Isolation,Identification And Cell Biological Characteristics Of Senecavirus A Chinese Strain

In order to demonstrate the biological characteristics of epidemic strain of Seneca virus A?SVA?where was isolated from Hubei area of China.In this study,the blisters'surface and it's liquid from nose kiss and hoof crown of the suspected pigs was collected,SVA is isolated by IBRS-2.Primers were designed based on the SVA SVV-001 strain sequence and amplification by PCR.Porcine kidney cell line?IBRS-2?,swine testis cells?ST?and porcine kidney-15 cells?PK-15?were infected with SVA repectively.Through tissue culture infective dose 50(TCID₅₀)experiment,Plaque assays,real-time PCR quantification and indirect immunofluorescence assays to research it's characteristics of biology.The results of SVA isolation and identification showed that virus-liquid of isolation produced CPE on IBRS-2 cells,and the degree of cytopathicity increased with the increase of the number of passages.The results of whole genome sequence alignment showed that compared with the strains which were reported in China,such as 2017?CH-HN-2017,CH-HNSL-2017,CH-FJ-2017?,2015?CH-01-2015,CH-03-2015,CH-04-2015?and 2016?CH-GX109-2016,CH-DL-01-2016?,the nucleotide homology was 98.5%-98.6%,96.4%-97.6%and 96.2%-96.4%,respectively.In addition,compared with foreign strains,suchasthestrainofAmerica?USA-IA44952-2015,USA-IA44662-2015,USA-SD41901-2015?,the strainof Brazil?BRA-G03-2015,BRA-MG2-2015,BRA-MG1-2015?andthestrainofCanada?Canada-MB-NCFAD-11-7-20,Canada-ON-FMA-2015?,the nucleotide homology was 98.5%,97.4%and 96.4%-96.6%,respectively.Thus,it was determined that the isolated strain belongs to the genus Seneca virus.The results of cell biological characteristics showed that the strain could be replicated and proliferated in IBRS-2,ST and PK-15,and both produced CPE after infection but IBRS-2cells were most susceptible to this strain.In IBRS-2 cells,obvious lesions appeared around 8h,and the TCID₅₀0 result was 10^-7.4/100?L.Compared to IBRS-2,ST cells and PK-15 cells showed not obvious lesions and viral titer is low.Based on the level of RNA,SVA had the highest gene replication level in IBRS-2 cells.Indirect immunofluorescence assay showed specific fluorescence in IBRS-2 cells,ST cells,and PK-15 cells.A SVA epidemic strain was successfully isolated in this experiment,and the biological characteristics of the strain were analyzed to provide the foundation for the in-depth study of the virus.