Isolation And Identification Of Senecavirus A,Establishment Of Detection Methods And Development Of Monoclonal Antibody

Senecavirus A(SVA),previously called Seneca Valley virus(SVV),a nonenveloped single-stranded RNA virus,belongs to the genus Senecavirus in the family Picornaviridae.It is one of the cause of porcine idiopathic vesicular disease(PIVD).Adult sows often manifeste as latent infection.The virus can cause elevated body temperature,vesicular lesions on the snouts and coronary bands in sows,even a acute death in neonatal piglets.Since the first outbreak of the disease in China in 2015,the virus has spreaded in many provinces,posing a serious challenge to the healthy development of the pig industry.In this study,a SVA strain was isolated and the following aspects were studied:1.Isolation and identification of a Senecavirus A.Vesicle fluid samples were collected from vesicles on suspected SVA-infected pigs.BHK-21 cells culture system was used for the virus isolation,and the designated virus was named as SVV-CH-SD.The virions,with a diameter of 27?30 nm,had an icosahedral symmetry.The virus strain could grown on BHK-21,Marc-145,ST,Vero,293T and 3D4 cells.The SVV-CH-SD strain was distributed in the cytoplasmic of the infected BHK-21 cells,and the titer of the virus reached the highest at 20 hour post-infection(h.p.i).The plaques were approximately 1.5 mm in diameter at 24 h.p.i.In a word,the isolation of the virus strain will be benefit the genetic evolution analysis of SVA,the study of the pathogenic mechanism as well as the design of vaccines.2.Establishment and application of a RT-PCR assay for detection of Senecavirus A.In this study,three pairs of primers which designed based on the conserved regions of SVA genome sequence were designed,and the best primer which had highest specificity and amplification efficiency was selected.The optimal reaction conditions are as follows:the primer concentration was 4 mM/L,the cycling conditions were 95? for 5 min,followed by 35 cycles of 95? for 30 s,52? for 30 s,72? for 15 s,and a final extension at 72? for 5 min.This method had a good specificity and sensitivity.And the sensitivity of the detection was 1 TCID50.100 samples of pig tissue from Shandong province were detected,and the positive rate was 2%.The establishment of this method could be as a technical means for SVA detection.3.Establishment and application of indirect ELISA assay based on VP1 for the identification of antibody to Senecavirus A.The purified SVA recombinant VP1 protein was as coated antigen for the development of indirect ELISA assay.The reaction conditions of the ELISA method were optimized.Also,the relative sensitivity and the coincidence rate were comparied with seroneutralization test.The optimal reaction conditions are as follows:the coating concentration of protein was 3 ug/ml,the coating condition was 37? 2 h,and the plate was blocked with 5%nonfat dry milk at 37? for 3 h,the dilution of blood sample was 1:100 at 37? for 1 h,the dilution of HRP-SPA was 1:5000 at 37? for 30 min,and the TMB was added at 37? for 8 min.The S/P ratio was as the criterion,if S/P>0.278,the serum sample was judged as positive;if S/P<0.186,it was judged as negative.And this method had a grest specification and repeatability.Comparing with seroneutralization test,it had the relative sensitivity of 98%.The positive rate of SVA antibodies was 22.1%of 276 clinical pig serum.In short,this method can be used as a rapid and simple serological diagnostic method for monitoring SVA infection and epidemiological investigation in pigs.4.Development of monoclonal antibody against VP1 protein and identification of epitopes of Senecavirus A.In this study,the purified SVV-CH-SD strain and recombinant VP1 protein were as immunogen to immune BALB/c mice.Through cell fusion and subcloning technology,ultimatly,one hybridoma cell line 1G9 was successfully obtained.1G9 can stably secreting monoclonal antibody(mAb),which could specifically recognize SVA,recognizing the epitope at 21GELAAP26 on VP1.The 1G9 mAb was of the IgG1 subclass,and the light chain type was Kappa.The characteristics of 1G9 mAb including antigenicity,spatial structure and conservative analysis were also studied in this paper.The development of the 1G9 mAb and the identification of epitope will be beneficial to the development of diagnostic technologies and the design of viral vaccines.