RESUMO
BACKGROUND: Senecavirus A (SV-A) is an RNA virus that belongs to the genus Senecavirus within the family Picornaviridae. This study aimed to analyze factors that can influence the molecular diagnosis of Senecavirus A, such as oligonucleotides, RNA extraction methods, and RT-qPCR kits. METHODS: Samples from suspected cases of vesicular disease in Brazilian pigs were analyzed for foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis. All tested negative for these diseases but positive for SV-A. RT-qPCR tests were used, comparing different reagent kits and RNA extraction methods. Sensitivity and repeatability were evaluated, demonstrating efficacy in detecting SV-A in clinical samples. RESULTS: In RNA extraction, significant reduction in Cq values was observed with initial dilutions, particularly with larger supernatant volumes. Trizol and Maxwell showed greater sensitivity in automated equipment protocols, though results varied in tissue tests. RT-qPCR kit comparison revealed differences in amplification using viral RNA but minimal differences with plasmid DNA. Sensitivity among methods was comparable, with slight variations in non-amplified samples. Repeatability tests showed consistent results among RT-qPCRs, demonstrating similarity between methods despite minor discrepancies in Cq values. CONCLUSIONS: Trizol, silica columns, and semi-automated extraction were compared, as well as different RT-qPCR kits. The study found significant variations that could impact the final diagnosis.
Assuntos
Infecções por Picornaviridae , Picornaviridae , RNA Viral , Doenças dos Suínos , Animais , Picornaviridae/genética , Picornaviridae/isolamento & purificação , Suínos , Infecções por Picornaviridae/diagnóstico , Infecções por Picornaviridae/veterinária , Infecções por Picornaviridae/virologia , RNA Viral/genética , Doenças dos Suínos/virologia , Doenças dos Suínos/diagnóstico , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase em Tempo Real/métodos , Doença Vesicular Suína/diagnóstico , Doença Vesicular Suína/virologia , Febre Aftosa/diagnóstico , Febre Aftosa/virologia , Brasil , Reprodutibilidade dos TestesRESUMO
Senecavirus A (SVA) has been a problem in Brazil since the end of 2014. The infections caused by SVA have disrupted the productive chain in Brazil, as it can be confused with foot-and-mouth disease. Although, the virus has remained endemic in the country, an increase in the number of cases of the disease was observed in 2018. The aim of the present study was to conduct the differential diagnosis of foot-and-mouth disease in an outbreak of vesicular disease in finishing swine. Animals (160-170 days old) were kept on a farm with 6000 pigs in Minas Gerais State, Brazil. The morbidity and mortality rates were 20% and 2.2%, respectively. The diagnosis was performed by RT-PCR, using primers that determine the amplification of an internal region of the 3D gene. Furthermore, samples were inoculated into BHK-21 cell culture for viral isolation. In the first passage under cultivation, a cytopathogenic effect compatible with SVA replication (rounding and detachment of the cell monolayer) was observed. The viral identity was confirmed using two additional assays: indirect immunofluorescence assay (IFA) and nucleotide sequencing. Both tests confirmed that the infection was caused by SVA. In summary, we described a method for the diagnosis and viral isolation of SVA, a virus that arrived in Brazil in 2014 and has become endemic in the country.(AU)
Senecavírus A (SVA) é um problema no Brasil desde o final de 2014. As infecções causadas pelo SVA têm causado problemas para a cadeia produtiva no Brasil, pois podem ser confundidas com febre aftosa. Embora o vírus permaneça endêmico no país, foi observado um aumento no número de casos em 2018. O objetivo do presente estudo foi realizar o diagnostico diferencial de febre aftosa em um surto de doença vesicular em suínos de terminação. Os animais (160 a 170 dias de idade) eram mantidos em uma granja com 6.000 suínos no estado de Minas Gerais, Brasil. As taxas de morbidade e mortalidade foram de 20% e 2,2%, respectivamente. O diagnostico foi realizado por RT-PCR, utilizando primers que determinam a amplificação de uma região interna do gene 3D. Além disso, as amostras foram inoculadas na cultura de células BHK-21 para isolamento viral. Na primeira passagem em cultivo, foi observado efeito citopatogênico compatível com a replicação do SVA (arredondamento e descolamento da monocamada celular). A identidade viral foi confirmada usando duas técnicas adicionais: ensaio de imunofluorescência indireta (IFA) e sequenciamento de nucleotídeos. Ambos os testes confirmaram que a infecção foi causada por SVA. Em resumo, descrevemos um método para o diagnóstico e isolamento viral do SVA, um vírus que chegou ao Brasil em 2014 e se tornou endêmico no país.(AU)