - Repositório Digital da UFSM |
repository.name.fl_str_mv |
Manancial - Repositório Digital da UFSM - Universidade Federal de Santa Maria (UFSM) |
repository.mail.fl_str_mv |
atendimento.sib@ufsm.br||tedebc@gmail.com |
_version_ |
1805922072980881408 |