Complete Genome Sequence Analysis And Establishment Of Reverse Genetic Operating System Of Senecavirus

Senecavirus A(SVA)is a pathogen that can cause in the mouth and hoof crown blisters in the infected pigs.In this study,the SVA/CH/ZZ/2016 isolated from Zhengzhou City were sequenced and performed the whole-genome analysis.Firstly,the whole genome of SVA/CH/ZZ/2016 strain was segmental amplificated by RT-PCR technology and was introduced the Nde I restriction site at 2176 position.The recombinant plasmid was transfected into BHK-21 cells for virus rescue,and it was blindly transmitted on PK-15 cells until cytopathic(CPE)appeared.Moreover,the CPE cells was taked supernatant for RT-PCR amplification,sequencing,indirect immunofluorescence test,Western-blotting test for identification.At last,after the plaque test and the one-step growth curve of the virus were compared between the identified rescued virus and the parental virus,the recombinant virus immunized animal is made into a polyclonal positive serum,and the cross-neutralization test is performed with the parent virus.The results showed that the sequence of SVA/CH/ZZ/2016 isolates amplified by RT-PCR was 7292 bp in length,The sequence of 9 reference strains domestically and internationally was selected to compare the nucleotides of the 12 genes in the coding region.It showed that the 3B gene had the highest nucleotide homology,the VP1 gene had the lowest homology.The genetic evolution analysis of the VP1 gene showed that the SVA/CH/ZZ/2016 strain was closely related to the 2016 US isolates,and belongs to the same evolutionary branch.The recombinant plasmid was successfully constructed and the recombinant plasmid was transfected into the BHK-21 cells.The typical SVA CPE appeared after the virus was blindly transferred to F5 generation.Indirect immunofluorescence test and westerning-blot test of CPE cells supernatant showed that the recombinant SVA virus was successfully rescued;The virus plaque test and growth curve results showed that the rescued strain and the parent strain had similar replication capabilities and value-added characteristics;the cross-neutralization test results show that the antigen matching relationship between the two strains r=0.98,and the two strains have a relatively high antigen matching relationship.