Isolation,Identification And Biological Characteristics Of Senecavirus A

Senecavirus A(SVA)was also known as Seneca Valley virus,belonging to the genus of Senecavirus within the Pricornaviridae family,and it is the only number of the Senecavirus genus.In 2007,SVA was first reported that SVA was identified as the pathogen of primary vesicular disease of pigs in Canada.Subsequently,it was reported that SVA caused disease in pigs in the United States,Canada,Colombia,Brazil,Vietnam and China.The continuous spread and repeated epidemic of SVA and its pathogens are also constantly mutating,making the prevention and control of SVA face serious challenges.In this research,the pathogen of blister disease in a pig farm and in Henan province 2020 was isolated and identified,and the biological characteristics of the SVA strain were studied and immunogenicity was preliminarily analyzed,providing data for the epidemiological study of SVA in China,and laying a foundation for further research on the pathogenesis and prevention and control technology of SVA.1 Isolation and identification of SVA/HN/2020 strainRNA was extracted from blister skin samples of infected pigs in a pig farm in HeNan province,and the pathogen was identified by RT-PCR.Then the virus was isolated and cultured with BHK-21 cells,and the isolated virus strains were identified by indirect immunofluorescence assay(IFA)and Western blot analysis.RT-PCR results showed that the pathogen of blister disease was SVA.The results of virus isolation and culture showed that the strain could induce significant cytopathic changes in BHK-21 cells.IFA and Western blot analysis showed that the isolated strain could react specifically with SVA rabbit polyresistance,indicating that a SVA epidemic strain was successfully isolated and named as SVA/HN/2020.2 Biological characteristics and genetic evolution analysis of SVA/HN/2020 strainThe proliferation characteristics of SVA/HN/2020 strain were analyzed by plaque formation test and one-step growth curve of virus.The results showed that the SVA/HN/2020 strain could form a clear edge plaque on BHK-21 cells.The one-step growth curve showed that the strain could replicate at a high level after infection with BHK-21 cells,and the virus titer reached 109.5 TCID50/100μL at 16h.The whole genome sequence of SVA/HN/2020 was amplified by RT-PCR,and the phylogenetic tree based on P1 gene was constructed.Full genome sequencing results showed that the full length of the genome of this strain was 7279bp(excluding Poly A),with an open reading frame of 6546bp,encoding A polymeric protein of 2181 amino acids,including 2574bp of P1 gene.The whole and P1 gene nucleotide sequence analysis showed that isolates had the highest homology with USA/MI16-038766/2016 and HB-CH-2016 CH-01-2015 in 99.6%-99.7%.The strains isolated from Guangdong,Guangxi,Henan,Hebei and other regions in China during 2017-2020 were far from each other,indicating that SVA was constantly evolving and mutating while prevalent in China.3 Preliminary immunogenicity analysis of SVA/HN/2020 strainSVA/HN/2020 strain was prepared and emulsified by diethylene-imine(BEI)inactivation.Pigs were immunized with the inactivated vaccine.Serum was collected on 7,14,21 and 28 day to detect neutralizing antibody.These results indicated that SVA/HN/2020 strain had good immunogenicity.In conclusion,an SVA epidemic strain SVA/HN/2020 was isolated in this study.Genetic evolution analysis of genome sequence showed that this strain had the highest homology with B-CH-2016 and CH-01-2015 in China.The results of immunizing piglets with inactivated vaccine showed that the strain had good immunogenicity.The research results lay a foundation for the research of SVA pathogenesis and prevention and control technology.