Establishment And Application Of An Indirect ELISA Antibody Detection Method For Getah Virus

Getah virus(GETV)is a serious threat to public health and safety.So far,it has spread to 12 countries in Eurasia and the Pan Pacific region.The high pathogenicity of GETV to horses and pigs has caused huge economic losses to the breeding industry.After 1962,there were seven large-scale outbreaks of GETV in horses all over the world.In 1965,Japan first reported the infection of pigs by GETV,and then the virus quickly spread to Asia.In recent years,the popularity of GETV has become worse and worse in China.The clinical manifestations of equine infection with GETV often include fever,systemic rash and hindlimb edema.The infected piglets showed depression and tremor,and sows would have high mortality and abortion after infection.Although the virus poses a great threat to society,the circulation,distribution and evolution of GETV are not very clear.Therefore,it is necessary to increase the research on the detection technology of GETV to reduce the risk.Considering that the traditional technologies such as virus isolation and cell culture take a long time,the operation is complex,the cost is high,and it is difficult to deal with a large number of samples,and ELISA is very suitable for the detection of a large number of samples in clinic with its advantages of sensitivity,simplicity and rapidity,this study constructs an ELISA detection method to provide a scientific basis for the development of later detection technology.This study includes the following three contents:1.Expression and purification of GETV-E2 proteinGETV-E2 protein is a key protein of GETV and is very stable.The gene marker of GETV pathogenicity is located in E2 protein,and the key protein identified by most detection techniques is also this protein.In this study,E2 protein was expressed and purified,which was about 46 KD and the concentration was 1.96 μg/μL.Western blot analysis showed that the E2 protein had immunogenicity and could be used as the coating antigen of ELISA.2.Establishment and application of pig-GETV indirect ELISA methodIn this study,an indirect ELISA method for porcine GETV antibody was established.This ELISA method has strong specificity,high sensitivity and good repeatability;By comparing ELISA with IFA,the coincidence rate was 94.96%.This study applies this method to analyze three aspects.Firstly,the relationship between sample source and pig GETV positive rate was analyzed.Overall,the total positive rate of 338 pig sera from 14 provinces was 34.72%,indicating that the infection rate of GETV is relatively high nationwide.Secondly,the relationship between season and pig GETV positive rate was analyzed.169 sera were collected in April,and the positive rate was 22.49%;The positive rate of168 sera collected in October was 47.02%,which was significantly higher than that in April.Finally,the relationship between age and pig GETV positive rate was analyzed.The positive rate of 7-21 days old was 38.75%,while the positive rates of 40-70 days old and 3-5 months old were 28.75% and 29.90% respectively,which decreased,but the positive rate of 8-10 months old was 42.50%.3.Establishment and application of horse-GETV indirect ELISA methodIn this study,horse GETV indirect ELISA antibody detection method was established,which has strong specificity,high sensitivity and good repeatability;By comparing ELISA with IFA,the coincidence rate was 97.87%.This study applies this method to analyze two aspects.First,the relationship between sample source and horse GETV positive rate was analyzed.328 horse sera were collected from the Netherlands,Japan,Ireland and Australia,and the total positive rate was 31.71%.The positive rates of the Netherlands and Ireland were lower,23.17% and 4.88% respectively,while those of Japan and Australia were higher,45.12% and 53.66% respectively.Second,the relationship between age and horse GETV positive rate was analyzed.The positive rate of 1-3 years old was 32.53%,while the positive rates of 3-4 years old and 4-5 years old were 23.00% and 27.14% respectively,which decreased,but the positive rate of 5-7 years old was 41.33%,showing an upward trend.In conclusion,this study established the indirect ELISA antibody detection methods of pig-GETV and horse-GETV,and preliminarily learned that the infection rate of GETV in Chinese pigs is high,and the positive rate of GETV in horses from four countries is not low,so the prevention and control and research of GETV should be increased.