Caracterização genômica de bvdv-1 subtipo i e vírus ‘HoBi’- like detectados no Brasil

O gênero Pestivirus, pertencente à família Flaviviridae, é constituído por espécies virais de importância na saúde animal no mundo todo, as quais podem afetar a economia dos países de forma impactante. São reconhecidas quatro espécies pelo Comitê Internacional de Taxonomia Viral (ICTV): vírus da peste suína clássica (Classical Swine Fever Virus – CSFV), vírus da doença da fronteira (Border Disease Virus- BDV), vírus da diarreia viral bovina tipo 1 (Bovine Viral Diarrhea Virus 1- BVDV-1) e 2 (BVDV-2). Algumas das espécies deste gênero- CSFV e BVDV- são de notificação obrigatória na Organização Mundial de Saúde Animal (OIE), causando sanções econômicas importantes quando presentes. Recentemente, possíveis novas espécies vêm sendo caracterizadas, porém ainda não foram reconhecidas como espécies do gênero Pestivirus. Com o objetivo de gerar mais informações acerca da diversidade genética de pestivírus no país, o presente trabalho descreve os genomas completos e a caracterização genômica e filogenética de uma cepa de BVDV-1 subtipo i e duas de vírus ‘HoBi’-like. Os genomas completos foram obtidos através de sequenciamento de nova geração; as anotações, predição da poliproteína viral e dos sítios de clivagem foram feitos através do software Geneious, e a análise filogenética foi realizada através do software MEGA 6. O BVDV-1 subtipo i foi pela primeira vez isolado no Brasil, sendo também a primeira descrição de genoma completo deste subtipo de BVDV. Também foi descrito o genoma completo de duas cepas de vírus ‘HoBi’-like isolados no Brasil, além da caracterização de outras cepas de ‘HoBi’-like disponíveis em bancos de dados. Os dados moleculares destes isolados foram comparados com aqueles das demais espécies do gênero Pestivirus, e estas informações deverão auxiliar na futura classificação deste como espécie. Os resultados apresentados na dissertação adicionam conhecimento sobre a diversidade genética de BVDV-1 no Brasil além de informações acerca do vírus ‘HoBi’-like, reforçando esta espécie ainda não reconhecida como um novo membro do gênero Pestivirus, os ‘HoBi’-like vírus.The genus Pestivirus, within the family Flaviviridae, includes species that are important pathogens affecting animal health that can cause impacting losses in the economy worldwide. According to the International Committee on Taxonomy of Virus (ICTV), there are four recognized species in this genus: Classical swine fever virus (CSFV), Bovine Viral Diarrhea Virus1 and 2 (BVDV -1, BVDV-2), and Border disease virus (BDV). Some of the species within this genus - CSFV and BVDV- are notifiable to the World Organization for Animal Health (OIE), and can cause exportation barriers or sanctions. Other putative new species have been characterized recently, but remain officially unrecognized. In order to generate data about the genetic diversity of pestivirus in Brazil, this study describes complete genomes and the genomic and phylogenetic characterization of an isolate of BVDV-1i and two isolates of ‘HoBi’-like virus. Complete genomes were sequenced through Next Generation Sequencing; genome annotations, polyprotein prediction and identification of cleavage sites were performed with software Geneious, and phylogenetic analysis with software MEGA 6. BVDV-1 subtype i was found in Brazil for the first time, and this is the first complete genome ever characterized for this subtype. Two strains of ‘HoBi-like’ virus isolated in Brazil were also described and characterized together with other ‘HoBi’-like strains available in databases. The molecular data obtained for these isolates were compared to those of other Pestivirus species. These data can help in future classification of these ‘HoBi’-like strains as a new recognized species. The knowledge on genetic diversity and the characterization of pestiviruses can contribute with surveillance programs and with appropriate animal health measures to control these viral diseases

Pestivírus em suínos e bovinos : uma abordagem sorológica, molecular e evolutiva

O gênero Pestivirus, pertencente à família Flaviviridae, compreende vírus de genoma RNA classificados em 11 espécies e na sua grande maioria (10 espécies) estão associadas a infecções em animais ungulados. No Brasil, assim como em vários países, os pestivírus podem afetar a atividades pecuária e gerar prejuízos econômicos, como infecção por Pestivirus C (peste suína clássica) e K (pestivírus atípico porcino) em suínos e por Pestivirus A, B (vírus da diarreia viral bovina 1, 2) e H (pestivírus hobi-like) em bovinos. Com o objetivo de contribuir com mais informações acerca da epidemiologia e diversidade dos pestivírus no Brasil e da caracterização antigênica entre cepas de pestivírus de bovinos, o presente trabalho será apresentado sob a forma de 4 capítulos. O objetivo e resultados alcançados nos trabalhos foram, respectivamente: 1) a caracterização de pestivírus detectados em suínos de criação de fundo de quintal no Rio Grande do Sul, através de soroneutralização para detecção de anticorpos contra vírus da diarreia viral bovina (BVDV) 1 e 2, além de RT-PCR para detecção de pestivírus seguida de sequenciamento e análise filogenética. Como resultado, 28 (4,4%) das amostras foram positivas na sorologia e duas amostras foram positivas na RT-PCR e classificadas como BVDV-1d e BVDV-2a; 2) a primeira descrição de Pestivirus K no Brasil, através de análise de amostras de leitões nascidos com tremores congênitos em duas granjas do Sul do país por RT-PCR. Como resultado, foi obtida a primeira detecção deste vírus na América do Sul;.3) o uso da Análise de Componentes Principais (PCA) como ferramenta estatística para caracterização de cepas de BVDV-1 e 2 através de soroneutralização. Os resultados demonstraram que a PCA gerou gráficos de fácil visualização e interpretação de diferenças e semelhanças entre agrupamentos antigênicos e que não houve mesmo padrão de antigenicidade entre isolados pertencentes a um mesmo subgenótipo; e 4) a caracterização de pestivírus detectados em amostras de bovinos recebidas no Laboratório de Virologia Veterinária-UFRGS entre 2016-2018, bem como inferência temporal (através da ferramenta relógio molecular) dos subgenótipos de pestivírus de bovinos já escritos no Brasil. Como resultado, foram descritas amostras de subgenótipos BVDV-1a, 1b e 2b (mais prevalentes no país) e 1d e 1e (subgenótipos já descritos, porém menos frequentes), e análise temporal da filodinâmica destas cepas e outras representando os subgenótipos já descritos no país pode ser inferida e explicada pela história da pecuária bovina no país. Os resultados aqui apresentados somam novo conhecimento acerca dos pestivírus e podem no futuro contribuir para planos de controle e erradicação das doenças causadas por pestivírus no país.The genus Pestivirus, belonging to the family Flaviviridae, comprises RNA genome viruses classified into 11 species, from which 10 species are associated with infection in ungulate animal hosts. In Brazil, as in several countries, pestiviruses can affect livestock and generate economic losses, such as Pestivirus C (classical swine fever virus) and K (atypical porcine pestivirus- APPV) swine infection and Pestivirus A, B (bovine viral diarrhea virus 1, 2- BVDV-1, 2) and H (HoBi-like virus) cattle infection. In order to contribute with data about pestiviruses in Brazil and antigenic characterization of bovine pestivirus strains, the present thesis will be presented in 4 chapters. The chapters present 1) the characterization of pestiviruses detected in backyard pigs in Rio Grande do Sul, 2) the first description of Pestivirus K in Brazil, 3) the use of Principal Component Analysis (PCA) as a statistical tool for antigenic characterization of strains of bovine pestiviruses and 4) the characterization of pestiviruses detected in bovine samples sent to the Veterinary Virology Laboratory-UFRGS between 2016-2018, as well as temporal inference (through the molecular clock tool) of subgenotypes detected in Brazil. In the first chapter, swine serum samples from backyard animals from Rio Grande do Sul state were tested through serum neutralization in order to detect antibodies against BVDV-1 and 2, as well as RT-PCR to detect pestivirus followed by sequencing and phylogenetic analysis. As a result, 28 (4.4%) of the samples were positive in serology and two samples were positive in RT-PCR and classified as BVDV-1d and BVDV-2a. In the second chapter, samples of piglets born with congenital tremors from two pig farms in South Brazil were analyzed using RT-PCR in an attempt to first detect the recently described Pestivirus K species in Brazilian swine herds. As a result, this was the first detection of APPV in South America. In the third chapter, PCA was applied to virus neutralization results in order to visualize antigenic relationships between vaccine strains and field isolates from several BVDV subgenotypes. As a result, PCA generated easy interpretation graphics for differences and similarities between antigenic clusters. The results also demonstrated that there are no grouping patterns between isolates belonging to the same subgenotype. In the fourth chapter, phylogeny of pestivirus samples received in the laboratory from 2016 to 2018 resulted in detection of BVDV-1a, 1b and 2b (most prevalent in the country) and 1d and 1e (less frequent) subgenotypes, and temporal analysis of subgenotypes in the country could be inferred. The results presented here contribute to pestiviruses knowledge and may in the future contribute to control and eradication of pestiviruses in the country

Homologous recombination in pestiviruses: Identification of three putative novel events between different subtypes/genogroups

AbstractViruses from the genus Pestivirus of the family Flaviviridae have a non-segmented, single-stranded RNA genome and can cause diseases in animals from the order Artiodactyla. Homologous recombination is rarely reported in this virus family. To detect possible recombination events, all complete pestivirus genomes that are available in GenBank were screened using distinct algorithms to detect genetic conversions and incongruent phylogenies. Three putative recombinant viruses derived from recombination from different pestivirus subtypes/genogroups were detected: Bovine viral diarrhea virus 1 (BVDV-1) strain 3156, BVDV-2 strain JZ05-1 and Classical swine fever virus (CSFV) strain IND/UK/LAL-290. The present study demonstrated that the pestivirus classification cannot be based only on the analysis of one fragment of the genome because genetic conversions can lead to errors. The designation of the recombinant forms (RF) provides a more informative structure for the nomenclature of the genetic variant. The present work reinforces that homologous recombination occurs in pestivirus populations under natural replication and describes the first evidence of recombination in BVDV-2

Multivariate analysis reveals that BVDV field isolates do not show a close VN-based antigenic relationship to US vaccine strains

Objective Evaluate bovine viral diarrhea virus (BVDV) antigenicity by using virus neutralization titers (VNT) analyzed using the principal component analysis (PCA) from antisera generated against US-based vaccine strains against both US-origin field isolates and non-US-origin field isolates. Results Data from both independent analyses demonstrated that several US-origin and non-US-origin BVDV field isolates appear to be antigenically divergent from the US-based vaccine strains. Results from the combined analysis provided greater insight into the antigenic diversity observed among BVDV isolates. Data from this study further support genetic assignment into BVDV subgenotypes, as well as strains within subgenotypes is not representative of antigenic relatedness. PCA highlights isolates that are antigenically divergent from members of the same species and subgenotype and conversely isolates that belong to different subgenotypes have similar antigenic characteristics when using antisera from US-based vaccine isolates

Use of multivariate analysis to evaluate antigenic relationships between US BVDV vaccine strains and non-US genetically divergent isolates

Bovine viral diarrhea virus (BVDV) comprises two species, BVDV-1 and BVDV-2. But given the genetic diversity among pestiviruses, at least 22 subgenotypes are described for BVDV-1 and 3-4 for BVDV-2. Genetic characterization is generally accomplished through complete or partial sequencing and phylogeny, but it is not a reliable method to define antigenic relationships. The traditional method for evaluating antigenic relationships between pestivirus isolates is the virus neutralization (VN) assay, but interpretation of the data to define antigenic relatedness can be difficult to discern for BVDV isolates within the same BVDV species. Data from this study utilized a multivariate analysis for visualization of VN results to analyze the antigenic relationships between US vaccine strains and field isolates from Switzerland, Italy, Brazil, and the UK. Polyclonal sera were generated against six BVDV strains currently contained in vaccine formulations, and each serum was used in VNs to measure the titers against seven vaccine strains (including the six homologous strains) and 23 BVDV field isolates. Principal component analysis (PCA) was performed using VN titers, and results were interpreted from PCA clustering within the PCA dendrogram and scatter plot. The results demonstrated clustering patterns among various isolates suggesting antigenic relatedness. As expected, the BVDV-1 and BVDV-2 isolates did not cluster together and had the greatest spatial distribution. Notably, a number of clusters representing antigenically related BVDV-1 subgroups contain isolates of different subgenotypes. The multivariate analysis may be a method to better characterize antigenic relationships among BVDV isolates that belong to the same BVDV species and do not have distinct antigenic differences. This might be an invaluable tool to ameliorate the composition of current vaccines, which might well be important for the success of any BVDV control program that includes vaccination in its scheme

Detecção de agentes enteropatogênicos associados à diarreia crônica em um gatil no Sul do Brasil

This study carried out a survey about enteropathogenic agents in domestic cats’ shelter as a stage of investigation for the intermittent chronic diarrhea. Individual fecal samples from 39 cats with free access to the external environment were submitted to parasitological examination, parvovirus, and coronavirus by PCR, and Cryptosporidium spp., Giardia spp. and Tritrichomonas foetus by real-time PCR. From the cats evaluated, 30 (76.9%) were positive for one or more enteric agents, and coinfections were observed in 11 cats samples (28.2%). Helminth eggs were observed in 48.7% of cats (19/30), 16 (41%) were positive for parvovirus or coronavirus and 25.6% (10/30) were infected by protozoa. From the positives for protozoa, five cats were positive to T. foetus (12.82%). The first finding of this protozoan through PCR was in the southern Brazil, and the second was in the whole country. Chronic diarrhea in cats may be multifactorial in shelter animals where the population density is high and the control of parasitic, and viral infections are deficient. Moreover, it is due to poor hygiene conditions in these shelters. The factors associated with the proliferation of infectious diseases in shelters are correlated with new pathogens infections such as T. foetus.Uma pesquisa de agentes enteropatogênicos em gatos domésticos de um abrigo foi realizado como etapa da investigação das causas de diarreias crônicas intermitentes. Amostras fecais individuais de 39 gatos, com livre acesso ao ambiente externo, foram obtidas para pesquisa de helmintos através do exame parasitológico, investigação de parvovírus e coronavírus e de Cryptosporidium spp., Giardia spp. e Tritrichomonas foetus através de PCR em tempo real. Dos gatos avaliados, 30 (76,9%) foram positivos para algum ou mais de um destes agentes entéricos. Desses, 11 (28,2%) apresentaram co-infecções parasitárias. Ovos de helmintos foram observados em 48,7% dos gatos (19/30), 16 felinos (41%) foram positivos para parvovírus ou coronavírus e 25,6% (10/30) estavam infectados por protozoários. Dos positivos para protozoários, cinco apresentaram Tritrichomonas foetus (12,82%), um organismo pouco relatado no Brasil, sendo este o primeiro relato de detecção deste protozoário através de PCR em fezes de gatos no Sul do Brasil e o segundo no país. A diarreia crônica em gatos pode ser multifatorial em animais de abrigo onde a densidade populacional é elevada e os meios de controle parasitário e viral são deficitários, além das condições de higiene precárias. Os fatores associados à proliferação de doenças infecciosas em abrigos promovem o surgimento de infecções por novos patógenos como o Tritrichomonas foetus, até então pouco relatado no Brasil

First Evidence of Bovine Viral Diarrhea Virus Infection in Wild Boars

Background: The farming of wild boars has growing due to the interest of the human consumption of this exotic meat. Such a development may pose an increased risk of disease transmission between boars and domestic animals. The wild boar population has increased in South America in the last years due the absence of predator causing economic losses due to direct damage to crops and risk of disease transmission. The genus Pestivirus within the family Flaviviridae are composed by four recognized species by the International Committee on the Taxonomy of Viruses (ICTV): classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhea virus type 1 (BVDV-1) and 2 (BVDV-2). Other putative species denoted as atypical pesitiviruses have been reported as ‘HoBi’-like virus, giraffe pestivirus, Bungowannah pestivirus, Pronghorn antelope virus, atypical porcine pestivirus (APPV), Norwegian rat pestivirus (NrPV) and Rhinolophus affinis bat pestivirus (RaPestV-1). CSFV is commonly detected in wild boars, but despite positive serology, bovine viral diarrhea virus (BVDV) was never detected in this animal species. Thereby, the present communication describes the first detection of BVDV in the lungs of captive boars using RT-PCR and DNA sequencing.Materials, Methods & Results: Forty lung samples from farmed wild boars were collected after slaughter in a commercial abattoir. The organs were crushed separately, centrifuged, and the supernatant was stored for further analysis. The total RNA was isolated using a phenol-based protocol and RT-PCR protocol that amplified 118 bp of 5’ untranslated region (5’UTR) was carried out. One out 40 samples resulted positive. The positive sample had partial fragments of 5’UTR and N terminal autoprotease (Npro) sequenced and analyzed. The strain LV Java/2012 presented 99% of identity in 5’UTR and 98% in Npro region with a BVDV-2 previously reported in bovines in Southern Brazil. In both 5’UTR and Npro phylogenetic analysis, the strain LV Java/2015 clustered with BVDV-2 strains and was most closely related to subtype 2b identified in bovines in Southern Brazil grouping in the same terminal node.Discussion: Wild boars are commonly associated to pathogen transmission to domestic animals. This animal species is considered a reservoir of the pestivirus CSFV and important keys in CSFV control and eradication programs in Europe. Despite indirect presence of BVDV was reported in wild boars by serology tests, the direct detection of the viral agent was never reported. The present study showed the presence of BVDV-2 genomic segments obtained by RT-PCR followed by DNA sequencing in captive wild boars. The reported data suggests a possible importance of this animal species in the epidemiology of ruminant pestiviruses which could interfere in control and eradication programs of these important pathogens for cattle worldwide. The strain LV Java/2012 was closely related to BVDV-2b and presented highest identity with a strain detected in cattle from Southern Brazil. This data suggests that wild boars and bovines could be sharing this pathogen due the similarity of the strains and that both were reported in the same region. It can lead to need of inclusion of wild swines in BVDV control programs since boars can circulate between different regions and carry this pathogen to different cattle herds. The present study reported the first molecular evidence of BVDV in wild boars in the literature. The data generated herein suggests a possible importance of boars in the epidemiology of ruminant pestiviruses

Detecção de Parvovírus e Anelovírus em pulmão de javalis em cativeiro

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Detecção de Parvovírus e Anelovírus em pulmão de javalis em cativeiro

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