基因2型牛病毒性腹泻病毒的分离与鉴定
摘要
牛病毒性腹泻病(BVD)是由牛病毒性腹泻病毒(BVDV)引起牛以腹泻,急、慢性黏膜病,持续性感染,母畜流产、死胎和畸形胎等为临床特征的一种重要的传染病。BVDV呈世界性分布,其宿主谱相当广,除了可以感染各个年龄段的牛之外,还能感染猪、绵羊、山羊、鹿、骆驼及多种野生动物,且其易感动物群正呈逐渐扩大的趋势。
BVDV有两个基因型,即BVDV-1和BVDV-2。BVDV-1的发现早,分布比较普遍,流行于世界各个国家,而BVDV-2在20世纪80年代末才首次分离于加拿大和美国爆发的急性BVD中。BVDV-1和BVDV-2感染表现的临床症状很相似,但BVDV-2还能引起严重的血小板减少症和出血综合症。我国在1980年首次分离报道了BVDV-1,随后在各地区都相继报道了该病毒的感染,目前,已有二十多个省市报道,说明BVDV-1在我国牛群流行普遍。2009年,我们实验室首次分离报道了BVDV-2,说明BVDV-2也开始出现在我国并可能流行。目前为止,关于BVDV-2在我国流行的详实情况还不是很清楚,该方面数据比较少。因此,本研究将集中如下三部分：一、目前国内BVDV的流行病学调查；二、对BVDV分离株的特性进行研究；三、对BVDV分离株进行基因测序分析及分型。上述研究旨在为目前我国牛病毒性腹泻病毒的流行提供资料,丰富该方面的数据,为进一步研究针对BVDV-1和BVDV-2的疫苗奠定基础。
研究一：从我国各省不同地区的养牛场采集疑似BVDV感染牛的临床病料组织(脾、淋巴结、心肌、血液等),共127份。将样品初步处理后,用我们实验室建立的双抗夹心ELISA检测抗原。经ELISA检测为阳性的样品接种于MDBK细胞,进行病毒分离。样品在MDBK细胞上经3次传代后取提RNA,设计针对BVDV-1、BVDV-2和针对BVDV-1和BVDV-2两型的特异性引物,用我们实验室建立的RT-PCR方法进行鉴定。结果表明,在所检测的样品中,BVDV-2阳性11份(8.66%),所检病料中没有检出BVDV-1。上述研究表明,BVDV-2在我国牛群中已有广泛流行。
研究二：经检测为BVDV2阳性的病料样品接种MDBK细胞,获得11株BVDV-2野毒株。分离到的11株BVDV-2分别盲传培养至18代时仍不见任何典型的细胞病变。间接免疫荧光试验证明分离的野毒株能与鼠源的重组BVDV-2E2蛋白抗血清发生免疫反应,产生特异性胞内荧光；而与针对BVDV-1E2蛋白的单抗不发生反应。将病毒感染的MDBK细胞制备超薄切片,电镜观察可见感染细胞的内质网中有约60nm大小的病毒粒子；经差速离心和20%蔗糖垫底超速离心纯化病毒,将提纯的病毒经磷钨酸染色,在电镜下能观察到大小约60nm有囊膜的病毒颗粒。
研究三：提取病毒RNA,并反转录为cDNA。设计针对BVDV5'-UTR、Npro、 E2和NS2-3的引物,以cDNA为模板分别扩增分离株的5’-UTR、Npro、E2和NS2-3基因片段。采用“TA”克隆法,将所扩增的片段连接到PMD18-T Vector上,经转化筛选出阳性克隆,并送去测序。通过5'-UTR序列进化树分析,结果表明11株分离株均为BVDV-2。比较这11株分离株的Npro、E2和NS2-3基因与GenBank中已发表的BVDV序列的同源性,结果表明,我们的这11株分离株的Npro、E2和NS2-3基因与890、XJ-04、p11Q、p24515、NewYork'93、NADL、Osloss、CD7、1373、 SD-1、KS86-lncp、Oregon C24V毒株的E2和NS2-3基因的核苷酸同源性分别为93.1%-67.1%,92.2%-63.2%,91.7%-64.2%,且这11株分离株均与XJ-04株(国内最早分离株)亲缘性最近。
Bovine viral diarrhea disease (BVD), characterized by fever, diarrhea, and acute/chronic form of mucosal disease, persistent infection and high abortion, is a worldwide distributed infectious disease of cattle caused by bovine viral diarrhea virus (BVDV). It is an important pathogen of cattle and also causes infection of sheep, pig, deer and other wild animals.
BVDV has two genotype, BVDV-1and BVDV-2. BVDV-1which has involved in cattle industry for many years prevenced worldwide, whereas BVDV-2, first reported by research groups in Canadia and America in1980s, can cause the acute diseases which are characterized by thrombocytopenia, hemorrhages and high abortion and mortality rates. After the first BVDV strain isolated in China in1980, many places, more than twenty provinces, had reported the cases of infection of BVDV. BVDV-2was first isolated from cattles in China by our laboratory in2009. However, the information about BVDV-2is scant. Therefore, in this study, we focus on the following three parts:first, epidemiological survey of BVDV in China. Second, study about the characteristic of the isolates. Third, sequencing and genotyping of the isolates. The purpose of this study is to investigate the epidemic status of BVDV in China, which will be important for the development of vaccine.
Study one:Altogether127samples, including spleen, lymph nodes, heart muscle, blood etc., were collected from different cattle farms in several province in China. After preparation, samples were detected by sandwich ELISA and RT-PCR. The viral RNA was extracted according to the manufacturer's instructions and onlyBVDV-1, only BVDV-2, BVDV-1and BVDV-2directed primers were designed for RT-PCR. The results showed that eleven BVDV-2isolates were discovered and no BVDV-1isolate was discovered.
Study two:we obtained11BVDV isolates from MDBK cells-inoculated positive blood samples, which couldn't cause characteristic of CPE for serial passages of18times in MDBK cells. Indirect immunofluorescence assay demonstrated that all the11isolates-infected MDBK cells reacted with the monoclonal antibody specific to BVDV-2with fluoresce lights within the cytoplasms, and no reactivity with MAb specific to BVDV-1. By preparing the ultra-thin sections from the isolates-infected MDBK cells, there were virus-like particles about roughly size of60nm in diameter located in endoplasmic reticulums observed, the morphology of negative staining of phosphotungstic acid for the purified virions were round, with size of60nm in diameter under the transmission electronic microscope.
Study three:primers were designed for the amplification of5'-UTR、Npro、E2and NS2-3genes. The PCR fragments were cloned into PMD18-T Vector and send to sequencing. The phylogenetic tree based on5'-UTR gene showed that the11isolates fall into BVDV-2. Comparation analysis of Npro、E2and NS2-3genes between the11isolates and other BVDV resulted that the nucleotide identity were93.1%～67.1%,92.2%～63.2%,91.7%～64.2%, respectively.
BVDV有两个基因型,即BVDV-1和BVDV-2。BVDV-1的发现早,分布比较普遍,流行于世界各个国家,而BVDV-2在20世纪80年代末才首次分离于加拿大和美国爆发的急性BVD中。BVDV-1和BVDV-2感染表现的临床症状很相似,但BVDV-2还能引起严重的血小板减少症和出血综合症。我国在1980年首次分离报道了BVDV-1,随后在各地区都相继报道了该病毒的感染,目前,已有二十多个省市报道,说明BVDV-1在我国牛群流行普遍。2009年,我们实验室首次分离报道了BVDV-2,说明BVDV-2也开始出现在我国并可能流行。目前为止,关于BVDV-2在我国流行的详实情况还不是很清楚,该方面数据比较少。因此,本研究将集中如下三部分：一、目前国内BVDV的流行病学调查；二、对BVDV分离株的特性进行研究；三、对BVDV分离株进行基因测序分析及分型。上述研究旨在为目前我国牛病毒性腹泻病毒的流行提供资料,丰富该方面的数据,为进一步研究针对BVDV-1和BVDV-2的疫苗奠定基础。
研究一：从我国各省不同地区的养牛场采集疑似BVDV感染牛的临床病料组织(脾、淋巴结、心肌、血液等),共127份。将样品初步处理后,用我们实验室建立的双抗夹心ELISA检测抗原。经ELISA检测为阳性的样品接种于MDBK细胞,进行病毒分离。样品在MDBK细胞上经3次传代后取提RNA,设计针对BVDV-1、BVDV-2和针对BVDV-1和BVDV-2两型的特异性引物,用我们实验室建立的RT-PCR方法进行鉴定。结果表明,在所检测的样品中,BVDV-2阳性11份(8.66%),所检病料中没有检出BVDV-1。上述研究表明,BVDV-2在我国牛群中已有广泛流行。
研究二：经检测为BVDV2阳性的病料样品接种MDBK细胞,获得11株BVDV-2野毒株。分离到的11株BVDV-2分别盲传培养至18代时仍不见任何典型的细胞病变。间接免疫荧光试验证明分离的野毒株能与鼠源的重组BVDV-2E2蛋白抗血清发生免疫反应,产生特异性胞内荧光；而与针对BVDV-1E2蛋白的单抗不发生反应。将病毒感染的MDBK细胞制备超薄切片,电镜观察可见感染细胞的内质网中有约60nm大小的病毒粒子；经差速离心和20%蔗糖垫底超速离心纯化病毒,将提纯的病毒经磷钨酸染色,在电镜下能观察到大小约60nm有囊膜的病毒颗粒。
研究三：提取病毒RNA,并反转录为cDNA。设计针对BVDV5'-UTR、Npro、 E2和NS2-3的引物,以cDNA为模板分别扩增分离株的5’-UTR、Npro、E2和NS2-3基因片段。采用“TA”克隆法,将所扩增的片段连接到PMD18-T Vector上,经转化筛选出阳性克隆,并送去测序。通过5'-UTR序列进化树分析,结果表明11株分离株均为BVDV-2。比较这11株分离株的Npro、E2和NS2-3基因与GenBank中已发表的BVDV序列的同源性,结果表明,我们的这11株分离株的Npro、E2和NS2-3基因与890、XJ-04、p11Q、p24515、NewYork'93、NADL、Osloss、CD7、1373、 SD-1、KS86-lncp、Oregon C24V毒株的E2和NS2-3基因的核苷酸同源性分别为93.1%-67.1%,92.2%-63.2%,91.7%-64.2%,且这11株分离株均与XJ-04株(国内最早分离株)亲缘性最近。
Bovine viral diarrhea disease (BVD), characterized by fever, diarrhea, and acute/chronic form of mucosal disease, persistent infection and high abortion, is a worldwide distributed infectious disease of cattle caused by bovine viral diarrhea virus (BVDV). It is an important pathogen of cattle and also causes infection of sheep, pig, deer and other wild animals.
BVDV has two genotype, BVDV-1and BVDV-2. BVDV-1which has involved in cattle industry for many years prevenced worldwide, whereas BVDV-2, first reported by research groups in Canadia and America in1980s, can cause the acute diseases which are characterized by thrombocytopenia, hemorrhages and high abortion and mortality rates. After the first BVDV strain isolated in China in1980, many places, more than twenty provinces, had reported the cases of infection of BVDV. BVDV-2was first isolated from cattles in China by our laboratory in2009. However, the information about BVDV-2is scant. Therefore, in this study, we focus on the following three parts:first, epidemiological survey of BVDV in China. Second, study about the characteristic of the isolates. Third, sequencing and genotyping of the isolates. The purpose of this study is to investigate the epidemic status of BVDV in China, which will be important for the development of vaccine.
Study one:Altogether127samples, including spleen, lymph nodes, heart muscle, blood etc., were collected from different cattle farms in several province in China. After preparation, samples were detected by sandwich ELISA and RT-PCR. The viral RNA was extracted according to the manufacturer's instructions and onlyBVDV-1, only BVDV-2, BVDV-1and BVDV-2directed primers were designed for RT-PCR. The results showed that eleven BVDV-2isolates were discovered and no BVDV-1isolate was discovered.
Study two:we obtained11BVDV isolates from MDBK cells-inoculated positive blood samples, which couldn't cause characteristic of CPE for serial passages of18times in MDBK cells. Indirect immunofluorescence assay demonstrated that all the11isolates-infected MDBK cells reacted with the monoclonal antibody specific to BVDV-2with fluoresce lights within the cytoplasms, and no reactivity with MAb specific to BVDV-1. By preparing the ultra-thin sections from the isolates-infected MDBK cells, there were virus-like particles about roughly size of60nm in diameter located in endoplasmic reticulums observed, the morphology of negative staining of phosphotungstic acid for the purified virions were round, with size of60nm in diameter under the transmission electronic microscope.
Study three:primers were designed for the amplification of5'-UTR、Npro、E2and NS2-3genes. The PCR fragments were cloned into PMD18-T Vector and send to sequencing. The phylogenetic tree based on5'-UTR gene showed that the11isolates fall into BVDV-2. Comparation analysis of Npro、E2and NS2-3genes between the11isolates and other BVDV resulted that the nucleotide identity were93.1%～67.1%,92.2%～63.2%,91.7%～64.2%, respectively.
引文
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