| As an international metropolis,Shanghai is the birthplace of China's earliest horse racing,dating back to 1850.With the people's pursuit of a better life,the development of horse racing and related leisure industry is developing day by day,the number of horses is increasing,so to strengthen the surveillance of equine diseases,especially new diseases,will help to promote the sustainable development of related industries.Getah virus(GETV)is a single-stranded RNA virus transmitted by blood-sucking arthropods and is a member of the Togaviridae virus family.GETV was first isolated from mosquitoes in Hainan Province of China in 1364,and has since spread widely and rapidly in China.GETV has been isolated from mosquitoes,pigs,horses,cattle and foxes in Hebei,Hainan,Sichuan,Gansu and Shanghai provinces.GETV has become an important equine pathogen,causing febrile disease,anorexia,hind limb edema stiff gait,urticaria,and lymphadenopathy.So far,there are still no effective prevention and treatment of vaccines and drugs for the diseases caused by Getah virus.Therefore,it is necessary to strengthen the monitoring and control of GETV and to establish an accurate and specific technique for the rapid detection of GETV.In view of the increasing epidemic tendency and potential harm of GETV in horses in recent years,the distribution of mosquito population in horse farm and GETV carried them were investigated for the first time,and the virus were isolated and identified,and the recombinant E2 protein antigen of GETV was prepared,in order to understand the situation of GETV infection in horses,a rapid detection method of GETV antibody with colloidal gold was established by coating with nitrocellulose membrane,and the serological investigation and analysis of GETV in horses were carried out in Shanghai,Confirmed the GETV infection in the horses.This study consists of the following four parts:1.Investigation on mosquitoes and their carriers of Getah virus in horse farms of ShanghaiIn order to investigate the population structure and distribution and Getah virus them carried of mosquito in horse farms in Shanghai,surveillance sites were selected from 7 horse farms in Songjiang District,Fengxian District,Pudong New Area and Jinshan District in July 2018,light trap was used to collect mosquitoes and fluorescent quantitative RT-PCR was used to detect the presence of Getah virus in mosquitoes.The results showed that 11,140 mosquitoes of 6 species and 3 Genera were caught in 7 horse farms in Shanghai,including 10,738 Culex tritaeniorhynchus of the third generation accounting for 96.4%,116 Culex bitaeniorhynchus of the second generation accounting for 1.0%,and 247 Anopheles sinensis accounting for 2.2%,the other 3 species(Culex pipiens pallens,culex pipiens quinquefasciatus and Culex Quinquefasciatus)were 37,accounting for 0.3%.A total of 111 specimens(every 100 Mosquitoes/pool)were detected by RT-qPCR.Four samples were found to be positive for Getah virus,with a positive rate of 3.6%.It can be seen that Culex tritaeniorhynchus of the third generation is the dominant species in horse farms in Shanghai,Moreover,culex tritaeniorhynchus of the third generation or Anopheles Sinensis in Shanghai horse farm carry Getah Virus,Surveillance and control of mosquito vectors and Getah virus in horse farms in Shanghai should be strengthened.2.Isolation,identification and genetic evolution analysis of E2 gene of mosquito-source Getah virus from Horse Farm in ShanghaiIn order to understand the molecular characteristics of mosquito-borne Getah virus in Shanghai,11,140 mosquito were collected from Songjiang District,Fengxian District,Pudong New Area and Jinshan District horse farm in Shanghai in July 2018,and BHK-21 and C6/36 cells were inoculated for virus isolation,the suspected positive isolates were identified by RT-PCR and IFA.The results showed that two strains of Getah virus,named SH201801 and SH201802,were isolated from Anopheles Sinensis and culex tritaeniorhynchus respectively.The E2 gene of the two GETV strains was cloned,sequenced and evolutionized.The results showed that the two GETV strains belonged to GroupIII type,and had high homology with M1,SH05-6,SH05-16 and other domestic isolates,but had a distant genetic relationship with the Malaysian original strain MM2021.This study provides a scientific basis for the epidemiological investigation of GETV and is of great significance for the control of mosquitoes and mosquito-borne diseases in Shanghai.3.Preparation of Horse GETV antibody Colloidal Gold Test StripIn order to establish a rapid method for the detection of GETV specific antibody,the recombinant E2 protein of getavirus was expressed and the colloidal gold immunochromatographic assay was established.According to the sequence of structural protein E2 of GETV SH05-6 strain(GenBank:EU015066),The main antigenic domains were selected according to the codon of E.coli to optimize and synthesize the gene sequence,which was cloned into the prokaryotic expression vector pCold I and transformed into BL21.The engineering strain BL21(pCold I-E2)was successfully constructed and IPTG was induced and expressed at low temperature.After purification,renaturation and dialysis,the recombinant E2 antigen was coated with cellulose nitrate membrane.The colloidal gold spot samples and the ratio of Colloidal Gold were optimized to establish a rapid detection method of Getah virus antibody with colloidal gold.The sensitivity,specificity,stability and coincidence rate of the method were evaluated.The results showed that the recombinant E2 protein mainly existed as inclusion body and its molecular weight was about 33 KD,the purity of the purified protein was over 96%by SDS-PAGE.The Colloidal Gold Strip constructed with the recombinant E2 protein could be detected within 20 minutes,and the negative and positive serum could be distinguished obviously,the sensitivity was 1:640,no cross-reaction was found with the positive serum of equine infectious anemia virus,equine arteritis virus and Japanese encephalitis virus,and the coincidence rate with microneutralization test was 94.0%.When The Test Strip was kept at 37? for 2 weeks or at room temperature for 4 months,the results remains the same.Therefore,the recombinant E2 protein prepared in this study has good antigenicity,and the colloidal gold antibody detection method has the characteristics of rapidity,sensitivity,specificity and stability,and is suitable for the rapid detection of GETV antibody in the field.4.Seroepidemiological investigation and analysis of horse Getah virus in ShanghaiIn order to understand the infection status and epidemic characteristics of horse Getah virus in Shanghai,Getah virus antibody was detected in 182 equine serum samples collected from 7 horse farms in Shanghai in 2018 by the established colloidal gold strip method,the correlation between the positive rate of GETV antibody and urban area,season,age and sex of horse was analyzed.The results showed that 52 positive serum samples were detected,the total positive rate was 28.6%;The positive rate of GETV antibody was significantly higher in Fengxian District area(42.5%)and Jinshan District(30.6%)than in pudong new area District(15.6%)and Songjiang District area(22.9%)(p<0.05).However,there were no significant differences among,gender and age(p>0.05).The results suggested that the infection rate of horse GETV in Shanghai was high,therefore,the monitoring and control of Horse GETV should be strengthened.In conclusion,this study,we identified the GETV carried by mosquitoes in horse farms in Shanghai and analyzed the molecular epidemic characteristics of Getah virus from Mosquito The Colloidal Gold Strip for GETV antibody was established and applied,and the results showed that the infection rate of GETV in some horse farms in Shanghai was high.Therefore,the monitoring and control of GETV should be strengthened,which provided scientific basis for the control of GETV in Shanghai. |
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