探针检测鸭黄病毒的地高辛标记DNA的制备与应用

利用RT-PCR方法扩增鸭黄病毒的NS3基因406bp的特异性片段,回收并纯化PCR产物,用地高辛标记,制备核酸探针。特异性试验结果表明,该探针仅与鸭黄病毒的核酸特异性杂交,而与鸭瘟病毒、H9N2禽流感病毒、新城疫病毒、传染性法氏囊病病毒、减蛋综合征病毒的核酸杂交均为阴性。敏感性试验表明,该探针对鸭黄病毒的RNA最低检出限量为100μg/L。对疑似黄病毒感染鸭的肝脏、肺脏、脾脏、输卵管、卵泡膜和泄殖腔棉拭子进行检测,以卵泡膜的检出率最高。该研究为鸭黄病毒感染的诊断和流行病学调查提供了一种可靠的方法。     

Effect of bursal cell number on the pathogenesis of infectious bursal disease in chickens

The pathogenesis of infectious bursal disease (IBD) in chickens neonatally chemically bursectomized (CB) by cyclophosphamide and subsequently inoculated with various numbers of bursal cells was examined. CB chickens inoculated with at least 62.5 X 10(6) bursal cells were as susceptible to IBD clinical manifestations (as determined by gross and microscopic evaluation of bursal tissues, virus recovery from spleen, and antibody titer) as intact chickens following inoculation with virus at 5 weeks of age. In contrast, CB chickens inoculated with 2.5 X 10(6) or fewer bursal cells were refractory to the IBD clinical manifestations compared with intact chickens or CB chickens inoculated with 62.5 X 10(6) or more bursal cells. Results from this study suggest that the availability of a large number of bursal cells is an essential factor in the development of IBD.     

A preliminary note on the prevalence of infectious bursal disease of poultry in Cameroon

The occurrence of infectious bursal disease (IBD) in industrial poultry flocks in Cameroon was investigated by serological and virological techniques. Antibody to IBD virus was detected in 118 (33.9%) out of 348 serum samples collected in seven randomly selected areas. On the other hand, virological examination was performed on bursa of Fabricius samples obtained post-mortem from flocks in which there was a high mortality among young birds. This virological examination revealed the presence of IBD virus antigen. These observations confirm the occurrence of infectious bursal disease in Cameronian industrial poultry flocks.     

鸡传染性法氏囊病病毒的PCR检测法

用聚合酶链反应（ＰＣＲ）技术直接检测鸡传染性法氏囊病病毒，可快速、特异及敏感地检出实验毒株和病鸡法氏囊内存在的微量病毒核酸。对病鸡、进口种鸡的实测结果表明，本法对诊断鸡传染性法氏囊病病毒具有重要的实用意义     

First report of an infectious bursal disease outbreak in a vaccinated chicken flock in Anambra State, Nigeria.

Infectious bursal disease was reported in a flock of 7-week old vaccinated chickens. Clinical findings and post-mortem changes were classical as well as the microscopic pathology of the bursa. Bursal homogenates from dead birds were positive for IBD virus antigen in agar gel diffusion test (AGDT). Convalescent sera obtained from birds 14 days following the onset of clinical signs were also positive for IBD virus antibody in AGDT. Seven-week old susceptible birds, each infected i/m with 0.1 ml of a bursal preparation from the outbreak, showed clinical signs of IBD on the 3rd day and were all dead by the 6th day. Their bursae were also positive for IBD virus antigen in AGDT. This is the first recorded outbreak of IBD in Southern Nigeria following inoculation with a locally produced vaccine.     

表达鸡传染性法氏囊病病毒VP2基因的重组火鸡疱疹病毒的构建及生物学特性分析

鸡传染性法氏囊病（IBD）是一种严重危害养禽业的高度致死性和免疫抑制性传染病。为研制IBD重组火鸡疱疹病毒（HVT）活载体疫苗,本研究构建了表达鸡传染性法氏囊病病毒（IBDV）保护性抗原VP2基因的重组HVT并对其体外生物学特性进行了分析。通过RT-PCR扩增IBDV超强毒株VP2基因并克隆入pCI载体,获得重组真核表达质粒pCI-VP2。用限制性内切酶将携带CMV启动子的VP2基因表达框架切下,连接于入门质粒pENTR,构建获得重组入门质粒pENTR-VP2。将pENTR-VP2与HVT重组黏粒H3-Kan/ccdB进行LR重组反应,构建重组表达黏粒H3-VP2。用H3-VP2与其他4个相互重叠并覆盖HVT全基因组的黏粒共同转染鸡胚成纤维细胞（CEF）,拯救获得重组病毒rHVT-VP2。将重组病毒在CEF中连续传至20代后用PCR、间接免疫荧光试验和免疫印迹试验进行检测,并绘制重组病毒体外生长曲线,分析其体外复制特性。结果表明,重组病毒rHVT-VP2能够稳定表达VP2蛋白,rHVT-VP2在CEF中的复制能力与亲本病毒无明显差异。重组病毒rHVT-VP2免疫鸡后能够诱导产生IBDV中和抗体,并对IBDV强毒株攻击引起的死亡提供90%免疫保护。重组病毒rHVT-VP2的构建为研制IBD重组HVT活载体疫苗奠定了基础,对IBD的防控具有重要意义。     

A novel oligonucleotide probe for the detection of Newcastle disease virus.

An approach that possesses high specificity and broad applicability was used to obtain a DNA probe with potential diagnostic value. By utilizing synthesized oligonucleotide DNA, termed NDV probe, all 14 strains of NDV tested under high-stringency conditions were recognized in a slot-blot hybridization assay. The sequence of the NDV probe was generated from a highly conserved region of the NDV genome. No hybridization was observed with RNA isolated from other avian viruses, including avian influenza, infectious bursal disease, and infectious bronchitis. The specificity inherent in using an oligonucleotide probe offers advantages over probes obtained from cloned DNA fragments.     

Tissue-print hybridization using a non-radioactive probe for the detection of infectious bursal disease virus.

Tissue-print hybridization was evaluated as a simplified means for detection of infectious bursal disease virus (IBDV) in the bursa of Fabricius from infected chickens. The assay employed a biotin-labeled synthetic oligonucleotide as a probe. The bound probe was detected using a color assay consisting of streptavidin conjugated to alkaline phosphatase. Bursae were imprinted onto nitrocellulose and then hybridized with the biotinylated probe. Bursal prints from IBDV-infected chickens were readily distinguished from control prints by color development and differences in signal intensity.     

Differentiation of infectious bursal disease viruses by restriction enzyme analysis of RT-PCR amplified VP1 gene sequence

In order to differentiate infectious bursal disease virus (IBDV) isolates/strains, a quick method of RT-PCR followed by restriction enzyme analysis of VP1 gene sequence is being reported for the first time. A 480 bp fragment, comprising one of the RNA dependent RNA polymerase motifs of VP1 gene sequence of an Indian classical virus, an attenuated vaccine strain, Georgia and two Indian field isolates, genetically similar to reported very virulent strains of IBDV, was amplified by RT-PCR. Restriction enzyme digestion of PCR products with Taq1 enzyme generated distinct profile for field isolates, different from the classical and attenuated viruses, whereas restriction profile with BstNI restriction enzyme was similar in all the viruses, irrespective of the pathotype. Therefore, the present results suggest that Taq1 digestion can be taken up for the differentiation of field isolates from the classical and vaccine strains. The sequence analysis of VPI gene of reported very virulent IBD viruses from Europe and Japan, using 'MapDraw' programme of Lasergene software, revealed similar restriction enzyme profile as in Indian field isolates.     

IBDV逆转录—聚合酶链反应和核酸杂交检测方法的研究

以建立的传染性法氏囊病病毒（ＩＢＤＶ）逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）和核酸杂交检测方法，检测了细胞培养物、病理组织、粪便和饲料中的ＩＢＤＶ，并与ＩＢＤＶ夹心ＥＬＩＳＡ、琼脂扩散试验（ＩＤ）和病毒分离方法进行了平行比较试验。结果表明，ＲＴ－ＰＣＲ和核酸杂交检测方法具有高度的敏感性和特异性，是深入研究ＩＢＤ流行病学，特别是传播途径的新手段     

鸡传染性法氏囊病病毒适应于Vero细胞的初步研究

将国内６个省市（广东、巾东、河北、辽宁、、新疆和北京）的７株鸡传染性法氏囊病（ＩＢＤ）的法氏囊组织处理后，直接在Ｖｅｒｏ细胞上盲传３～４代，均能不同程度的产生特征性细胞病变效应（ＣＰＥ），并通过选用具有广谱性的抗法氏囊病病毒的单克隆抗体（ＩＢＩ）Ｖ－ＭｃＡｂ）采用间接免疫荧光（ＩＦＡ）和碱性磷酸酶—抗碱性磷酸酶桥联酶显色技术（ＡＰＡＡＰ）的方法对７株分离毒的Ｖｅｒｏ第８代细胞进行鉴定，又用逆转—聚合酶链式反应（ＲＴ－ＰＣＲ）对７株分离毒的第１４代Ｖｅｒｏ细胞毒进行进一步证实，结果表明这７株ＩＢＤ囊毒均适应于Ｖｅｒｏ细胞上，这是国内首次报道将组织毒直接适应于Ｖｅｒｏ传代细胞上。     

用SPF鸡制备IBD诊断抗原及血清的研究

将IBDV接种SPF鸡，死者取其法氏囊，经超声波粉碎或匀浆，反复冻融制备IBDAGP诊断原，未死者，再用IBD组织灭活苗免疫制备IBD诊断血清。如此制备的IBD诊断液特异性强、灵敏度高、成本低．适于禽病检验室推广应用。     

Digoxigenin-labeled nucleic acid probe for the detection of infectious bursal disease virus in infected cells.

A cDNA probe was synthesized from the VP-4 region of a virulent field isolate of infectious bursal disease virus (IBDV). The probe was labeled during synthesis with a non-radioactive steroid hapten, digoxigenin. The probe was used to develop a hybridization assay to detect the presence of IBDV in infected cell-culture and tissue suspensions from the bursa of Fabricius of infected chickens. The test was rapid, reproducible, and sensitive, and it could detect four serologic subtypes of IBDV, including the GLS-5 isolate.     

鸡传染性法氏囊病新型疫苗的研究进展

鸡传染性法氏囊病(IBD)是由鸡传染性法氏囊病病毒(IBDV)引起的雏鸡的一种高度接触性传染病,自1962年报道以来,世界上主要养禽国家和地区均有流行,给养禽业带来严重经济损失。目前,IBD防控的主要方法是采用疫苗接种,使易感雏鸡获得主动或被动免疫保护,因此疫苗的质量对临床上IBD的防控起着至关重要的作用。虽然各国均有较好的商品化疫苗,但随着IBDV毒株的不断变异,商品化疫苗的抗原性与流行毒株不能完全匹配,临床上免疫失败时有发生,因此迫切需要研发与临床流行毒株相匹配的新型疫苗用于IBD的防控。对近期IBD的基因缺失苗、亚单位疫苗、DNA疫苗以及活载体疫苗等新型疫苗的研究进展进行概述,以此为IBD新型疫苗的研究提供参考。     

应用生物素标记的NDV－cDNA探针检测感染尿囊液中NDV－RNA的初步研究

本文应用聚合酶链反应（ＰＣＲ）技术从构建的新城疫病毒（ＮＤＶ）ｃＤＮＡ文库中扩增含编码Ｆ糖蛋白前体──Ｆｏ酶切位点序列的３５９ｂｐ的Ｆ蛋白基因ｃＤＮＡ片段。将此３５９ｂｐｃＤＮＡ片段经光敏生物素标记后,即成ＮＤＶ－ｃＤＮＡ探针。该探针能特异性地从感染的尿囊液中检测出ＮＤＶ强毒株和疫苗毒株的基因组ＲＮＡ,而不与ＩＢＤｖ－ｄｓＲＮＡ、ＡＩＢｖ－ｓｓＲＮＡ、ＥＤＳ７６－ｄｓＤＮＡ、ＭＤＶ－ｄｓＤＮＡ,ＦＰＶ－ｄｓＲＮＡ及ＡＩＬＶ－ｄｓＤＮＡ发生交叉杂交反应。试验结果表明：尽管该探钎含有编码Ｆｏ蛋白酶切位点序列的碱基顺序,但它还是不能把ＮＤＶ的强、弱毒株区分开。这说明ＮＤＶ强、弱毒株比区域内的碱基存在着相当大的同源性。不过,此探针对ＮＤＶ来说具有特异性,这就为ＮＤＶ的诊断技术开创了基因水平检测的新途径。