Serological Epidemiological Investigation Of Equine Getah Virus Disease In Xinjiang And Establishment Of Indirect ELISA

Getah disease is an mosquito-borne infectious disease caused by Getah virus(GETV).Due to the lack of related vaccines,the epidemic trend of Getah disease is becoming increasingly serious worldwide,especially in China,posing a potential threat to animal safety and public health.However,there are few reports about the epidemiological investigation of Getah disease in China.In addition,there is no mature diagnostic methods for GETV antibody,which makes it very difficult to understand the prevalence of Getah disease.Therefore,the establishment of a rapid,sensitive and suitable for large-scale sample detection of GETV antibody detection method is an urgent need to fully understand the prevalence of Getah disease.In this study,serum neutralization test was used to investigate the seroepidemiology of GETV disease among horses in some regions of Xinjiang(Karamay city,Aksu Prefecture,Changji Hui Autonomous Prefecture and Ili Kazak Autonomous Prefecture).Meanwhile,the E2 domain protein of GETV(GETV-E2d)was expressed by prokaryotic expression system.A checkerboard method was used to optimize the reaction conditions by using the purified recombinant protein as the coated antigen whereas the screened GETV negative and positive horse serum as the primary antibody,and the HRP labeled rabbit anti-horse serum as the secondary antibody.An indirect ELISA method was established for the detection of GETV protein antibody of horse.The results are as follows:(1)Among 732 equine serum samples from four regions in Xinjiang,465 positive serum samples were detected,and the positive rate of neutralizing antibody was 63.5%.The geometric mean titer(GMT)of neutralizing antibody in positive serum was 1:149.3.It is suggested that GETV infection is prevalent in horses in these areas.(2)The recombinant protein GETV-E2 d was mainly expressed as inclusion body,and the optimal induction conditions were as follows: 37℃,0.8 mmol/L IPTG,induced expression for 12 h.After purification and dialysis,the concentration of recombinant protein was 0.5 g/L.The prepared recombinant protein showed good reactivity and immunogenicity,and the Ig G titer of GETV-E2 d of rabbit polyantisera was 1: 1600000.(3)An indirect ELISA method of antibody against equine GETV using GETV-E2 d as antigen was successfully established.The optimal reaction conditions were as follows: the antigen concentration was 4μg/m L and the antigen was coated overnight at 4℃,followed by blocked with 1% BSA for 2 h;the dilution of serum to be tested and enzyme-conjugate secondary antibody was 1:40 and 1:3000,and the incubation time was 60 min and 90 min,respectively.The reaction time of TMB substrate was 20 min.This method has good specificity,sensitivity and repeatability.The indirect ELISA method was used to detect 210 equine serum samples,and compared with serum neutralization method,it had a high overall coincidence rate(92.4%)and a decent consistency(Kappa value 0.775).