Aspectos epidemiológicos e moleculares do atypical porcine pestivirus (APPV)
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2018-12-07
Autores
Gatto, Igor Renan Honorato [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
O gênero Pestivirus é composto por agentes geneticamente distintos, responsáveis por infecções em suínos, bovinos, ovinos, caprinos e outros animais domésticos e silvestres. O atypical porcine pestivirus (APPV), é um pestivirus geneticamente atípico recentemente identificado e associado a casos de tremor congênito (CT) em leitões recém-nascidos em quatro continentes, sugerindo uma distribuição mundial do APPV. Assim, os objetivos deste estudo foram: 1) detectar a presença do APPV em sêmen, líquidos prepuciais e swabs prepuciais de cachaços adultos por reação em cadeia da polimerase via transcriptase reversa quantitativa (qRT-PCR), 2) caracterizar geneticamente um subgrupo de amostras positivas de sêmen para melhor entender a ecologia do APPV em cachaços comerciais e o risco potencial de transmissão do APPV via sêmen, 3) investigar a presença do APPV em leitões com CT no Brasil e (4) caracterizar geneticamente um subconjunto de amostras do APPV oriundas de leitões. Um total de 597 amostras de sêmen, fluido prepucial e swabs prepuciais cada amostra representando um cachaço diferente foram obtidas de quatro granjas comerciais (sêmen) localizadas em três estados diferentes nos Estados Unidos e 13 leitões com CT de quatro granjas em dois estados do Brasil foram amostrados. RNA viral foi detectado por qRT-PCR em 15,08% das amostras (90/597), sendo o maior percentual 23,81% de positivo em swabs prepuciais (5/21) seguido de 22,81% no fluído prepucial (26/114) e de 12,91% no sêmen (59/457). Com base na análise filogenética do gene Npro do APPV, diferentes estirpes virais circulavam na mesma granja. O RNA viral foi detectado por qRT-PCR no cerebelo ou cerebelo e medula espinhal em 100% dos leitões com CT, e o APPV não foi detectado em nenhuma amostra de tecido de leitões clinicamente não afetados. Um subconjunto de amostras positivas foi selecionado para o sequenciamento de parte do gene NS3. A análise filogenética revelou que as sequências brasileiras do gene NS3 formaram um cluster independente e tiveram a maior identidade de sequência de nucleotídeos com uma amostra dos Estados Unidos. Este é o primeiro relato de detecção do APPV em sêmen de cachaços comerciais e a primeira identificação da infecção por APPV em leitões com CT na América do Sul.
The genus Pestivirus is composed of genetically distinct viruses, responsible for infections in swine, cattle, sheep, goats and other domestic and wild animals. The atypical porcine pestivirus (APPV) is a genetically atypical pestivirus recently identified and associated with cases of congenital tremor (CT) in newborn piglets from four continents, suggesting a worldwide distribution of the APPV. Accordingly, the objectives of this study were to 1) detect the presence of APPV in semen, preputial fluids and preputial swabs from adult boars by quantitative reverse transcription PCR (qRT-PCR), 2) genetically characterize a subset of positive semen samples to better understand the ecology of APPV in commercial boar studs and the potential risk of transmission of APPV via semen, 3) investigate the presence of APPV in piglets with CT in Brazil and (4) genetically characterize a subset of APPV samples from CT-piglets. A total of 597 samples of semen, preputial fluid and preputial swabs each samples representing a different boar were obtained from four commercial boar studs located in three different states in the United States and 13 CT-piglets from four farms in two different states in Brazil were sampled. Viral RNA was detected by qRT-PCR in 90 samples (15.08%; 90/597), with the greatest per cent positive from preputial swabs (23.81%; 5/21) followed by preputial fluid (22.81%; 26/114) and semen (12.91%; 59/457). Based on phylogenetic analysis of the APPV Npro gene, different viral strains can be on the same farm at the same and different times. Viral RNA was detected by qRT-PCR in the cerebellum or cerebellum and spinal cord in the 100% of the piglets with CT, and APPV was not detected in any tissue sample from clinically non-affected piglets. A subset of positive samples was selected for sequencing of part of the NS3 gene. Phylogenetic analysis revealed that Brazilian sequences of the NS3 gene formed an independent cluster and had the highest nucleotide sequence identity with a sample from the United States. This is the first report of detection of APPV in semen from commercial boar studs and the first identification of APPV infection in piglets with CT in South America.
The genus Pestivirus is composed of genetically distinct viruses, responsible for infections in swine, cattle, sheep, goats and other domestic and wild animals. The atypical porcine pestivirus (APPV) is a genetically atypical pestivirus recently identified and associated with cases of congenital tremor (CT) in newborn piglets from four continents, suggesting a worldwide distribution of the APPV. Accordingly, the objectives of this study were to 1) detect the presence of APPV in semen, preputial fluids and preputial swabs from adult boars by quantitative reverse transcription PCR (qRT-PCR), 2) genetically characterize a subset of positive semen samples to better understand the ecology of APPV in commercial boar studs and the potential risk of transmission of APPV via semen, 3) investigate the presence of APPV in piglets with CT in Brazil and (4) genetically characterize a subset of APPV samples from CT-piglets. A total of 597 samples of semen, preputial fluid and preputial swabs each samples representing a different boar were obtained from four commercial boar studs located in three different states in the United States and 13 CT-piglets from four farms in two different states in Brazil were sampled. Viral RNA was detected by qRT-PCR in 90 samples (15.08%; 90/597), with the greatest per cent positive from preputial swabs (23.81%; 5/21) followed by preputial fluid (22.81%; 26/114) and semen (12.91%; 59/457). Based on phylogenetic analysis of the APPV Npro gene, different viral strains can be on the same farm at the same and different times. Viral RNA was detected by qRT-PCR in the cerebellum or cerebellum and spinal cord in the 100% of the piglets with CT, and APPV was not detected in any tissue sample from clinically non-affected piglets. A subset of positive samples was selected for sequencing of part of the NS3 gene. Phylogenetic analysis revealed that Brazilian sequences of the NS3 gene formed an independent cluster and had the highest nucleotide sequence identity with a sample from the United States. This is the first report of detection of APPV in semen from commercial boar studs and the first identification of APPV infection in piglets with CT in South America.
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Palavras-chave
Pestivirus, qRT-PCR, Atypical porcine pestivirus, Sêmen, Análise filogenética