Isolation And Identification Of Seneca Valley Virus In Sichuan Province,Complete Gene Sequence Analysis And Eukaryotic Expression Of Its 3C Protein

Seneca Valley virus?SVV?can cause blisters and ulcerated wounds in pigs'noses and hoofs,leading to lameness and even lead death.SVV is contagious and harmful for piglets.In recent years,SVV had frequently erupted around the world,but its clinical symptoms are similar to foot-and-mouth disease,so it is of great significance for the systematic study of tissue culture characteristics,pathological characteristics and molecular biological characteristics of the virus.In this study,blister fluids and diseased tissues from suspected SVV-infected pigs in areas such as Meishan and Suining in Sichuan province were used as the original material.After detection,SVV-positive materials were selected and aseptically treated.PK-15 cells were inoculated for virus isolation.The CPE of PK-15 cells appeared in the process of isolation.To the 10th generation,typical CPE appeared stably after detoxification for24h,which showed that individual cells became round.And as time goes on,a large number of cells pile up and float,floating on the liquid surface.The isolations were purified by picking the spot caused by virus.It was confirmed by RT-PCR that two strains of SVV viruses were successfully isolated and named as SVV-MS and SVV-SN strains.The TCID₅₀of the SVV-MS strain and SVV-SN strain were determined to be 10^-5.66/0.1ml and 10^-6.12/0.1ml respectively.The virus-liquid of SVV-SN were injected into 2-3 day old and 7-9day old suckling mice.It was identified that two groups of suckling mice could not pass the SVV-SN strains.The two SVV strains obtained in this study provided raw materials for the pathology,molecular biology and vaccine production of Sichuan SVV epidemic strains.Based on GenBank's published strain SVV-001?accession number:DQ641257?,two SVV strains was amplification by 8 pairs of primers.Both the sequences of SVV-MS stain and SVV-SN stain were constituted by 7312 nt.And these two strains have typical structural characteristics of small RNA viruses.The whole gene sequence analysis of these two SVV strains showed the SVV-SN strain had a higher identity with the American strain while the SVV-MS strain had higher identity with Chinese strains,which reflects the diversity characteristics of SVV gene in Sichuan province.At the same time,both strains and most of the other SVV strains have low identity with the first strain SVV-001,suggesting the continued evolution of SVV.In addition,the sequence analysis of the VP1 gene of two SVV strains showed that there was only one mutation in the amino acid in the SVV-MS strain,but there were three multiple sites in the SVV-SN strain for VP1.In conclusion,these dates enriched the molecular epidemiology and genetic evolution of SVV in China,and provided a reference for subsequent research on SVV virology.Using software to analysis and compare the SVV strains isolated in this experiment with the world reference strains,a pair of specific primers for 3C gene were designed.After sequencing,the correct fragment was subjected to bioinformatics analysis.It can be seen that SVV 3C can encode a relatively stable non-secreted protein,and it is predicted that the protein has 16 phosphorylation sites,which proves that it has higher activity,and then it can be supported after predicting its secondary structure.Then pMD19-SVV-3C and eukaryotic expression vector pcDNA3.1?+?were digested and ligated,the recombinant plasmid which identified to be the positive sample will be named as pcDNA3.1?+?-SVV-3C.pcDNA3.1?+?-SVV-3C was transformed into BHK-21 for 24h,then collecting the sample for Western-blot.The predicted size band appeared,which proved that the recombinant plasmid could be expressed in BHK-21.pcDNA3.1?+?-SVV-3C and pEGFP-N1 plasmid which expressing GFP protein in a cap-dependent mechanism were co-transfected into BHK-21.After 24h and48h,comparing with the controlled group,more obvious fluorescence of the experimental group was observed under fluorescence microscope.Collecting the samples to Western-blot.The results were consistent with the results showed under fluorescence microscope.It was proved that SVV-3C protein can increase the expression level of protein which express in eukaryotic cells by cap-dependent mechanism.