Generation Of Neutralizing Monoclonal Antibodies Against Seneca Valley Virus And Idetification Of Their Neutralizing Epitopes

Seneca Valley virus?SVV?,the only member of the genus Senecavirus of the Picornavirus.Researchers isolated SVV-001 from pollutants in human embryo retinal cell cultures in 2002.And in 2007,it was reported for the first time that SVV infection in pigs is related to pig idiopathic vesicular disease.After than,there are many papers of infection about SVV have also been reported in the United States,Brazil,China,Thailand,Colombia,and Vietnam.Infections mainly cause pigs oral mucosa,nasal kiss and coronet blisters and ulcer lesions,and then lameness,anorexia,lethargy.The mortality of SVV infection in newborn piglets within 7 days is about 30%to 70%,often accompanied by diarrhea.The structural proteins in SVV play an significant role in infection and they can induce n Abs,but there are no reports on the preparation and research of SVV with n Abs.And it is significant in the further use of neutralizing antibody for immune prevention and treatment of SVV.In this study,the SVV-LNSY01-2017 strain was used as the research object.Six-week-old BALB/C female mice were immunized with the purified virus.The mice's serum showed positive by indirect ELISA,then made fusion of mouse splenocytes and myeloma cells.After detection of virus neutralization test,indirect immunofluorescence verification,chromosome counting and Western-blot identification,10 monoclonal antibodies with neutralizing effect on SVV-LNSY01-2017 strain were successfully obtained.The antibodies with higher ELISA titer were 2C8,3E4,4C3,6D7,6E3,6E5,and 7C11,and the antibodies with lower ELISA titer were 5A1,5D4,and 7H7.Eukaryotic expression plasmids were constructed and identified by Western-blot.Seven high ELISA titer m Abs were specifically bound to the structural protein VP2,indicating that VP2 contained some neutralizing epitopes.In order to further identify the epitope of SVV,the truncated fragment primers of the VP2 gene sequence of SVV-LNSY01-2017 strain were designed first.Constructed the V1-V8 recombinant expression plasmid.The fusion proteins expressing GST and truncated proteins were obtained by using these plasmids for inducible expression of recombinant proteins.Western-blot analysis indicated that 8 truncated proteins were showed up,and 7 m Abs with high ELISA titer recognized V5?129aa-166aa?and V8?143aa-200aa?truncated proteins,indicating that the neutralizing epitope was present in the VP2 protein between143aa-166aa.Then the truncated expression of this region was continued.By Western-blot identification,the linear neutralizing epitope was determined to be the VP2 protein 153aa-158aa(¹⁵³QELNEE¹⁵⁸).By comparing the amino acid sequence of sequence ¹⁵³QELNEE¹⁵⁸with twenty-six SVV strains of VP2,which was downloaded from Gen Bank,the neutralizing epitope ¹⁵³QELNEE¹⁵⁸is highly conserved in different isolate strains,and only one mutation site Q¹⁵³E exists in the SVA-HLJ-CHA-2016 strain.By constructing a single point mutation plasmid of the VP2 protein neutralizing epitope,Q¹⁵³,E¹⁵⁴,E¹⁵⁷and E¹⁵⁸were detected by Western-blot as the key amino acid sites of the neutralizing antibody.In summary,this study successfully prepared 10 neutralizing antibodies with neutralizing anti-SVV-LNSY01-2017 strain,7 m Abs specifically recognized the VP2protein,and identified a neutralizing epitope located at the VP2 protein ¹⁵³QELNEE¹⁵⁸.Four key amino acid positions have been identified,which will be helpful in the further study of the structure and function of SVV genome.