应对日益严峻的挑战：中国禽传染性支气管炎研究

自1937年禽传染性支气管炎病毒在美国被首次分离以来,该病毒的传播给世界养禽业带来了严重的经济损失。中国地域辽阔、气候多样,国内该病毒的流行情况十分复杂。本文就传染性支气管炎在国内的病原分离、分子流行病学、检测技术、疫苗及综合防控技术等方面的研究与实践进行总结。目前,该病毒在中国多种类型毒株并存,优势流行毒株为QX基因型毒株。除广泛使用的H120等Mass血清型疫苗外,4/91血清型疫苗和LDT3-A株疫苗也被逐步使用,多采用弱毒疫苗和灭活疫苗联合免疫的方法有效控制了其对养禽业造成的经济损失。     

微生物介导的新城疫病毒疫苗的研制现状

新城疫病毒引起的新城疫是一种禽类急性接触性传染病,采用疫苗防治该病是当前研究的热点领域之一。HN/F蛋白是2种有效的疫苗候选分子,乳酸乳球菌、鼠伤寒沙门菌、单增李斯特菌、根癌农杆菌、鸡痘病毒、牛痘病毒、鸽痘病毒、禽红白血病病毒、禽副粘病毒3型、传染性法氏囊病病毒和传染性气管炎病毒等微生物经过改造后可成为理想的疫苗载体。本文综述了重组乳酸乳球菌(rLL-HN)、重组鼠伤寒沙门菌(rSt-F和rSt-HN)、重组单增李斯特菌(rLm-F)、重组根癌农杆菌(rAt-F)、重组鸡痘病毒(rFPV-HN、rFPV-F-gB和rFPV-F-HN-gB)、重组牛痘病毒(rVV-F)、重组鸽痘病毒(rPPV-F)、重组禽红白血病病毒(rAEV-HN)、重组禽副粘病毒3型(rAPMV-F)、重组传染性法氏囊病病毒(rIBDV-HN)和重组传染性气管炎病毒(rIBV-HN)等疫苗的构建及其免疫机制的研制现状。     

传染性支气管炎病毒N蛋白CTL表位与BF2*15鸡MHC I基序的相互作用

【目的】近年来鸡传染性支气管炎病毒在国内鸡群呈现流行趋势,尤其是变异毒株的出现,加剧了对鸡群的危害。鸡主要组织相容复合体蛋白(MHC I)通过特定的基序结合抗原表位多肽,进而识别感染病毒的细胞并引起免疫反应,达到清除病毒的效果。鉴定BF2*15鸡主要组织相容复合体(MHC I)的结合基序。【方法】采用同源建模、分子动力学和分子对接等计算方法,构建了传染性支气管炎病毒(IBV)N蛋白表位多肽和鸡MHC I BF2*15之间的复合物结构,来探索BF2*15识别抗原表位多肽的潜在结合基序。【结果】通过对该复合物的相互作用关系分析,鉴定出BF2*15鸡主要组织相容复合体(MHC I)的一条潜在结合基序"x-Arg-xx-x-Arg"。【结论】解释了传染性支气管炎病毒N蛋白CTL表位与BF2*15鸡MHC I基序的相互作用原理,该研究结果对了解传染性支气管炎病毒免疫机制以及基于特定基序筛选和设计通用疫苗具有借鉴意义。     

传染性喉气管炎病毒gB基因和新城疫病毒F基因在重组禽痘病毒中共表达

在重组禽痘病毒中表达多个禽类病原的主要免疫原基因是构建多价基因工程疫苗的前提 ,但相关研究很少。在表达传染性喉气管炎病毒 (ILTV)gB基因重组禽痘病毒的转移载体的基础上 ,构建了含有ILTVgB基因和新城疫病毒 (NDV)F基因的重组禽痘病毒转移载体pSY-gB-F ,采用脂质体转染禽痘病毒感染的鸡胚成纤维 (CEF)细胞后 ,通过蓝斑试验筛选出重组禽痘病毒 (rFPV-gB-F) ,并进行了 6轮蚀斑纯化。Western blot试验和间接免疫荧光试验证明ILTVgB基因和NDVF基因在rFPV-gB-F感染的CEF细胞中获得表达。为传染性喉气管炎、新城疫与鸡痘活载体多价疫苗的研制奠定基础。     

传染性喉气管炎病毒gB基因和新城疫病毒F基因在重组禽痘病毒中共表达

在重组禽痘病毒中表达多个禽类病原的主要免疫原基因是构建多价基因工程疫苗的前提,但相关研究很少。在表达传染性喉气管炎病毒(ILTV)gB基因重组禽痘病毒的转移载体的基础上,构建了含有ILTV gB基因和新城疫病毒(NDV)F基因的重组禽痘病毒转移载体pSY-gB-F,采用脂质体转染禽痘病毒感染的鸡胚成纤维(CEF)细胞后,通过蓝斑试验筛选出重组禽痘病毒(rFPv-gB-F),并进行了6轮蚀斑纯化。Western-blot试验和间接免疫荧光试验证明ILTV gB基因和NBVF基因在rFPV-gB-F感染的CEF细胞中获得表达。为传染性喉气管炎、新城疫与鸡痘活载体多价疫苗的研制奠定基础。     

鸡传染性法氏囊病病毒研究进展

传染性法氏囊病(Infection bursal disease, IBD)是由鸡传染性法氏囊病毒(Infectious bursal disease virus, IBDV)引起的鸡和火鸡的一种高度接触性传染病,给世界各国的禽养殖业带来了巨大损失.自IBDV发现至今新的变异株不断出现,分子结构的改变导致病毒致病力的改变及宿主对疫苗应答的改变,使得传统的疫苗已不能控制其流行,因此各国学者对其基因组结构和功能进行了广泛深入的研究,并积极研制新型有效的疫苗以达到防治的目的.     

重组新城疫病毒疫苗研制现状

新城疫病毒引起的新城疫是一种常见的禽类传染病。新城疫病毒可诱导感染的宿主产生细胞、体液和黏膜免疫应答,是一种理想的疫苗载体,可表达病毒和细菌的多种蛋白。这些病原体包括禽流感病毒、人副流感病毒3型、犬瘟热病毒、禽巨肺病毒、呼吸道合胞病毒、Nipah病毒、水疱性口炎病毒、狂犬病毒、牛短崭热病毒、传染性支气管炎病毒、重症急性呼吸综合征冠状病毒、埃博拉病毒、马尔堡病毒、肠病毒71型、脊灰病毒、Ⅰ型人类免疫缺陷病毒、传染性法氏囊病毒、诺如病毒、非洲猪瘟病毒、牛疱疹病毒1型、西尼罗病毒、鹅细小病毒、猪圆环病毒Ⅱ型、传染性喉气管炎病毒、鸡毒支原体和博氏疏螺旋体等。本文简要综述重组新城疫病毒的构建及其免疫机制的研制现状。     

表达绿色荧光蛋白的重组鸡传染性支气管炎病毒的构建

为探讨鸡传染性支气管炎病毒(IBV)作为载体表达外源基因的可行性,本研究根据IBV H120疫苗株的全基因组序列设计引物,采用RT-PCR方法分10个片段对其基因组进行扩增,并克隆至pMD19-T载体中;同时构建IBV基因组5a基因编码区被增强型绿色荧光蛋白(EGFP)基因替换的重组质粒。采用体外拼接策略,将BsaI酶切处理的10个基因片段顺序连接,构建5a基因编码区被EGFP基因替换的基因组全长cDNA,其5’端具有完整的T7 RNA聚合酶启动子核心序列,3’端具有polyA尾巴结构。然后通过T7 RNA聚合酶体外转录系统合成病毒基因组RNA,脂质体转染BHK-21细胞进行病毒拯救。结果表明成功的从基因组全长cDNA拯救出重组病毒H120-5a/EGFP株,其在鸡胚中能有效的复制和传代,并表达绿色荧光蛋白;5a基因的缺失并不影响病毒对鸡胚的致病性。本研究为进一步开展IBV的分子致病机理、载体疫苗等研究奠定了基础。     

腮腺炎病毒疫苗株与野毒株基因鉴别的研究进展

腮腺炎病毒的分子生物学特性已基本清楚。接种腮腺炎减毒活疫苗后的主要问题是Ｕｒａｂｅ株疫苗可引起脑膜炎,故许多国家已改用ＪｅｒｙｌＬｙｎｎ株疫苗。鉴别不同病毒株的可靠方法是分析比较病毒基因组中某些基因区域的核苷酸序列,此法有助于腮腺炎疫苗质量的监控及腮腺炎病毒分子流行病学研究。     

痘病毒载体构建及其在病毒疫苗研制上的应用

概述了痘苗病毒载体和禽痘病毒载体的构建及其在动物病毒疫苗与医学病毒疫苗研制上应用的进展,进而分析了痘病毒载体疫苗尤其是禽痘病毒载体疫苗的发展趋势。     

Development,standardization and assessment of PCR systems for purity testing of avian viral vaccines

The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method.Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL).     

The Efficacy of a Live Attenuated TW I-Type Infectious Bronchitis Virus Vaccine Candidate

Virologica Sinica - Infectious bronchitis (IB) is a highly contagious avian disease caused by infection with infectious bronchitis virus (IBV), which seriously affects the development of the global...     

Elucidation of the avian nucleolar proteome by quantitative proteomics using SILAC and changes in cells infected with the coronavirus infectious bronchitis virus

The nucleolus is a dynamic subnuclear compartment involved in ribosome subunit biogenesis, regulation of cell stress and modulation of cellular growth and the cell cycle, among other functions. The nucleolus is composed of complex protein/protein and protein/RNA interactions. It is a target of virus infection with many viral proteins being shown to localize to the nucleolus during infection. Perturbations to the structure of the nucleolus and its proteome have been predicted to play a role in both cellular and infectious disease. Stable isotope labeling with amino acids in cell culture coupled to LC‐MS/MS with bioinformatic analysis using Ingenuity Pathway Analysis was used to investigate whether the nucleolar proteome altered in virus‐infected cells. In this study, the avian nucleolar proteome was defined in the absence and presence of virus, in this case the positive strand RNA virus, avian coronavirus infectious bronchitis virus. Data sets, potential protein changes and the functional consequences of virus infection were validated using independent assays. These demonstrated that specific rather than generic changes occurred in the nucleolar proteome in infectious bronchitis virus‐infected cells.     

Development of a porcine epidemic diarrhea virus M protein-based ELISA for virus detection

A membrane (M), protein-based ELISA was developed to detect porcine epidemic diarrhea virus (PEDV). The M gene of PEDV was expressed in Escherichia coli. The purified recombinant M protein was used to immunize rabbits to generate a polyclonal antibody. Immunofluorescence analysis indicated that the anti-PEDV-M antibody reacted with PEDV-infected cells. The antibody was utilized to develop an indirect ELISA to detect PEDV. Other viruses, porcine transmissible gastroenteritis coronavirus, avian infectious bronchitis coronavirus, porcine reproductive and respiratory syndrome virus, classic swine fever virus and porcine pseudorabies virus, were unreactive.     

Stabilizing Effect of Magnesium Sulfate on Avian Infectious Bronchitis Virus Propagated in Chicken Embryo Kidney Cells

The Beaudette strain of avian infectious bronchitis virus propagated in chicken embryo kidney cells is stabilized by exposure to 1 M MgSO(4) at 50 C for 80 min, at pH values ranging from 4 to 10.     

pH Stability Studies with Avian Infectious Bronchitis Virus (Coronavirus) Strains

A comparison of 17 infectious bronchitis virus strains, using the same test procedure and assay system, demonstrated that stability at an acid pH is a variable characteristic of the avian coronaviruses.     

Infectious disease survey of lesser prairie chickens in north Texas

Lesser prairie chicken (Tympanuchus pallidicinctus) abundance, like that of most grassland birds, has declined rangewide for decades. Although habitat loss and degradation are likely ultimate causes for this decline, infectious agents, particularly microparasites, could be proximate contributors. No surveys of pathogenic bacteria or viruses have been published for this species. We surveyed 24 free-living lesser prairie chickens from Hemphill County, Texas (USA), for evidence of exposure to Salmonella typhimurium, S. pullorum, Mycoplasma gallisepticum, M. synoviae, Chlamydophila psittaci, and the avian influenza, Newcastle disease, infectious bronchitis, and reticuloendotheliosis viruses. Two of 18, and eight of 17 samples were seropositive for the Massachusetts and Arkansas serotypes of infectious bronchitis virus, respectively. Five of the eight positive individuals were juveniles, two of which were seropositive for both serotypes. All other serologic and genetic tests were negative. Because the ecological significance of these results is unknown, the pathogenesis, transmission, and/or population-level influences of infectious bronchitis and related avian coronaviruses for lesser prairie chickens deserves further study.     

一种更灵敏的特异性检测H9亚型禽流感抗体的间接ELISA方法的建立

H9亚型禽流感在全球范围内广泛流行,控制其传播需监测H9亚型禽流感病毒的感染情况及疫苗的免疫效果。为了建立便于检测且灵敏特异的H9亚型禽流感抗体间接ELISA方法,本实验利用不同亚型之间变异较大的H9亚型禽流感病毒HA蛋白的头部球状区作为包被抗原,确定了最佳复合封闭液和抗体稀释液,提高了其特异性。结果显示建立的ELISA方法灵敏度高于血凝抑制试验(HI),且与H3N2、H5N2、H7N9亚型流感病毒及新城疫病毒(NDV)、鸡传染性支气管炎病毒(IBV)、鸡传染性法氏囊病毒(IBDV)和产蛋下降综合征病毒(EDSV)的阳性血清均无交叉反应。另外,利用该方法及HI试验对200份临床鸡血清样本进行检测,两种检测方法的符合率达97%,且存在较高的相关性(R2=0.981 1)。