Development And Genetic Stability Evaluation Of Swine Senecavirus A Attenuated Strain

Senecavirus A(SVA),as a member of the picornavirus family,causes primary blister infection in swine.The disease associated with SVA was first confirmed in China in 2015.So far,SVA-associated epidemics have spread to more than half of China’s Provinces.The resulting primary blister infection can cause lameness in piglets and death in newborn piglets.At present,vaccine immunization is the development direction of preventing and controlling SVA vesicular diseases.Therefore,epidemiological study and vaccine development associated with SVA is of high importance for preventing and controlling the disease.This study characterized the current genetic evolution of SVA.Then bioinformatics software was used to analyze the sites affecting virus replication in SVA non-structural proteins.SVA attenuated strain was prepared by reverse genetic operation technique,and the titer and genetic stability of the generated mutation strains were measured.The specific experimental content and results of this study were as follows:1 Characterize the genetic history of SVA and evaluate the application potential of CHN/SVA/HB/2018 attenuated strainsThe complete SVA genome sequences from 1989 to 2019 were evaluated for genetic recombination events,followed by genetic evolution analysis based on SVA strains without recombination events.Subsequently,SVA’s linear genetic evolutionary route was defined by using molecular programm,and its phylogenetic history was characterized.The possibility of CHN/SVA/HB/2018 strain recombination was ruled out,and its potential as a wild parent strain of the SVA attenuated vaccine was evaluated from the perspective of linear evolution and phylogeny.2 Construction of SVA reverse genetic systemThe CHN/SVA/HB/2018 strain was amplified by RT-PCR,and the Hepatitis D ribozyme and hammerhead ribozyme sequences were added at the 3’ and 5’ terminus of the complete genome.The complete SVA genome fragments and ribozyme were cloned to the eukaryotic expression vector pc DNA3.1 with CMV promoter by restriction endonuclease and DNA ligation methods.The reverse genetic system of SVA was constructed,and the virus was rescued successfully.3 Effect of mutation of SVA non-structural proteins on viral replication and generation of attenuated strainsThe sites affecting viral replication in non-structural proteins of CHN/SVA/HB/2018 strains were analyzed by using bioinformatics software.And a reverse genetic system for mutations on the selected amino acids was constructed using CMV promoter-driving plasmid.Finally,the SVA attenuated strains were rescued based on D84 N and C160 G single site mutant in 3C protein,respectively.Compared to the wild-type strain CHN/SVA/HB/2018,the two mutant strains presented significantly lower viral titer post-infection in IBRS-2 cells.4 The genetic stability of SVA mutant attenuated strains was testedIBRS-2 cell lines were inoculated with the two mutant attenuated strains in vitro,and viral passage was performed.The genetic stability of the attenuated mutant strains was evaluated by gene sequencing.The results showed that the genetic stability of the SVA 3C protein D84 N mutant was unfavorable,and atavism appeared after ten passages.However,the SVA 3C protein C160 G mutant attenuated strain did not have a return mutation at its mutation site after 20 passages,which means that the SVA 3C protein C160 G mutant attenuated strain had good genetic stability.In this study,the mutation of SVA 3C protein antagonistic innate immunity-related sites was carried out,and the attenuated mutant strain was successfully prepared under the background of mutual application of reverse genetics and bioinformatics.Compared with the wild-type parent strain,the virus titer of the attenuated strain was significantly reduced,and the genetic stability was tentatively high.The results of this study provide important application references for the research and development of SVA attenuated live vaccine and also provide a reference for the application and transformation of the research results of SVA molecular virology.