Isolation And Identification Of A Seneca Valley Virus Strain And Devolopment Of A QRT-PCR Detection Method

Seneca Valley virus(SVV),also known as Senecavirus A(SVA),can induce porcine idiopathic vesicular disease(PIVD).Its clinical manifestations are blisters and ulceration in the mouth,nose and hoof nails of swine,which are difficult to distinguish from Foot-and-Mouth disease and Vesicular Stomatitis of pigs.In recent years,the epidemic scope of SVV has been expanding,spreading from North America(the United States,Canada)to Southeast Asia(Thailand,Vietnam).It was introduced into China in 2015.At present,infection and prevalence of SVV have been reported in Guangdong,Guangxi,Fujian,Henan and other regions in China.In this study,positive samples were collected from a diseased swine farm in Nanyang,Henan Province,and identified as SVV infection by RT-PCR.PK-15 cells were infected by mechanical grinding pathologic materials,then subcultured continuously and observed the pathological changes.SVV strain was successfully isolated,named HeNNY-1-2018,and proliferated stably.It was verified and purified by indirect immunofluorescence and viral plaque assay.The virus growth curve showed that the highest viral titer in PK-15 cells was 107.6TCID50/mL.The nucleotide homology analysis showed that the isolated strain has 93.6%homology with SVV-001 and 96.4%homology with the first isolated strain in China.The phylogenetic tree based on the genome length and VP1 gene showed that the isolated strain had a far genetic relationship with the classical strain SVV-001 and was in the same branch of evolution with the epidemic strain in Guangxi.In order to establish an efficient,rapid and specific detection method,we compared VP1 genes of different SVV strains,designed specific primers and probes for conserved regions,and established a kind of fluorescence quantitative detection method.Specificity analysis showed that the detection method could specifically recognize SVV without cross reaction with other common porcine viral nucleic acids;sensitivity analysis showed that the sensitivity of the detection method could reach 278 copies/?L,stability analysis showed that the intra group coefficient of variation of the established detection method was 0.3%?1.0%,and the inter group coefficient of variation was 0.8%?2.0%.When the sample concentration was between 107?103 copies/?L,there was a good linear relationship.According to 16 clinical samples,this method can be used for the detection of clinical samples.In this study,a SVV strain was successfully isolated,and its biological characteristics and genomic genetic characteristics were preliminarily analyzed,which enriched the molecular epidemiological data of SVV in China.A kind of specific,sensitive and stable qRT-PCR method was established to provide technical support for clinical monitoring and diagnosis of SVV.