脑心肌炎病毒双抗原夹心ELISA建立、血清学调查、毒株分离及RNA干扰研究
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摘要
脑心肌炎病毒(Encephalomyocarditis virus, EMCV)可以感染多种哺乳动物、啮齿类动物、节肢动物和人并引发脑炎、心肌炎和心肌周围炎等症状；能引致母猪繁殖障碍和仔猪致死性心肌炎，致死率可达100%；而人多以亚临床形式存在。目前EMCV流行范围越来越广，在亚洲、非洲、美洲、欧洲和澳洲均有发现，且感染率逐年增加，不仅给养殖业造成严重的影响，同时也危害着人类健康。
本研究以脑心肌炎病毒为对象，进行了以下实验研究：
1.研发了EMCV双抗原夹心ELISA试剂盒并获得了国家发明专利授权：试剂盒以EMCV重组VP1、VP2和2C蛋白作为包被抗原，HRP标记的EMCV为酶标抗原，最适包被浓度为6μg/mL，最适酶标抗原浓度为1:400，最佳样品作用时间为45min，最佳显色时间为30min。与间接ELISA比较，符合率93.5%，特异性为95%，敏感性为92.9%，与FMD、HEV、PPV、BVDV等病毒抗血清无交叉反应，连续生产3批次，批间和批内CV%均小于6%。因此所研发的试剂盒重复性、特异性和稳定性较好，灵敏度较高，可以作为EMCV临床抗体检测和血清学调查有效的方法。
2. EMCV血清病学调查：⑴从东北、华北、西北等7个地区采集健康人群血清，用双抗原夹心ELISA检测抗体，结果EMCV抗体平均阳性率为30.56%，东北地区最高，阳性率为43.23%，华中地区最低，阳性率为11.26%，各区域之间存在显著性差异；通过分析，抗体阳性率与性别之间不存在关联，与年龄存在可能的线性关系。⑵从上述7个地区专门采集20±5岁青年人群血清进行检测，结果显示抗体阳性率平均为46.00%，高于全年龄段样品平均值，可能与这个年龄段的非家庭性流动、群居性及饮食有直接关系。⑶从这7个地区采集猪血清进行检测，结果抗体平均阳性率为77.00%，明显高于人源EMCV抗体，各区域之间差异不显著。⑷从甘肃某猪场选取30头猪，从出生开始采集血清，之后15d、30d、60d、75d、90d、120d、150d时分别采集血清并检测，结果显示30日龄仔猪抗体阳性率最高为90%，60日龄下降到6.67%，之后抗体阳性率随着时间逐步升高，到150日龄抗体阳性率达到86.67%，经分析，0-60仔猪日龄存在母源抗体保护，60d之后失去母源抗体保护，可自然感染EMCV，群体抗体阳性率又逐步升高。
3.首次分离到EMCV甘肃毒株，命名为GS01，对其全基因组进行测序，基因全长为7717bp，CDS区6879bp， Genbank登记号为：KJ524642，与国内其他分离毒株同源性99.4%-99.9%。病毒在BHK-21细胞培养3代后TCID50为10-5.8/0.1mL，病毒对小鼠具有极高致死性，LD50为10-3.75/0.5mL。
4.针对EMCV VP1序列，设计合成了4对编码siRNA的序列并构建真核表达载体，脂质体介导转染至BHK-21细胞并接种EMCV，结果4组siRNA均能抑制脑心肌炎病毒增值，其中X109-2组抑制作用最显著，下降了103.5倍；各组均不能抑制FMDV、JEV等病毒，表明RNAi技术可以抑制EMCV病毒在BHK-21细胞中增殖，特异性好；X109-2组质粒转染至CHO-K1细胞中，还可以抑制pEGF-C1-EMCV-VP1的表达，表明RNAi具有明确靶向性。
综上，本研究建立了双抗原夹心ELISA检测方法，积累了EMCV流行病学数据，首次分离到了EMCV甘肃毒株，探索了RNAi技术抑制病毒复制的效果，为进一步研究EMCV致病机制和防治等奠定基础。
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.
本研究以脑心肌炎病毒为对象，进行了以下实验研究：
1.研发了EMCV双抗原夹心ELISA试剂盒并获得了国家发明专利授权：试剂盒以EMCV重组VP1、VP2和2C蛋白作为包被抗原，HRP标记的EMCV为酶标抗原，最适包被浓度为6μg/mL，最适酶标抗原浓度为1:400，最佳样品作用时间为45min，最佳显色时间为30min。与间接ELISA比较，符合率93.5%，特异性为95%，敏感性为92.9%，与FMD、HEV、PPV、BVDV等病毒抗血清无交叉反应，连续生产3批次，批间和批内CV%均小于6%。因此所研发的试剂盒重复性、特异性和稳定性较好，灵敏度较高，可以作为EMCV临床抗体检测和血清学调查有效的方法。
2. EMCV血清病学调查：⑴从东北、华北、西北等7个地区采集健康人群血清，用双抗原夹心ELISA检测抗体，结果EMCV抗体平均阳性率为30.56%，东北地区最高，阳性率为43.23%，华中地区最低，阳性率为11.26%，各区域之间存在显著性差异；通过分析，抗体阳性率与性别之间不存在关联，与年龄存在可能的线性关系。⑵从上述7个地区专门采集20±5岁青年人群血清进行检测，结果显示抗体阳性率平均为46.00%，高于全年龄段样品平均值，可能与这个年龄段的非家庭性流动、群居性及饮食有直接关系。⑶从这7个地区采集猪血清进行检测，结果抗体平均阳性率为77.00%，明显高于人源EMCV抗体，各区域之间差异不显著。⑷从甘肃某猪场选取30头猪，从出生开始采集血清，之后15d、30d、60d、75d、90d、120d、150d时分别采集血清并检测，结果显示30日龄仔猪抗体阳性率最高为90%，60日龄下降到6.67%，之后抗体阳性率随着时间逐步升高，到150日龄抗体阳性率达到86.67%，经分析，0-60仔猪日龄存在母源抗体保护，60d之后失去母源抗体保护，可自然感染EMCV，群体抗体阳性率又逐步升高。
3.首次分离到EMCV甘肃毒株，命名为GS01，对其全基因组进行测序，基因全长为7717bp，CDS区6879bp， Genbank登记号为：KJ524642，与国内其他分离毒株同源性99.4%-99.9%。病毒在BHK-21细胞培养3代后TCID50为10-5.8/0.1mL，病毒对小鼠具有极高致死性，LD50为10-3.75/0.5mL。
4.针对EMCV VP1序列，设计合成了4对编码siRNA的序列并构建真核表达载体，脂质体介导转染至BHK-21细胞并接种EMCV，结果4组siRNA均能抑制脑心肌炎病毒增值，其中X109-2组抑制作用最显著，下降了103.5倍；各组均不能抑制FMDV、JEV等病毒，表明RNAi技术可以抑制EMCV病毒在BHK-21细胞中增殖，特异性好；X109-2组质粒转染至CHO-K1细胞中，还可以抑制pEGF-C1-EMCV-VP1的表达，表明RNAi具有明确靶向性。
综上，本研究建立了双抗原夹心ELISA检测方法，积累了EMCV流行病学数据，首次分离到了EMCV甘肃毒株，探索了RNAi技术抑制病毒复制的效果，为进一步研究EMCV致病机制和防治等奠定基础。
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.
引文
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