BHV-1、BRSV、BPIV-3和BVDV四重荧光PCR检测方法的建立与应用

为建立检测牛疱疹病毒(BHV-1)、牛呼吸道合胞体病毒(BRSV)、牛副流感病毒3型(BPIV-3)和牛病毒性腹泻病毒(BVDV)单一或混合感染的荧光PCR检测方法,根据BHV-1 gB基因、BRSV F基因、BPIV-3 M基因和BVDV 5'UTR基因保守区序列分别设计特异性引物和TaqMan荧光探针,经条件优化,...     

新城疫病毒分离株的蚀斑纯化及影响蚀斑形成的主要因素

从西北地区1979～1999年发生新城疫的鸡群中分离和收集的15株新城疫病毒(NDV)，经蚀斑纯化，共得到11个克隆毒株；对其进行了蚀斑形成能力的测定，证明蚀斑形成能力与毒力成正比；探讨了温度对蚀斑形成能力的影响，证明NDV蚀斑形成能力在41C比37C强，表现为蚀斑出现早、蚀斑大、蚀斑形成单位(PFU)高，同时在41C时细胞生长状况良好，不易污染，故认为在41C进行NDV的蚀斑纯化值得推荐。     

新城疫病毒LaSota疫苗株的蚀斑纯化

通过蚀斑克隆技术,对新城疫病毒LaSota疫苗株进行连续纯化,在原病毒株的基础上筛选到1株免疫原性较好的病毒纯化株,血凝效价从9 log2提高到11 log2,鸡胚半数感染量(E ID50)从10-9.1/0.1 mL上升到10-9.5/0.1 mL。通过对纯化株的F基因和HN基因进行测序,并与GenBank中已发表的新城疫病毒LaSota株基因序列进行比较发现,F和HN基因核苷酸序列的同源性分别在99.5%和98.6%,氨基酸同源性分别为99.1%和96.9%;F基因裂解位点区(112~117)氨基酸组成与原疫苗株完全一致。纯化株的鸡胚平均死亡时间(MDT)在109 h、1日龄雏鸡脑内接种致病指数(ICPI)为0.375,与原疫苗株进行生物学特性比较,表明该纯化株比原疫苗株更适合用于疫苗生产。     

BPIV-3和BVDV双重RT-PCR快速检测方法的建立

参照GenBank中登录的牛副流感病毒3型(BPIV-3)和牛病毒性腹泻病毒(BVDV)全基因序列,分别针对BPIV3特异性NP蛋白保守基因和BVDV保守区段E2基因设计2对引物,经优化反应条件建立了快速鉴别BPIV-3和BVDV的双重RT-PCR诊断方法。最佳扩增条件为94℃30s,56.2℃30s,72℃1min,循环30次;72℃延伸5min,16℃10min;BVDV引物浓度为1.0μmol/L,BPIV-3引物浓度为0.5μmol/L。采用该方法检测BPIV-3和BVDV参考病毒株,能同时扩增出预期为425bp和294bp大小的特异性片段,而扩增牛传染性鼻气管炎病毒、牛合胞体病毒、猪瘟病毒以及牛支原体、致病性大肠埃希菌、多杀性巴氏杆菌A型、化脓隐秘杆菌和鼠伤寒沙门菌等均呈阴性反应。对参考病毒株进行梯度稀释检测,结果证明该方法检测BPIV-3的灵敏度可达10-3 TCID50/0.1mL,而BVDV的灵敏度达102 TCID50/0.1mL。     

采用蚀斑技术克隆纯化口蹄疫病毒的研究

本研究应用BHK₂₁细胞通过蚀斑试验克隆和再次克隆牛AsiaⅠ型口蹄疫病毒AKT03毒株并对克隆前和第1次、第2次克隆的口蹄疫病毒毒株的细胞半数感染量（TCID₅₀）、乳鼠半数致死量（LD₅₀）进行比较测定,结果表明,通过蚀斑试验成功克隆了牛AsiaⅠ型口蹄疫病毒AKT03毒株,第1次克隆的毒株和第2次克隆的毒株TCID₅₀和LD₅₀效价明显提高。     

一株c基因型牛副流感病毒3型病毒的分离鉴定

从黑龙江省某牛场当中患有呼吸系统疾病的病牛鼻拭子中分离到一株病毒,该病毒在MDBK细胞上能够产生典型的病变。经免疫荧光鉴定表明成功分离到一株牛副流感病毒3型,命名为HLJ1。将病毒的M基因与GenBank中已发表BPIV3毒株的M基因进行同源性比较及系统进化分析,结果表明HLJ1与BPIV3c型参考毒株NX49的同源性最高为99.7%,因此HLJ1分离株属于c基因型BPIV3。我们的结果将为牛副流感病毒3型检测试剂盒及疫苗的研发工作奠定基础。     

1株B基因型牛副流感3型病毒的分离鉴定及全基因组序列分析

为探究新疆某集约化牛场1～6月龄犊牛群中牛副流感病毒3型(BPIV3)的感染情况,对采集的60份犊牛鼻拭子样品进行RT-PCR检测、病毒分离鉴定及全基因组测序和遗传进化分析。结果显示,样品中BPIV3核酸阳性率为11.67%。从核酸阳性样品中分离获得1株BPIV3,并命名为XJ21032-1(45),其基因组全长为15 512 bp;遗传进化分析表明,XJ21032-1(45)属于BPIV3 B基因型,该分离株与澳大利亚的BPIV3 B基因型参考株Q5592(EU277658)的同源性最高为93.4%。本研究成功分离得到了1株B基因型BPIV3,证实B型毒株在我国的存在和流行,为BPIV3疫苗研发提供了原材料,也有助于我国BPIV3分子进化规律及溯源的进一步研究。     

鸡传染性法氏囊病毒蚀斑克隆纯化CV01株生物学特性研究

通过蚀斑克隆技术,对野外分离的鸡传染性法氏囊病毒(IBDV)进行纯化后,筛选到1株病毒含量高、传代稳定的蚀斑纯化毒CV01株。生物学特性分析发现,IBDV能在鸡胚成纤维细胞(DF-1)上形成大小不同的蚀斑,且蚀斑大小与病毒的毒力有一定的相关性,大蚀斑的病毒含量高(108.0TCID50/m L以上),小蚀斑的病毒含量低(107.0l~108.0TCID50/m L)。蚀斑克隆VP2基因序列分析显示大蚀斑克隆毒具有IBDV超强毒株的特性。选病毒含量高(108.5TCID50/m L)的大蚀斑CV01进行进一步试验,结果发现CV01对10日龄SPF鸡胚的毒力达到106.7/0.2 m L以上,能致40%的30日龄SPF鸡感染。蚀斑克隆毒的免疫原性好,血清中和抗体水平高达15.6 log2,能100%抵抗超强毒株的攻击,表明该纯化株适合用于疫苗生产。     

北方三省(区)牛副流感病毒3型的血清学调查

为了解我国北方三省(区)牛副流感病毒3型(BPIV3)的感染状况,从内蒙古、吉林、山西3个省采集牛血清样品482份。采用病毒中和试验方法进行BPIV3抗体检测。结果显示,482份血清中BPIV3阳性共439份,阳性率为91.08%。吉林、内蒙、山西3省区中血清BPIV3阳性率分别97.35%、84.31%、95.15%。研究表明,中国北方三省(区)牛群中普遍存在BPIV3的感染。     

猪伪狂犬状病毒S1株的分离与鉴定

从上海事某猪场发效仔猪体内分离到1株病毒，该毒株能在鸡胚成纤维细胞，RK，Vero,BHK21,ST,PK15细胞上增殖并产生细胞病变，通过蚀斑克隆对其进行纯化，克隆毒株在BHK21细胞上的TCID50为50μL10^-6.68稀释液，该病毒能被伪狂犬病毒（PRV）阳性血清中和，接种细胞经PRV荧光抗体染色呈阳性反应，电镜观察可见直径110－180nm的典型疱疹病毒粒子，用该病毒接种兔仔猪均出现典型的伪狂犬病症状，表明该分离毒株为PRV。     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in argentina

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

新城疫病毒DF-1细胞蚀斑纯化方法的建立

本研究对DF-1细胞的培养条件及新城疫病毒（NDV）DF-1细胞的蚀斑纯化方法进行了优化摸索,结果显示相较于DMEM培养基,DF-1细胞更适宜在含15%胎牛血清的DMEM/F12培养基中生长,DF-1细胞以3×10⁵/孔的剂量覆盖6孔板能获得最佳的铺板效果,吸附病毒后1.5 h覆盖第1层琼脂,48 h后覆盖第2层琼脂能获得最佳的蚀斑形成效果。通过对蚀斑纯化株F和HN基因测序分析,结果表明蚀斑纯化后无任何变异。通过此方法可在1个星期之内获得稳定的纯化毒株,方法简单、可重复性强、纯化效率高。     

Restriction endonuclease analysis of BHV-1 and BHV-5 strains isolated in Argentina.

The genomes of 10 bovine herpesvirus 1 and 5 strains isolated in Argentina from 1989 to 1994, recovered from animals showing different clinical signs, and two reference strains (Los Angeles and A663) were compared by restriction endonuclease analysis. Four restriction enzymes, HindIII, BamHI, EcoRI and PstI, were used and analysis of the restriction patterns used to assign the isolate to either the BHV-1.1, BHV-1.2 or BHV-5 genotype. There was a correlationship between restriction pattern and clinical signs in six out of ten Argentinian isolates.     

Research on antibodies against BHV-1, BHV-2, BHV-4, BVD-MD virus, bovine adenovirus A and B, rotavirus and coronavirus in cattle in western Zaire: complementary results

Two-hundred bovine sera from western Zaire were screened for antibodies to 8 viruses: BHV-1, BHV-2, BHV-4, BVD-MD virus, bovine adenovirus A and B, bovine rotavirus and bovine coronavirus. Positive sera were found to all these viruses. For animals whose origin was undoubted, the main features were the high prevalence of infections by rotavirus and BHV-4 and the low prevalence of infections by coronavirus and BVD-MD virus.     

First serological evidence of BHV-1 virus in Algerian dromedary camels: Seroprevalence and associated risk factors

Infectious bovine rhinotracheitis (IBR), caused by bovine herpesvirus-1 (BHV-1), is a major livestock health concern in many countries of the world. The objectives of this cross-sectional study were (i) to estimate the seroprevalence of BHV-1 infection and (ii) to assess risk factors associated with this disease in dromedary camels in four districts of Algeria. Blood samples were taken from 865 camels from 84 randomly selected herds, and serum was analyzed for presence of antibodies against BHV-1 by indirect enzyme linked immunosorbent assay (ELISA). Logistic regression was used to determine associations between seroprevalence and potential risk factors (collected using a questionnaire). Antibodies against BHV-1 were detected in 3.7 % (32/865) of samples. Eighteen of 84 camel herds had at least one BHV-1 seropositive camel, giving a herd seroprevalence of 21.4 %. Based on univariate analysis, the introduction of purchased animals and contact with others animal herds appeared as major risk factors. By using multivariate analysis, the only important risk factor was introduction of new animals. This study provided, for the first time, evidence of BHV-1 infection in dromedary camels in Algeria; it also provided estimates of seroprevalence of this disease and suggests that camels may serve as a reservoir of BHV-1 for spread to other species.     

牛病毒性腹泻病毒、牛呼吸道合胞体病毒和牛副流感病毒3型三重RT-PCR检测方法的建立及应用

根椐GenBank中牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)、牛呼吸道合胞体病毒(bovine respiratory syncytial virus,BRSV)和牛副流感病毒3型(bovine parainfluenza virus type 3,BPIV-3)3种病毒基因序列,设计合成引物,建立3种病毒的三重RT-PCR方法。用这3对引物对同一样品中的BVDV、BRSV和BPIV-3核酸模板进行三重RT-PCR扩增,结果显示:可同时扩增BVDV的466 bp,BRSV的735 bp和BPIV-3的258 bp的特异性片段,而对其他4种病原的PCR扩增结果均为阴性;敏感性测定结果表明,该三重RT-PCR技术能检出10 pg的BVDV、1 pg的BPIV-3和10 pg的BRSV模板。用37份临床病料对本研究多重RT-PCR技术和单项RT-PCR技术进行对比验证,结果显示:两者的总符合率为100%。结果表明:建立的多重RT-PCR检测方法,具有特异、快速、准确的特点,可用于对这3种病毒的同时检测和鉴别诊断。     

Plaque assay of equine influenza virus

ESK cells, a stable cell line derived from a swine embryo kidney, were found to be a good medium for plaque formation of the Prague and Miami strains of equine influenza virus. Factors influencing the plaque formation were investigated and a plaque assay for these viruses was worked out. The method is not only simple enough for routine use, but also is as sensitive as the egg inoculation method. The method was readily adapted for a neutralization test.     

表达牛呼吸道合胞体病毒G蛋白基因的重组Ⅰ型牛疱疹病毒的构建与鉴定

为构建表达牛呼吸道合胞体病毒(BRSV)G蛋白基因的牛疱疹病毒Ⅰ型(BHV-1)重组病毒,本研究将人工合成的BRSV全长G蛋白基因编码序列插入到巨细胞病毒(CMV)启动子之下构建TK基因缺失转移载体。利用磷酸钙-DNA沉淀法将该转移载体与亲本病毒BHV-1/TK-/LacZ+的基因组DNA共转染牛鼻甲细胞后收获增殖的病毒。通过反向蚀斑筛选,得到重组病毒BHV-1/TK-/G+。PCR检测结果证实G蛋白基因已经插入到了亲本病毒BHV-1/TK-/LacZ+的基因组中,间接免疫荧光试验和western blot证实BHV-1/TK-/G+中的G蛋白基因在感染的细胞中获得了表达。本研究为研制BRSV及其他重要牛传染病的BHV-1病毒活载体疫苗奠定了基础。     

A comparison of polypeptides and restriction endonuclease sites of BHV-1 isolates and identification of IPV virus in Japan

Bovine herpesvirus 1 (BHV-1) isolates from respiratory tract and from vagina of bovine in Japan were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and the DNA restriction endonuclease cleavage pattern, and compared with European BHV-1 strains. Both protein profile and DNA cleavaged pattern of BHV-1 isolates from respiratory tract were the same as those of European infectious bovine rhinotracheitis (IBR) virus, whereas the protein profile and DNA cleavage patterns of one isolate (M1) from vagina was the same as those of the European infectious pustular vulvovaginitis (IPV) virus. The facts indicate that IPV virus has existed in Japan.