Isolation And Identification Of Seneca Valley Virus And The Construction Of A Full-length CDNA Infectious Clone

Senecavirus A(SVA),also known as Seneca valley virus(SVV),belongs to the family Picornaviridae.SVA is an emerging infectious pathogen that is susceptible to pigs of all ages and can cause porcine idiopathic vesicular disease(PIVD)and Epidemic Transient Mortality(ETNL)for newborn pigs.Since the end of 2014,the SVA epidemic has spread rapidly.SVA outbreaks have occurred in many countries,including Canada,the United States,Brazil,Thailand,China and Colombia.Since the beginning of 2015,SVA has been detected in diseased pig farms in Guangdong,Hubei,Fujian,Henan,Heilongjiang and Guangxi provinces.The SVA epidemic caused severe neonatal piglet deaths and caused large economic losses to the pig industry.The prevention and treatment of SVA in China faces great challenges.In this study,samples of vesicular fluid were collected from a diseased herd in a large-scale pig farm in Guangxi province.Conventional RT-PCR was used to exclude common vesicular pathogens.The results show tissue samples are negative for VSV,FMDV and SVDV,but positive for SVA.Tissue samples tested SVA positive are inoculated into BHK-21 cells.Cytopathic effects(CPEs)are observed in cell inculated at 24 hours post inoculation.Subsequently,indirect immunofluorescence assay(IFA)is conducted by using the SVA VP1 polyclonal antibody as primer antibody.The VP1 expression is obserbed in the infected cells.The sequence of VP1 gene of isolate comparison of the VP1 gene of isolate with other strains show that it is highly homologous with the known SVA strain.Our result demonstrates that the SVA virus named SVA CH-GX-01-2018,is successfully isolated.To amplify the whole-genome sequence of the strain,multiple specific primers covering the whole genome were designed for PCR amplification.The amplified fragments were cloned into T vector,sequenced and assembled.The complete genome of SVA CH-GX-01-2018 is submitted to Gene Bank under the accession number(MK039162).Phylogenetic tree analysis show that SVA CH-GX-01-2018 and prototype virus SVV-001 are in two completely different evolutionary branches,but in the same evolutionary branch with the 2017 Guangdong isolate.The whole genome sequence alignment results show that the nucleotide similarity between SVA CH-GX-01-2018 and US isolate SVV-001 is 93.1%,while with Guangdong isolate in 2017 is as high as 98.7%.To construct a full-length infectious cDNA clone of SVA,a serial of overlapping primers was designed based on the complete sequence of the SVA CH-GX-01-2018 genome and used for amplification of the full-length genome of SVA CH-GX-01-2018.The amplified fragments were cloned into the p Bluescript vector according to the corresponding restriction sites.Finally,CMV promoter and ribozyme elements were introduced at the 5’ and 3’ ends of viral genome,respectively.The resulting construct p SVA GX01 was transfected into BHK-21 cells for virus rescue.The recovered virus was identified by methods such as specific PCR detection,cytopathic effects(CPE),indirect immunofluorescence assay(IFA)and growth curves.The results show that the infectious clone p SVA GX01 is recovered.The isolation of the SVA CH-GX-01-2018 strain lays a foundation for the development of diagnostic reagent and vaccine.Meanwhile,the establishment of the reverse genetic manipulation platform of SVA plays an important role in the study of viral replication,pathogenic mechanism and novel vaccine development.