| Senecavirus A(SVA),a member of the family Picornaviridae,genus Senecavirus,is a novel foreign virus of swines.The clinical presentation of SVA resembles that of other vesicular diseases,such as foot-and-mouth disease(FMD),swine vesicular disease(SVD),and vesicular stomatitis(VS),swine vescular exanthema(VES).SVA is easily confused and misdiagnosed with these diseases,which can lead to great economic losses.However,there was few researches on pathogenic mechanism,epidemic situation,diagnosis and prevention and control technology of SVA and lack of materials for research.How to into China,occurrence and epidemic,genetic evolutionary need further study.Therefore,this study carried out experiments,including the isolation and identification of SVA in Guangdong and the prokaryotic expression of VP1 gene,which has practical significance.Isolation and identification of SVA strains in Guangdong Province.In this study,108 clinical samples collected and preserved from multiple farms in Guangdong by the laboratory in 2016-2018 were screened by triple fluorescent RT-PCR method,and two samples were suspected.After two sterile SVA-positive samples were sterile filtered,Swine Testis Cell(ST)and Baby Hamster Syrian Kidney cells(BHK-21)were inoculated for virus isolation.After passage 6 passage,typical cytopathic lesions appeared within 48 hours.After continuous subculture,the cell culture supernatant was subjected to sucrose density gradient centrifugation purification,subjected to RT-PCR amplification and sequencing,and the sequencing knot was submitted to NCBI Blastn alignment,and the results showed that the two strains were SVA,named SVA CH-GDBL1-2016 and SVA CH-GDBL2-2016,respectively.The cell culture supernatant was subjected to electron microscopic examination,and virions having a diameter of about 25 nm were observed.The titers were 106.8 and 106.7,respectively,as determined by TCID50.The whole geneome sequence was determined and compared for the analysis.According to the SVA sequence registered by NCBI,three pairs of specific primers were designed,and two SVA viruses were amplified and analyzed.The NCBI Blastn website and the analysis software DNAStar were used to analyze the whole gene sequence of the virus.The results showed that the homology of the two viruses was between 93.8% and 99.3%.Among them,the American strain ATCC PTA-5343 has the lowest homology,93.9%,93.8%,respectively,and the highest homology with Chinese strain CH/GXI09/2016,up to 99.3%.Furthermore,the homology between the SVA CH-GDBL1-2016 strain and the SVA CH-GDBL2-2016 strain was 99.8%.In the phylogenetic tree,the two strains in this study have the closest relationship with the American strains ATCC PTA-5343 and SVV-001,and the variation is large.Among them,the SVV-001 strain is the classic prototype of SVA;and the South China 2016 strains.The CH/GXI09/2016 strain has the closest genetic relationship.Thus,the two strains isolated and identified in this study belong to the genus Senecavirus.Prokaryotic expression assay of the VP1 gene.In order to establish an indirect ELISA kit for SVA,the VP1 gene of the isolate SVA CH-GDBL1-2016 was prokaryotically expressed and successfully expressed to obtain a fusion protein of about 48 KDa,which was confirmed by SDS-PAGE.With Western Blot text,the VP1 protein was identified to have good antigenicity.Under the optimization of multiple conditions,a fusion protein was expressed with better effect which can provide materials for the subsequent establishment of an indirect ELISA kit.In summary,this study isolated 2 strains of Senecavirus in Guangdong,which homology analysis showed that the 2 strains are closer to the Vietnamese and Chinese strain CH/GXI09/2016 rather than strains in the United States,Colombia,Brazil.And this study successfully obtained the VP1 fusion protein of SVA,which has good antigenicity and is intended to be used to establish an ELISA diagnostic method.It provides key materials and lays the foundation for the diagnosis,prevention of SVA. |
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