禽呼肠孤病毒检测方法的建立及病毒分离鉴定
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摘要
呼肠孤病毒(reovirus)可分为两个主要的群，即哺乳动物来源呼肠孤病毒(MRV)和禽类来源呼肠孤病毒(ARV)。ARV属于呼肠孤病毒科中的正呼肠孤病毒属（Orthoreovirus genus）。ARV感染可引起多种疾病，常见的有病毒性关节炎/腱鞘炎、吸收障碍综合征（MAS）、免疫抑制等。近年来，随着ARV感染的增加、流行趋势和临床疾病表现形式的多样化，ARV的防制难度不断增大。由于本病主要是早期感染，而且多数情况感染症状不明显，因此对本病的早期确诊就显得尤为重要。为了快速有效的检测ARV的感染情况，分析其在国内的流行情况，本研究分别建立了检测抗原和抗体的两种检测方法，并且分离了2株ARV病毒。
根据GenBank中发布的ARV S1基因序列设计１对针对σC基因的引物和１条特异性TaqMan探针，建立了检测ARV病毒载量的Real-time RT-PCR方法。该方法具有良好的敏感性、特异性和重复性，可用于ARV的定量检测，为该病的诊断及基础性研究提供了技术手段。利用建立的Real-time RT-PCR方法分析了ARV病毒在体内的复制情况。结果表明：在肝、脾、肾、胸腺、胰腺等器官中均可检测到病毒，各器官病毒载量并无明显差异，在攻毒后5d体内病毒量达到最高峰，随后开始下降。
利用pET-30a原核表达系统成功表达了ARV σC蛋白，将纯化后的重组σC蛋白作为包被抗原，建立检测ARV抗体的间接ELISA方法。经过对反应条件的优化，确定了最佳反应条件：蛋白最佳包被浓度为7.2μg/mL、包被条件为4℃过夜、最佳封闭液为3%脱脂乳、一抗最佳稀释度为1:400、作用条件为室温作用60min、二抗最佳稀释度为1:2000、作用条件为室温作用60min、显色时间为室温15min。阳性判断标准为：OD_(450)>0.314判定为阳性。该方法特异性、重复性、符合性良好，具有较高的临床使用价值，为试剂盒的研发奠定了基础。
采用鸡胚接种及鸡胚成纤维细胞接种两种方式从病料中分离出两株ARV毒株，并通过电镜观察、RT-PCR、测序及间接免疫荧光试验等方法对其进行鉴定。对分离毒株的S1基因全长进行了测序，序列分析结果显示：两株病毒（命名为ARV11-01株和ARV11-02株）S1核苷酸同源性为99.7%，与大部分的引起关节炎的ARV毒株核苷酸同源性性较高（90%以上），与分离自美国的发育障碍的AVS-B毒株同源性较低，核苷酸为62.8%和62.7%，与国际标准毒株S1133的核苷酸同源性较高，为99.1%和99.2%，表明这两株分离毒株可能源自S1133株。动物回归实验表明，两株病毒的致死率分别为18.2%和72.7%。
本研究建立了基于S1基因的Real-time RT-PCR方法，该方法不仅可以用于ARV临床样品的检测，也可以应用与该病毒的基础性研究。利用大肠杆菌表达了能够诱导产生中和抗体的σC蛋白，纯化后，做为包被抗原，建立了检测ARV抗体的间接ELISA方法，该方法特异性、重复性、符合性良好，具有较高的临床使用价值，可进一步开发为诊断试剂盒。此外，分离鉴定了2株ARV，对其S1基因序列进行了测序，丰富了ARV的基因组序列。
Reovirus can be divided into two main groups: mammalian reovirus (MRV) and avian reovirus(ARV). ARV belongs to the family Reoviridae of the genus Orthoreovirus. ARV causes many diseasessuch as viral arthritis/tenosynovitis, malabsorption syndrome, and immune suppression in chickens.Due to increasing of ARV infection and diversification of clinical disease, the controlling of ARV isbecoming more difficulty. Because the infection is in earlier period and the symptom is unobviously,the early diagnosis of ARV is looked to be more important. In this study, two detection methods fordetecting of antigens and antibodies were established, which can be used for detecting the infection ofARV more efficiently and analysising the prevalence in China. Besides, two strains of ARVwereisolated and identified.
The S1gene of ARV was chosen as the target region. The real-time RT-PCR method wasdeveloped for detecting of ARV using a pair of primers and a specific TaqMan probe. This method issensitivitive, specific and reproducible and can be used to detect ARV quantificationally, whichprovides the technical means for ARV diagnosis and basic research. By using the method, replication ofARV in the SPF chickens was determined. The results showed that the ARV could be detected in theliver, spleen, kidney, thymus, pancreas and other organs and no obviously difference of the viral loadsin these organs.What’s more, the viral loads reached a peak in5days post-infection and then decreased.
The σC gene was cloned into pET-30a vector and was expressed in E.coli. An indirect ELISAmethod was developed based on purified σC protein as detecting antigen. The optimum reactionconditions were determined: the protein concentration is7.2μg/mL; the coating condition is4°Covernight; the confining liquid is3%skim milk; the serum dilution is1:400and the working conditionis room temperature for60min; the HRP-labeled IgG dilutions1:2000and the working condition isroom temperature for60min; the TMB reaction time is15min. The cut-off value is OD_(450)>0.31. Thismethod was specific, reproducible and has good correlation with other commercial ELISA kit, whichcan be used for clinical survey of ARV infection and immunization.
Two ARV strains were isolated from chicken’s samples of arthritis symptom by SPF chickenembryos and chicken embryo fibroblast (CEF) cells, and identified by RT-PCR, sequences analysis,IFA and electron microscope. The whole S1genes were sequenced of two ARV isolates (namedARV11-01and ARV11-02). The results suggested that the S1nucleotide homology was99.7%betweenthe two strains. The homologies are62.8%and62.7%with the American isolate (AVS-B strain).However, the homology is more than90%with those of most ARV caused arthritis symptom and thehomology with the S1133strain are99.1%and99.2%. It implied that the two isolates may come fromthe S1133strain. Animal regression results show that the mortality of the two viruses were18.2%and72.7%。
In this study, a real-time RT-PCR method was developed and this method not only can be usedfor the detection ARV from clinical samples but also can be applied in basic research. Using therecombination σC protein as detecting antigen an indirect ELISA method were established for detection the ARV antibody which can be used for clinical survey of ARV infection and immunization. Besides,two ARV strains were isolated and identified and the S1gene were sequenced and analyzed which mayhelp to enrich the ARV genome sequence information.
根据GenBank中发布的ARV S1基因序列设计１对针对σC基因的引物和１条特异性TaqMan探针，建立了检测ARV病毒载量的Real-time RT-PCR方法。该方法具有良好的敏感性、特异性和重复性，可用于ARV的定量检测，为该病的诊断及基础性研究提供了技术手段。利用建立的Real-time RT-PCR方法分析了ARV病毒在体内的复制情况。结果表明：在肝、脾、肾、胸腺、胰腺等器官中均可检测到病毒，各器官病毒载量并无明显差异，在攻毒后5d体内病毒量达到最高峰，随后开始下降。
利用pET-30a原核表达系统成功表达了ARV σC蛋白，将纯化后的重组σC蛋白作为包被抗原，建立检测ARV抗体的间接ELISA方法。经过对反应条件的优化，确定了最佳反应条件：蛋白最佳包被浓度为7.2μg/mL、包被条件为4℃过夜、最佳封闭液为3%脱脂乳、一抗最佳稀释度为1:400、作用条件为室温作用60min、二抗最佳稀释度为1:2000、作用条件为室温作用60min、显色时间为室温15min。阳性判断标准为：OD_(450)>0.314判定为阳性。该方法特异性、重复性、符合性良好，具有较高的临床使用价值，为试剂盒的研发奠定了基础。
采用鸡胚接种及鸡胚成纤维细胞接种两种方式从病料中分离出两株ARV毒株，并通过电镜观察、RT-PCR、测序及间接免疫荧光试验等方法对其进行鉴定。对分离毒株的S1基因全长进行了测序，序列分析结果显示：两株病毒（命名为ARV11-01株和ARV11-02株）S1核苷酸同源性为99.7%，与大部分的引起关节炎的ARV毒株核苷酸同源性性较高（90%以上），与分离自美国的发育障碍的AVS-B毒株同源性较低，核苷酸为62.8%和62.7%，与国际标准毒株S1133的核苷酸同源性较高，为99.1%和99.2%，表明这两株分离毒株可能源自S1133株。动物回归实验表明，两株病毒的致死率分别为18.2%和72.7%。
本研究建立了基于S1基因的Real-time RT-PCR方法，该方法不仅可以用于ARV临床样品的检测，也可以应用与该病毒的基础性研究。利用大肠杆菌表达了能够诱导产生中和抗体的σC蛋白，纯化后，做为包被抗原，建立了检测ARV抗体的间接ELISA方法，该方法特异性、重复性、符合性良好，具有较高的临床使用价值，可进一步开发为诊断试剂盒。此外，分离鉴定了2株ARV，对其S1基因序列进行了测序，丰富了ARV的基因组序列。
Reovirus can be divided into two main groups: mammalian reovirus (MRV) and avian reovirus(ARV). ARV belongs to the family Reoviridae of the genus Orthoreovirus. ARV causes many diseasessuch as viral arthritis/tenosynovitis, malabsorption syndrome, and immune suppression in chickens.Due to increasing of ARV infection and diversification of clinical disease, the controlling of ARV isbecoming more difficulty. Because the infection is in earlier period and the symptom is unobviously,the early diagnosis of ARV is looked to be more important. In this study, two detection methods fordetecting of antigens and antibodies were established, which can be used for detecting the infection ofARV more efficiently and analysising the prevalence in China. Besides, two strains of ARVwereisolated and identified.
The S1gene of ARV was chosen as the target region. The real-time RT-PCR method wasdeveloped for detecting of ARV using a pair of primers and a specific TaqMan probe. This method issensitivitive, specific and reproducible and can be used to detect ARV quantificationally, whichprovides the technical means for ARV diagnosis and basic research. By using the method, replication ofARV in the SPF chickens was determined. The results showed that the ARV could be detected in theliver, spleen, kidney, thymus, pancreas and other organs and no obviously difference of the viral loadsin these organs.What’s more, the viral loads reached a peak in5days post-infection and then decreased.
The σC gene was cloned into pET-30a vector and was expressed in E.coli. An indirect ELISAmethod was developed based on purified σC protein as detecting antigen. The optimum reactionconditions were determined: the protein concentration is7.2μg/mL; the coating condition is4°Covernight; the confining liquid is3%skim milk; the serum dilution is1:400and the working conditionis room temperature for60min; the HRP-labeled IgG dilutions1:2000and the working condition isroom temperature for60min; the TMB reaction time is15min. The cut-off value is OD_(450)>0.31. Thismethod was specific, reproducible and has good correlation with other commercial ELISA kit, whichcan be used for clinical survey of ARV infection and immunization.
Two ARV strains were isolated from chicken’s samples of arthritis symptom by SPF chickenembryos and chicken embryo fibroblast (CEF) cells, and identified by RT-PCR, sequences analysis,IFA and electron microscope. The whole S1genes were sequenced of two ARV isolates (namedARV11-01and ARV11-02). The results suggested that the S1nucleotide homology was99.7%betweenthe two strains. The homologies are62.8%and62.7%with the American isolate (AVS-B strain).However, the homology is more than90%with those of most ARV caused arthritis symptom and thehomology with the S1133strain are99.1%and99.2%. It implied that the two isolates may come fromthe S1133strain. Animal regression results show that the mortality of the two viruses were18.2%and72.7%。
In this study, a real-time RT-PCR method was developed and this method not only can be usedfor the detection ARV from clinical samples but also can be applied in basic research. Using therecombination σC protein as detecting antigen an indirect ELISA method were established for detection the ARV antibody which can be used for clinical survey of ARV infection and immunization. Besides,two ARV strains were isolated and identified and the S1gene were sequenced and analyzed which mayhelp to enrich the ARV genome sequence information.
引文
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