我国部分地区猪源牛病毒性腹泻病毒研究
摘要
1.猪源牛病毒性腹泻病毒分离与鉴定将处理后BVDV阳性仔猪组织样品接种MDBK细胞,分离到1株猪源BVDV命名为SD0803株。通过细胞培养、直接免疫荧光、5'-UTR与NproPCR扩增、电镜观察、TCID50测定及对其分子进化特征加以分析。结果表明,该毒株在MDBK细胞上盲传至13代未出现细胞病变。在直接免疫荧光试验中呈阳性荧光信号。PCR扩增分别获得5'-UTR与Npro预期大小DNA片段。电镜观察,病毒粒子略呈圆形,有囊膜,直径约50nm。病毒滴度为10-6.5TCIDso·0.2mL-1。 SD08035'-UTR、 Npro序列进化分析显示,该分离株属于BVDV-1,与己知BVDV-1各亚型之问同源性较低,单独成一分支。以上结果推断,成功分离鉴定一株猪源BVDV SD0803,该毒株为非致病变型BVDV-1,极有可能为BVDV-1新的亚型。
2.猪源牛病毒性腹泻病毒SD0803株全长基因序列的测定及生物信息学分析采用RT-PCR方法分10个片段扩增SD0803株全基因序列；病毒5’-末端与3’-末端序列采用RACE方法扩增,扩增得到的各片段克隆测序。结果,将测序得到的个片段序列采用Seqman软件拼接后,得到了SD0803分离株全长序列,其长度为1227Int,包括5'-UTR (388nt)3'-UTR (186nt)以及一个编码3898个氨基酸的大ORF,不含外源基因序列。成功获得BVDV SD0803株全基因序列,丰富了猪源BVDV流行病学研究资源。采用MEGA4.1、 DNAStar、 I-TASSER Server、 CLC RNA Workbench等生物信息学软件对SD0803全长序列进行分析,结果表明：SD0803株全长序列与BVDV-1参考株在一簇,与ZM-95亲缘关系最近,二者同源性仅为83.2%；该毒株各基因与瘟病毒参考株同源性为42.7%-91.7%；5'-UTR存在三个高变区、两个oligopyrimidine-rich tracts,形成4个(A、B、C、D)功能区的二级结构；3'-UTR的一级结构存在一个高变区与一个相对保守区,在高变区里少了约40nt,二级结构与牛源参考株差异较大,仅形成3个茎-环结构；结构蛋白区含有14个潜在糖基化位点；I-TASSER Server预测SD0803分离RdRp高级结构,该毒株RdRp含有50.9%氨基酸形成(366/719)卷曲结构(coil,c)；8.6%(62/719)氨基酸形成β-片状折叠结构(S)；剩余的的氨基酸(40.5%,291/719)形成螺旋结构(helix, H);三级结构具有典型的核酸复制酶“手状结构”,与BVDV1S4F结构相似。本研究分析和预测了SD0803株全长序列的进化、结构与功能的关系,为进一步研究其生物学功能及其应用奠定基础。
3.猪源牛病毒性腹泻病毒套式PCR检测方法的建立根据GenBank中BVDV-1、 BVDV-2、CSFV及新出现瘟病毒的基因序列,设计2对特异引物,建立了能鉴别诊断BVDVs与CSFV的Nested-PCR检测方法；确定其方法的特异性、敏感性、稳定性。建立的Nested-PCR能从BVDVs标准毒株、猪源BVDV毒株中扩增198bp特异性片段,对猪瘟病毒、猪繁殖与呼吸综合征病毒检测结果为阴性,此结果表明,该方法具有较好特异性；其敏感性可检测到0.195fg RNA模板量；经重复性试验表明该方法具有良好稳定性。初步应用检测表明,猪BVDV阳性率较高,达36.0%；牛血清及其制品、猪瘟活疫苗BVDVs污染严重。本试验建立的Nested-PCR具有敏感、特异和稳定等特点,可用于临床诊断和流行病学调查。
4.我国部分地区猪源牛病毒性腹泻病毒分子流行病学研究采用Nested-PCR方法检测了2007年至2010年问从北京、河南、新疆、山东、江西、浙江、安徽、湖南、广西、江苏和上海等地采集的猪病料,同时采用该方法对我国部分地区采集牛场的牛血清、国产商品胎牛血清、进口商品胎牛血清、猪瘟活疫苗、猪繁殖与呼吸综合征活疫苗、猪伪狂犬病活疫苗、MDBK细胞进行了检测。结果显示,猪病料中BVDV总阳性率为11.88%(146／1229),2007年至2010年感染率呈增长趋势；牛场的牛血清阳性率26.0%(130／500)；国产商品胎牛血清96.4%(53／55)；进口商品胎牛血清100%(10／10)；猪瘟活疫苗52.0%(26／50)；猪繁殖与呼吸综合征活疫苗和猪伪狂犬病活疫苗未检测出BVDV; MDBK细胞均呈阳性。应用生物信息学软件对不同来源的毒株5'-UTR序列进行遗传进化分析,结果表明,来自牛场的牛血清中,4株为BVDV-1m,2株为BVDV-1b,还有1株属于BVDV-1a;商品胎牛血清中,BVDV-1b (FS2、FS4), BVDV-1c(FS3),BVDV-1m(DFBS2/3、FS5),BVDV-1p(FS6), BVDV-2a (FS1),还有一株暂不确定型；猪活疫苗中,2株属于BVDV-1m,2株为BVDV-1o,1株为BVDV-1k,1株为BVDV-1b;在猪源BVDV分离株中,1株(BVDV-1a),6株(BVDV-1b),6株(BVDV-1m),1株(BVDV-1o)还有6株暂不能确定亚型；猪源BVDV毒株与牛血清(牛场)毒株同源性为87.3%-100%,与国产商品血清(除了FSl为BVDV-2外)为77.1%～98.8%,所有猪源BVDV毒株与进口商品血清80.5%～97.5%。猪源BVDV毒株与猪瘟活疫苗源BVDV之间,在病毒最保守区(5'-UTR)均有变异,这两种不同来源毒株之间核苷酸的变异无规律性。
5.猪源牛病毒性腹泻病毒荧光定量PCR检测方法的建立根据BVDVs与CSFV5'-UTR保守序列,设计一套引物和特异TaqMan探针,以本实验室构建的猪源BVDV5'-UTR阳性重组质粒为标准品,通过优化反应条件,建立了标准曲线。以构建的标准品为模板进行了特异性、敏感性、重复性试验、并应用于临床检测,结果,该方法检测CSFV、 PRRSV均为阴性；最低可检测到每个反应相当于10拷贝的标准品DNA;重复性试验的批内变异系数为0.20%-0.95%；应用所建立方法对临床样品进行检测,其结果与普通RT-PCR结果的符合率为86.7%。本研究建立的实时荧光定量PCR具有特异、敏感、快速、重复性好等优点,可用于猪感染BVDV、猪瘟疫苗污染BVDVs的监测。
6.猪源牛病毒性腹泻病毒E2蛋白表达及抗体制备表达去除猪源牛病毒性腹泻病母(BVDV) SD0803株E2基因跨膜疏水区(sE2)的蛋白并制备其抗体,RT-PCR扩增SD0803株sE2基因,构建重组原核表达载体pGEX-4T-1-sE2(rsE2),转化大肠埃希菌BL21(DE3), IPTG诱导蛋白表达,将纯化的重组蛋白为抗原免疫家兔制备多克隆抗体。采用间接ELISA、 Western-blot、间接免疫荧光等方法鉴定获得的抗体。结果显示,抗体效价最高可达1：256000；制备的抗体可与rsE2蛋白特异性结合；该抗体可特异性识别BVDV感染MDBK细胞表达的E2蛋白,显示抗体高度的特异性。
1. Isolation and Identification of a pig Bovine Viral Diarrhea Virus. Virus were isolated from BVDV positive sample from the piglet and used to study pig bovine viral diarrhea virus. BVDV positive sample from the piglet were inoculated in MDBK cells and a pig BVDV SD0803were obtained. In the study, cell culture, direct immunofluorescence assay, PCR amplification of5'-UTR, electron microscopy and TCID50were performed, moreover, bioinformatics software were used to analyze the characteristics of molecular evolution. The results showed that the virus strain hadn't caused cytopathic effect (CPE) until thirteenth passage on MDBK cell. SD0803infected cells showed strong fluorescent signal. DNA fragment of307bp and428bp were amplified by PCR from5'-UTR and Npro of SD0803.Under electron microscopy, virus particles were near round and enveloped with size of50nm in diameter. Virus titer of SD0803was10-6.5TCID5o·0.2mL-1. The homology comparison and phylogenetic analysis of5'-UTR and Npro between SD0803and representative BVDV isolates showed that SD0803shares the same group with BVDV-1reference isolates; thereby the isolate belongs to BVDV-1. However, SD0803have an obvious distance from the subtypes of BVDV-1and formed a separate group according to the phylogenetic relationship and this showed that the isolate might be a new subtype of BVDV-1. So a pig BVDV SD0803was successfully isolated and identified, and belongs to NCP BVDV-1and was a probably new subtype of BVDV-1.
2. Sequencing and Bioinformation Analysis for complete genome of pig Bovine viral diarrhea virus SD0803strain. Complete genome of Bovine viral diarrhea virus (BVDV) SD0803isolate were sequenced and used to study swine BVDV. RT-PCR assay was respectively applied to obtain10fragments of SD0803strain. Sequence of both5'-and3'-ends were performed with the Rapid-amplification of cDNA ends (RACE). All fragments were spliced with Seqman software and the complete genome of the isolate obtained was12272nt. The genomic RNA contained5'-untranslated region (5'-UTR),3'-untranslated region (3'-UTR), and one open reading frame (ORF) encoding a large polyprotein of3899amino acids but not exogenetic genomic sequences. Complete genome of BVDV SD0803strain was successfully obtained. The study on molecular epidemiology of pig BVDV in China, to some extent, was promoted.The purpose of this present study was to analyze the complete genome of Bovine viral diarrhea virus SD0803strain and predict the structure-function relationship. Bioinformationatic softwares such as MEGA4.1, DNAStar, I-TASSER Server, CLC RNA Workbench were applied to analyzed the complete genome. The results showed:SD0803isolate belongs to BVDV-1and the highest homology identity was83.2, compared with ZM-95, the homology identity of each gene is42.7%～91.7%with pestivirus strains. There were three high variable domains four functional ones (A、B、C、D) and two oligopyrimidine-rich in5'-UTR. There were one high variable and one conserved domain and contains3stem-loop structures in3'-UTR. The structural proteins contain14Potential N-linked glycosylation sites. There are50.9%amino acids (AAs) forming coil,8.6%AAs forming P-sheet, and the remainders posing helix. Tertiary structure possessed replicas'structures.
3. Establishment of Nested-PCR for Detecting pig Bovine Viral Diarrhea Viruses. Two pairs of primers were designed according to these genomic sequences of BVDV-1, BVDV-2, CSFV and a new member of the pestivirus genus, and a Nested-PCR were developed for the differentiation of pig BVDVs and CSFV. This PCR assay could respectively amplify a198bp fragment from BVDV NADL, pig BVDV, but not from CSFV, porcine reproductive and respiratory syndrome virus (PRRSV). Sensitivity was determined as0.195fg RNA. And in the field trail of100pig specimen,36%of them were positive. These results showed that the Nested-PCR with high sensitivity and specificity provided a new and alternative tool for the detection of pig BVDVs and CSFV.
4. Molecular epidemiological characterization of pig bovine viral diarrhea virus in some region of China. Known about molecular epidemiological characterization of pig bovine viral diarrhea virus in some region of China and primary explore of evolution of pig BVDV and the relationship among BVDV from bovine and vaccines, to establish the base of prevent and control pig BVDV. Samples from Beijing, Henan, Xinjiang, Shandong, Jiangxi, Zhejiang, Anhui, Hunan, Guangxi, Jiangsu and Shanghai and so on were detected by Nested-PCR. Also, Bovine serums come from cattle farm; commercial fetal serum made in China and imported fetal serum, live vaccine for Classical swine fever virus, PRRSV, PRV, and MDBK cells. The results showed, the positive rate of pig were11.88%(146/1229) and was increasing tendency. Serum from cattle farm26.0%(130/500), commercial domestic fetal serum96.4%(53/55); imported fetal serum100%. BVDV were not detected in vaccine for PRRSV and PRV. The sequences of BVDV5'-UTR of different source were analyzed by Bioinformatics software. The results revealed, all sequences but FS1(commercial domestic fetal serum), belonged to BVDV-1; among serum from cattle farm4isolates were BVDV-1m,2isolates BVDV-1b,1one BVDV-1a. among imported fetal serum,2ones BVDV-1b.1one BVDV-1c,3ones BVDV-1m,1one BVDV-1p,1one BVDV-2a and1one can't be ensured. Among CSF live vaccine,2strains were BVDV-1m,2ones BVDV-1o,1one BVDV-1k,1one BVDV-1b.20pig BVDV strains,1strainBVDV-1a,6ones BVDV-1b,6ones BVDV-1m,1one BVDV-1o, and6ones can't be ensured. The homology between pig BVDV and serum from cattle farm was87.3%～100%, and commercial domestic fetal serum77.1%～98.8%. imported fetal serum80.5%～97.5%. there are mutant sites in of5'-UTR of all BVDV strain and not regular.
5. Development of real-time fluorescence quantitative PCR assay for detection of Bovine viral diarrhea virus in pigs. According to the conserved gene sequence of5'-UTR of BVDVs and CSFV, pairs of primer and specific TaqMan probe were designed. The positive standard plasmids were used as quantitative template to make the standard curves, through the method was optimized. Specificity, sensitivity, and conformity were tested. By sensitivity analysis, the FQ-PCR indicated that a minimum of10copies of plasmid DNA was detected. The assay exhibited reproducibility and specificity, and all negative controls such as classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) showed negative detection. As a result of the sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time FQ-PCR will be used as a routine assay for the diagnosis of pig BVDV infection and CSFV vaccine polluted by BVDVs.
6. Expression and Antibody Preparation of E2Protein of Pig Bovine Viral Diarrhea Virus. To provide the method about the diagnosis on pig bovine viral diarrhea virus, sE2gene, which removed C-terminal transmernbrane of E2gene of BVDV SD0803isolate (sE2), and sE2protein were got and anti-sE2antibody was prepared. sE2gene which was amplified from SD0803strain by PCR was cloned into pGEX-4T-1and transformed into BL21(DE3). After induction with IPTG, the recombinant protein was obtained. Rabbits were immunized with the purified protein as antigen, and antiserum was acquired. The polyclonal antibodies were analyzed by indirect ELISA, Western-blot and indirect immunofluorescence assay. The antiserum titer was determined by indirect ELISA and was1:256000. The results of Western-blot and indirect immunofluorescence assay confirmed the antibodies reacted with specifically to the protein expressed. A recombinant SD0803sE2protein and the specific antibodies have been obtained.
2.猪源牛病毒性腹泻病毒SD0803株全长基因序列的测定及生物信息学分析采用RT-PCR方法分10个片段扩增SD0803株全基因序列；病毒5’-末端与3’-末端序列采用RACE方法扩增,扩增得到的各片段克隆测序。结果,将测序得到的个片段序列采用Seqman软件拼接后,得到了SD0803分离株全长序列,其长度为1227Int,包括5'-UTR (388nt)3'-UTR (186nt)以及一个编码3898个氨基酸的大ORF,不含外源基因序列。成功获得BVDV SD0803株全基因序列,丰富了猪源BVDV流行病学研究资源。采用MEGA4.1、 DNAStar、 I-TASSER Server、 CLC RNA Workbench等生物信息学软件对SD0803全长序列进行分析,结果表明：SD0803株全长序列与BVDV-1参考株在一簇,与ZM-95亲缘关系最近,二者同源性仅为83.2%；该毒株各基因与瘟病毒参考株同源性为42.7%-91.7%；5'-UTR存在三个高变区、两个oligopyrimidine-rich tracts,形成4个(A、B、C、D)功能区的二级结构；3'-UTR的一级结构存在一个高变区与一个相对保守区,在高变区里少了约40nt,二级结构与牛源参考株差异较大,仅形成3个茎-环结构；结构蛋白区含有14个潜在糖基化位点；I-TASSER Server预测SD0803分离RdRp高级结构,该毒株RdRp含有50.9%氨基酸形成(366/719)卷曲结构(coil,c)；8.6%(62/719)氨基酸形成β-片状折叠结构(S)；剩余的的氨基酸(40.5%,291/719)形成螺旋结构(helix, H);三级结构具有典型的核酸复制酶“手状结构”,与BVDV1S4F结构相似。本研究分析和预测了SD0803株全长序列的进化、结构与功能的关系,为进一步研究其生物学功能及其应用奠定基础。
3.猪源牛病毒性腹泻病毒套式PCR检测方法的建立根据GenBank中BVDV-1、 BVDV-2、CSFV及新出现瘟病毒的基因序列,设计2对特异引物,建立了能鉴别诊断BVDVs与CSFV的Nested-PCR检测方法；确定其方法的特异性、敏感性、稳定性。建立的Nested-PCR能从BVDVs标准毒株、猪源BVDV毒株中扩增198bp特异性片段,对猪瘟病毒、猪繁殖与呼吸综合征病毒检测结果为阴性,此结果表明,该方法具有较好特异性；其敏感性可检测到0.195fg RNA模板量；经重复性试验表明该方法具有良好稳定性。初步应用检测表明,猪BVDV阳性率较高,达36.0%；牛血清及其制品、猪瘟活疫苗BVDVs污染严重。本试验建立的Nested-PCR具有敏感、特异和稳定等特点,可用于临床诊断和流行病学调查。
4.我国部分地区猪源牛病毒性腹泻病毒分子流行病学研究采用Nested-PCR方法检测了2007年至2010年问从北京、河南、新疆、山东、江西、浙江、安徽、湖南、广西、江苏和上海等地采集的猪病料,同时采用该方法对我国部分地区采集牛场的牛血清、国产商品胎牛血清、进口商品胎牛血清、猪瘟活疫苗、猪繁殖与呼吸综合征活疫苗、猪伪狂犬病活疫苗、MDBK细胞进行了检测。结果显示,猪病料中BVDV总阳性率为11.88%(146／1229),2007年至2010年感染率呈增长趋势；牛场的牛血清阳性率26.0%(130／500)；国产商品胎牛血清96.4%(53／55)；进口商品胎牛血清100%(10／10)；猪瘟活疫苗52.0%(26／50)；猪繁殖与呼吸综合征活疫苗和猪伪狂犬病活疫苗未检测出BVDV; MDBK细胞均呈阳性。应用生物信息学软件对不同来源的毒株5'-UTR序列进行遗传进化分析,结果表明,来自牛场的牛血清中,4株为BVDV-1m,2株为BVDV-1b,还有1株属于BVDV-1a;商品胎牛血清中,BVDV-1b (FS2、FS4), BVDV-1c(FS3),BVDV-1m(DFBS2/3、FS5),BVDV-1p(FS6), BVDV-2a (FS1),还有一株暂不确定型；猪活疫苗中,2株属于BVDV-1m,2株为BVDV-1o,1株为BVDV-1k,1株为BVDV-1b;在猪源BVDV分离株中,1株(BVDV-1a),6株(BVDV-1b),6株(BVDV-1m),1株(BVDV-1o)还有6株暂不能确定亚型；猪源BVDV毒株与牛血清(牛场)毒株同源性为87.3%-100%,与国产商品血清(除了FSl为BVDV-2外)为77.1%～98.8%,所有猪源BVDV毒株与进口商品血清80.5%～97.5%。猪源BVDV毒株与猪瘟活疫苗源BVDV之间,在病毒最保守区(5'-UTR)均有变异,这两种不同来源毒株之间核苷酸的变异无规律性。
5.猪源牛病毒性腹泻病毒荧光定量PCR检测方法的建立根据BVDVs与CSFV5'-UTR保守序列,设计一套引物和特异TaqMan探针,以本实验室构建的猪源BVDV5'-UTR阳性重组质粒为标准品,通过优化反应条件,建立了标准曲线。以构建的标准品为模板进行了特异性、敏感性、重复性试验、并应用于临床检测,结果,该方法检测CSFV、 PRRSV均为阴性；最低可检测到每个反应相当于10拷贝的标准品DNA;重复性试验的批内变异系数为0.20%-0.95%；应用所建立方法对临床样品进行检测,其结果与普通RT-PCR结果的符合率为86.7%。本研究建立的实时荧光定量PCR具有特异、敏感、快速、重复性好等优点,可用于猪感染BVDV、猪瘟疫苗污染BVDVs的监测。
6.猪源牛病毒性腹泻病毒E2蛋白表达及抗体制备表达去除猪源牛病毒性腹泻病母(BVDV) SD0803株E2基因跨膜疏水区(sE2)的蛋白并制备其抗体,RT-PCR扩增SD0803株sE2基因,构建重组原核表达载体pGEX-4T-1-sE2(rsE2),转化大肠埃希菌BL21(DE3), IPTG诱导蛋白表达,将纯化的重组蛋白为抗原免疫家兔制备多克隆抗体。采用间接ELISA、 Western-blot、间接免疫荧光等方法鉴定获得的抗体。结果显示,抗体效价最高可达1：256000；制备的抗体可与rsE2蛋白特异性结合；该抗体可特异性识别BVDV感染MDBK细胞表达的E2蛋白,显示抗体高度的特异性。
1. Isolation and Identification of a pig Bovine Viral Diarrhea Virus. Virus were isolated from BVDV positive sample from the piglet and used to study pig bovine viral diarrhea virus. BVDV positive sample from the piglet were inoculated in MDBK cells and a pig BVDV SD0803were obtained. In the study, cell culture, direct immunofluorescence assay, PCR amplification of5'-UTR, electron microscopy and TCID50were performed, moreover, bioinformatics software were used to analyze the characteristics of molecular evolution. The results showed that the virus strain hadn't caused cytopathic effect (CPE) until thirteenth passage on MDBK cell. SD0803infected cells showed strong fluorescent signal. DNA fragment of307bp and428bp were amplified by PCR from5'-UTR and Npro of SD0803.Under electron microscopy, virus particles were near round and enveloped with size of50nm in diameter. Virus titer of SD0803was10-6.5TCID5o·0.2mL-1. The homology comparison and phylogenetic analysis of5'-UTR and Npro between SD0803and representative BVDV isolates showed that SD0803shares the same group with BVDV-1reference isolates; thereby the isolate belongs to BVDV-1. However, SD0803have an obvious distance from the subtypes of BVDV-1and formed a separate group according to the phylogenetic relationship and this showed that the isolate might be a new subtype of BVDV-1. So a pig BVDV SD0803was successfully isolated and identified, and belongs to NCP BVDV-1and was a probably new subtype of BVDV-1.
2. Sequencing and Bioinformation Analysis for complete genome of pig Bovine viral diarrhea virus SD0803strain. Complete genome of Bovine viral diarrhea virus (BVDV) SD0803isolate were sequenced and used to study swine BVDV. RT-PCR assay was respectively applied to obtain10fragments of SD0803strain. Sequence of both5'-and3'-ends were performed with the Rapid-amplification of cDNA ends (RACE). All fragments were spliced with Seqman software and the complete genome of the isolate obtained was12272nt. The genomic RNA contained5'-untranslated region (5'-UTR),3'-untranslated region (3'-UTR), and one open reading frame (ORF) encoding a large polyprotein of3899amino acids but not exogenetic genomic sequences. Complete genome of BVDV SD0803strain was successfully obtained. The study on molecular epidemiology of pig BVDV in China, to some extent, was promoted.The purpose of this present study was to analyze the complete genome of Bovine viral diarrhea virus SD0803strain and predict the structure-function relationship. Bioinformationatic softwares such as MEGA4.1, DNAStar, I-TASSER Server, CLC RNA Workbench were applied to analyzed the complete genome. The results showed:SD0803isolate belongs to BVDV-1and the highest homology identity was83.2, compared with ZM-95, the homology identity of each gene is42.7%～91.7%with pestivirus strains. There were three high variable domains four functional ones (A、B、C、D) and two oligopyrimidine-rich in5'-UTR. There were one high variable and one conserved domain and contains3stem-loop structures in3'-UTR. The structural proteins contain14Potential N-linked glycosylation sites. There are50.9%amino acids (AAs) forming coil,8.6%AAs forming P-sheet, and the remainders posing helix. Tertiary structure possessed replicas'structures.
3. Establishment of Nested-PCR for Detecting pig Bovine Viral Diarrhea Viruses. Two pairs of primers were designed according to these genomic sequences of BVDV-1, BVDV-2, CSFV and a new member of the pestivirus genus, and a Nested-PCR were developed for the differentiation of pig BVDVs and CSFV. This PCR assay could respectively amplify a198bp fragment from BVDV NADL, pig BVDV, but not from CSFV, porcine reproductive and respiratory syndrome virus (PRRSV). Sensitivity was determined as0.195fg RNA. And in the field trail of100pig specimen,36%of them were positive. These results showed that the Nested-PCR with high sensitivity and specificity provided a new and alternative tool for the detection of pig BVDVs and CSFV.
4. Molecular epidemiological characterization of pig bovine viral diarrhea virus in some region of China. Known about molecular epidemiological characterization of pig bovine viral diarrhea virus in some region of China and primary explore of evolution of pig BVDV and the relationship among BVDV from bovine and vaccines, to establish the base of prevent and control pig BVDV. Samples from Beijing, Henan, Xinjiang, Shandong, Jiangxi, Zhejiang, Anhui, Hunan, Guangxi, Jiangsu and Shanghai and so on were detected by Nested-PCR. Also, Bovine serums come from cattle farm; commercial fetal serum made in China and imported fetal serum, live vaccine for Classical swine fever virus, PRRSV, PRV, and MDBK cells. The results showed, the positive rate of pig were11.88%(146/1229) and was increasing tendency. Serum from cattle farm26.0%(130/500), commercial domestic fetal serum96.4%(53/55); imported fetal serum100%. BVDV were not detected in vaccine for PRRSV and PRV. The sequences of BVDV5'-UTR of different source were analyzed by Bioinformatics software. The results revealed, all sequences but FS1(commercial domestic fetal serum), belonged to BVDV-1; among serum from cattle farm4isolates were BVDV-1m,2isolates BVDV-1b,1one BVDV-1a. among imported fetal serum,2ones BVDV-1b.1one BVDV-1c,3ones BVDV-1m,1one BVDV-1p,1one BVDV-2a and1one can't be ensured. Among CSF live vaccine,2strains were BVDV-1m,2ones BVDV-1o,1one BVDV-1k,1one BVDV-1b.20pig BVDV strains,1strainBVDV-1a,6ones BVDV-1b,6ones BVDV-1m,1one BVDV-1o, and6ones can't be ensured. The homology between pig BVDV and serum from cattle farm was87.3%～100%, and commercial domestic fetal serum77.1%～98.8%. imported fetal serum80.5%～97.5%. there are mutant sites in of5'-UTR of all BVDV strain and not regular.
5. Development of real-time fluorescence quantitative PCR assay for detection of Bovine viral diarrhea virus in pigs. According to the conserved gene sequence of5'-UTR of BVDVs and CSFV, pairs of primer and specific TaqMan probe were designed. The positive standard plasmids were used as quantitative template to make the standard curves, through the method was optimized. Specificity, sensitivity, and conformity were tested. By sensitivity analysis, the FQ-PCR indicated that a minimum of10copies of plasmid DNA was detected. The assay exhibited reproducibility and specificity, and all negative controls such as classical swine fever virus (CSFV) and porcine reproductive and respiratory syndrome virus (PRRSV) showed negative detection. As a result of the sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time FQ-PCR will be used as a routine assay for the diagnosis of pig BVDV infection and CSFV vaccine polluted by BVDVs.
6. Expression and Antibody Preparation of E2Protein of Pig Bovine Viral Diarrhea Virus. To provide the method about the diagnosis on pig bovine viral diarrhea virus, sE2gene, which removed C-terminal transmernbrane of E2gene of BVDV SD0803isolate (sE2), and sE2protein were got and anti-sE2antibody was prepared. sE2gene which was amplified from SD0803strain by PCR was cloned into pGEX-4T-1and transformed into BL21(DE3). After induction with IPTG, the recombinant protein was obtained. Rabbits were immunized with the purified protein as antigen, and antiserum was acquired. The polyclonal antibodies were analyzed by indirect ELISA, Western-blot and indirect immunofluorescence assay. The antiserum titer was determined by indirect ELISA and was1:256000. The results of Western-blot and indirect immunofluorescence assay confirmed the antibodies reacted with specifically to the protein expressed. A recombinant SD0803sE2protein and the specific antibodies have been obtained.
引文
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