| Encephalomyocarditis virus can infect a variety of mammals, rodents, arthropods and humans,which has been found in Asia, Africa, America, Europe and Australia. It can cause brain myocarditis,myocarditis and myocardial inflammation around the shape, as well as reproductive failure in sows,acute fatal myocarditis in piglets, and the mortality rate could reach up to100%in infected pigs.However, it is usually presented subclinical symptoms when humans are infected. In recent years,EMCV becomes more and more widespread all over the world, and the infection rate is also evaluated.EMCV infection not only threatens the cultivation industry, but also becomes a big threaten to thehuman health.A double antigen sandwich ELISA (Ds-ELISA) method was developed, and then used to conductepidemiological survey. At the same time, EMCV Gansu strain was isolated and identified, then weinhibited virus replication in BHK-21cells by RNAi technology. The results are as follows:1. The developed EMCV double antigen sandwich detection ELISA kit won the national inventionpatent: it was coated with recombinant EMCV viral proteins VP1, VP2and2C as coating antigen, andHRP-labeled EMCV as enzyme-labelled antigen. The optimal concentration of coating antigen was6μg/mL, and the optimal concentration of enzyme-lablled antigen was1:400dilutied. The best reactiontime was45minutes, and the color reaction time was30minutes. Compared to indirect ELISA, theagreement rate was93.5%, specificity was95%, and sensitivity was92.9%, and didn’t havecross reaction with FMD、HEV、PPV、BVDV antiserum. The kit was made in3batches continuously,CV%between different batches and inside the batch was less than6%. As a result, this kit provided amore effective tool for the investigation of encephalomyocarditis virus antibody surveillance andepidemiology survey with good repeatability, specificity and stability.2. Epidemiology survey was conducted as follows:(1) the human serum samples were collected fromseven places including northeast、northwest and North China. The average positive rate of EMCVantibody of humans was30.56%with regional difference that was43.23%in northeast as the highest,and was11.26%in central China as the lowest. There is no correlation between antibody positive rateand gender, but antibody positive rate may be linear with age.(2) The positive rate among20±5adolescents from these seven places was46.00%on average, significantly higher than the total average,and this may have relation to the sexual flow, social and family diet.(3) The average positive rate was77.00%in pigs, which was higher than that in humans and there was no obvious difference betweendifferent areas.(4) We also selected30pigs from a farm in Gansu province, and collected their serumsin15d、30d、60d、75d、90d、120d、150d after birth. At30d, the antibody titer was the highest, about90%, and at60d, the antibody was decreased to6.67%, and then increased to86.67%at150d. Ouranalysis showed that the piglets were protected by maternal antibody in0-60days, and then lost it after60days, so the positive rate increased gradually.3. This is the first time to isolate encephalomyocarditis virus strain of Gansu, and named as GS-01. Wesequenced the genome of GS01and found out its complete genome was7717bp, with CDS region of 6879bp. Its Genbank accession no. is KJ524642., and has high homology with other domestic isolatedstrains, the homology was between99.4%-99.9%. The virus TCID50was10-5.8/0.1mL after propagationin BHK-21cells and it had high pathogenicity in mice with an LD3.7550value of10-/0.5mL.4. Selecting VP1as target gene, the present study was performed to test the influence of VP1shRNA onencephalomyocarditis virus (EMCV) replication. Four interfering vectors ofpcDNA6.2-GW/EmGFP-miR, namely X109-1, X109-2, X109-3and X109-4, were constructed andwere transfected into BHK-21cells by Lipofectamine2000. After inoculation of EMCV, TCID50andVP1gene expression in BHK-21cells was calculated by TaqMan real-time PCR. Results showed thatall the recombinant VP1shRNAs inhibited viral replication, of which VP1shRNA in X109-2mostseverly interfered with the replication of EMCV. Further investigation showed that viral titer of LgTCID50for EMCV in BHK-21cells was3.5, whereas viral titers of food-mouth disease virus (FMDV)and Japanese encephalitis virus (EJV) were greater than or equal to7.0, demonstrating VP1shRNAspecificly inhibited the replication of EMCV in BHK-21cells. Co-transfection of eukaryotic expressionvector pEGFP-VP1and X109-2plasmid into CHO-K1cell line was carried out. The levels of VP1genetransciption and VP1protein expression in pEGFP-VP1and X109-2cotransfected cells were obviouslylower than those in pEGFP-VP1cells, further confirming the inhibition specificity of VP1shRNA.These findings laid a foundation for further researches of treatment, infection and replicationmechanism of EMCV using VP1siRNA interference technique.In this study, we established Ds-ELISA detection method, and obtained the epidemiology data aboutEMCV, isolated Gansu strain for the first time and explored the inhibition effect on virus replicationwith RNAi technology. It laid a foundation for the further study of EMCV pathogenesis and prevention. |
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