Isolation And Identification Of Senecavirus A And Preparation Of VP3 Protein Monoclonal Antibody

Senecavirus A(SVA)is the only member of the genus Piconavirus,Senecavirus genus.SVA infection causes lameness,lethargy,anorexia,acute death,and characteristic blistering symptoms in newborn piglets.At present,many countries in Canada,Colombia,the United States,Brazil,China,Vietnam,Thailand and other countries have reported the SVA epidemic.Since 2015,14 provinces such as Guangdong,Hubei,Fujian,and Hubei have reported SVA outbreaks in succession.Studies have shown that the currently popular SVA strains in China are mutating,and some strains have been reorganized.The latest research found that SVA seems to have acquired a stronger pathogenicity and ability to spread than before.However,the epidemiological information of SVA strains in China still needs to be supplemented,especially the information of SVA pathogenic strains is still very lacking.Therefore,in this study,two Chinese SVA strains were isolated and identified,and the differences in the strains were analyzed in terms of genetic relationship,genomic level,and pathogenicity.A monoclonal antibody capable of specifically recognizing VP3 protein was prepared,and its epitope was identified,which provided a powerful immunological tool for the development of SVA new diagnostic reagents and related research work of the virus.The details are as follows:1 Identification,sequencing and evolution analysis of Senecavirus A strainsIn this study,the suspected disease materials from several pig farms in Guangdong Province from 2017 to 2018 were analyzed,virus isolation and whole genome sequencing,and the two SVA strains were named SVA GD04/2017 and SVA GD-S5/2018(GenBank login No.:MH316113,MK463618).The growth kinetics curve showed that the two strains had similar biological characteristics.At 12?60 h after infection,the titer of GD-S5/2018 was slightly higher than that of GD04/2017.The plaque assay showed that the two strains had similar biological characteristics,and the GD-S5/2018strain had slightly larger plaque.The ORF genome and polypeptide sequences of GD-S5/2018,GD04/2017,SVA SD15-26 and SVV-001 were analyzed and compared.The phylogenetic tree based on ORF and VP1 coding region shows that GD-S5/2018and SVA SD15-26 belong to the same genetic branch,GD04/2017 belong to another branch,and SVV-001 has the farthest phylogenetic relationship with other SVA strains.GD-S5/2018and SVA SD15-26 showed the highest similarity.Compared with other reference strains,GD04/2017has 11 nucleotides inserted at 5'UTR of genome.2 Pathogenicity analysis of Senecavirus AIn order to study the pathogenicity difference of SVA strains(GD-S5/2018and GD04/2017),the virus infection experiment of pigs was carried out.The results showed that both SVA strains successfully infected all pigs.In the GD-S5/2018 group,blisters and ulcerative lesions on the tongue and gums were observed.However,no other clinical symptoms were observed except for malaise in the GD04/2017 group.Within 1-3 days after infection,the virus load in the nasal swab of GD-S5/2018 infection group was higher than that of GD04/2017 infection group.Within 1-6 days after infection,the body temperature of GD-S5/2018 group was higher than that of GD04/2017 group.The GD04/2017,GD-S5/2018 and GD05/2017 were neutralized by SVA-infected pig sera.The results showed that all the SVA-infected pigs generated neutralizing antibodies against SVA strains,and there were antigen-specific differences between the strains.3 Preparation and epitope identification of VP3 McAb against Senecavirus AIn this study,the truncated VP3 fragment was inserted into the procaryotic expression vector of pCold ? and transferred into E.coli BL21(DE3).The fusion protein was induced and expressed by IPTG.The purified protein was immunized to mice.After three times of immunization,the mouse polyclonal serum was positive by IFA test.The spleen lymphocytes of mice were fused with hybridoma cells.Hybridoma cell lines which could stably secrete monoclonal antibodies against SVA VP3 protein were obtained by positive screening and subcloning.The results of Western blot and IFA showed that the ascites McAb could specifically recognize SVA VP3 protein.Further,the epitopes recognized by the McAb were identified.The gene sequence of the antigen protein was amplified and inserted into the vector pEGFP-C3 for expression.Then the smallest fragment that can be recognized by monoclonal antibody was identified by Western blot.The results showed that the McAb recognized the 191-201 amino acid sequence of SVA VP3 protein(191WFSLHKLTK201).This epitope sequence was compared with the amino acid sequence of VP3 of 65 SVA strains in different regions.The results showed that this epitope was highly conserved in each strain sequence,and there was no amino acid difference in this epitope in all strains.The preparation of the monoclonal antibody and the identification of B cell epitope will provide a powerful tool for development of SVA diagnostic reagents.