Enzyme-linked immunosorbent assay for the detection of antibodies to infectious bronchitis virus

An enzyme-linked immunosorbent assay (ELISA) for antibodies to avian infectious bronchitis (IB) virus is described. The immune response of chickens following vaccination with IB virus was monitored using this test, and the titers were compared with those obtained by serum neutralization. The ELISA appears to be suitable for IB serology.     

鸡传染性支气管炎病毒Real-time PCR方法的建立及其对感染鸡体内病毒的检测

本研究针对传染性支气管炎病毒（IBV）tl／CH／LDT3／03毒株的N基因（GenBank登录号为AY702975）设计并合成了一对引物,构建重组质粒作为阳性标准品,建立了检测IBV核酸的SYBRGreenⅠ荧光定量PCR方法。该方法可检测到初始模板中6．45×10copies／μL的病毒核酸。与常规PCR相比,敏感性高100倍。该检测方法特异性强,与其它禽源病毒如新城疫病毒（NDV）、传染性喉气管炎病毒（ILTV）、传染性法氏囊病毒（IBDV）、禽流感病毒（AIV）、马立克氏病毒（MDV）均不发生交叉反应;重复性试验的变异系数小于2．6％。结果表明,本试验建立的荧光定量PCR检测方法灵敏度高、特异性强、重复性好,可用于传染性支气管炎的临床诊断。同时应用本方法对人工感染IBV的SPF鸡的主要脏器盲肠扁桃体、肾脏、肺和气管进行了病毒RNA定量检测,结果表明,肾脏的病毒含量最高,盲肠扁桃体中病毒持续的时间最长,从而揭示了IBV在SPF鸡体内复制的动态变化,证实了感染鸡的临床表现与病毒滴度的时间相关性。     

Coronavirus avian infectious bronchitis virus

Infectious bronchitis virus (IBV), the coronavirus of the chicken (Gallus gallus), is one of the foremost causes of economic loss within the poultry industry, affecting the performance of both meat-type and egg-laying birds. The virus replicates not only in the epithelium of upper and lower respiratory tract tissues, but also in many tissues along the alimentary tract and elsewhere e.g. kidney, oviduct and testes. It can be detected in both respiratory and faecal material. There is increasing evidence that IBV can infect species of bird other than the chicken. Interestingly breeds of chicken vary with respect to the severity of infection with IBV, which may be related to the immune response. Probably the major reason for the high profile of IBV is the existence of a very large number of serotypes. Both live and inactivated IB vaccines are used extensively, the latter requiring priming by the former. Their effectiveness is diminished by poor cross-protection. The nature of the protective immune response to IBV is poorly understood. What is known is that the surface spike protein, indeed the amino-terminal S1 half, is sufficient to induce good protective immunity. There is increasing evidence that only a few amino acid differences amongst S proteins are sufficient to have a detrimental impact on cross-protection. Experimental vector IB vaccines and genetically manipulated IBVs--with heterologous spike protein genes--have produced promising results, including in the context of in ovo vaccination.     

Detection and quantification of antibodies to infectious bronchitis virus by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for detecting and quantifying antibodies to infectious bronchitis virus (IBV) is described. Purified antigen, prepared on sucrose density gradients, was required to decrease the nonspecific background, and saline was found to be superior to bicarbonate buffer for coating the cuvettes with antigen. The sensitivity of the test in measuring antiserum titers could be altered greatly and linearly by adjusting the protein content of the antigen. The ELISA was able to detect an antibody response to IBV infection earlier than the virus-neutralization (VN) test. Antibody titers obtained by ELISA were considerably higher than those obtained by VN. Serotypes of IBV could not be differentiated with ELISA because of extensive antiserum cross-reactivity. The utility of ELISA in studies on IBV is discussed.     

传染性支气管炎病毒RT-PCR方法的建立

传染性支气管炎病毒 (IBV)的主要结构蛋白有 3种 ,纤突蛋白 S、膜蛋白 M和核蛋白 N。S蛋白由 S1和 S2 2部分组成 ,其中 S1蛋白的进化最为活跃 ,是 IBV具有众多血清型的基础。 S1蛋白的活泼表现源于 S1基因容易发生插入、缺失和不同毒株基因间重组。因此 ,围绕着 S1基因的研究工作就成为 IBV研究的热点 [1～ 3 ]。研究 IBV基因组结构的基本手段之一是建立有效的反转录聚合酶链反应 (RT- PCR)以扩增出所需要的基因片段 ,由于 IBV基因组结构比较特殊 (核酸片段较大 ,碱基分配不均匀 ,G C含量较低 ) ,其 RT-PCR方法的建立十分困…     

A comparison of infectious bronchitis virus hemagglutination-inhibition test procedures

Replicate hemagglutination-inhibition (HI) tests of selected convalescent and hyperimmune infectious bronchitis virus (IBV) antisera were run against five IBV antigens: Massachusetts 41, Connecticut 46, H52, Arkansas 99, and JMK. Six different HI test procedures were included in this comparison to evaluate the effect of procedural differences on HI titers. The results were similar among procedures to the extent that positive and negative reactions were differentiated by each procedure with each antigen. This indicated that specificity was not affected by the procedure. However, the mean titers did differ among procedures in tests of the same serum-antigen combination, and the range of the titer differences was frequently fourfold or greater. Generally, high or low titers were associated with specific procedures, regardless of which test antigen was used.     

传染性支气管炎病毒的鉴定

试验采用鸡胚接种和气管环培养相结合的方法,对IBV毒株进行培养鉴定,IBV经鸡胚传一代后再上鸡胚气管环培养可引起气管环纤毛运动停止,应用此法可以快速地进行IBV检测。     

禽传染性支气管炎病毒M基因在毕赤酵母中表达与蛋白反应活性检测

将禽传染性支气管炎病毒(IBV)M基因克隆到毕赤巴斯德表达载体pPIC9K中,构建了重组表达载体pPIC9K-M,电击转化毕赤巴斯德酵母GS115,经MD和MM平板筛选和PCR鉴定后,采用G418抗性梯度筛选得到高拷贝重组菌株GS115/pPIC9K-M His~+Mut~+,重组菌株用1%的甲醇诱导分泌表达,并对表达产物进行SDS-PAGE、Western-blot分析.结果表明,IBV M基因在毕赤酵母中成功获得了表达,表达蛋白的相对分子质量约为33 000,能与IBV阳性血清特异性结合.将表达蛋白初步纯化后作为包被抗原,ELISA检测结果显示表达蛋白与特异性抗体反应良好,表明表达的M蛋白生物学活性良好.本研究表达的IBV M蛋白可作为制备IB诊断抗原的基础材料,对IB的防治有重要理论和实用价值.     

单抗介导的银加强金标免疫技术检测传染性支气管炎病毒

利用胶体金标记纯化的羊抗鼠IgG,建立了一种银加强金标免疫技术(SECGA)检测IB抗原.检测抗原时先将抗原点样于硝酸纤维素膜(NCM)中央,封闭后浸于1:40 IBV单抗中作用10 min,洗涤后再浸于1:4金标羊抗鼠IgG液中作用1 h,银染10 min后观察,在膜上出现灰黑斑点的为阳性结果,反之,不出现斑点为阴性结果.SECGA对IB抗原最低检测量为0.703 ng/点(2 μL),对含毒尿囊液的最低检测量为102.9 EID50,检测IBV M41在鸡胚中繁殖时以36～54 h含毒量最高;检测SPF鸡气管粘液,至少于感染后3 d内检出病毒.结果表明,SECGA检测抗原可用于IBV早期诊断,具有群特异性、快速、敏感、简便,不需特殊设备、结果易判定等优点,尤适于基层实验室或现场应用.     

Standardization and application of the enzyme-linked immunosorbent assay for infectious bronchitis

The indirect enzyme-linked immunosorbent assay (ELISA) was used to detect antibody to infectious bronchitis virus in chickens. The serum-neutralization test for infectious bronchitis (SNIB) was used as a reference serologic test. The ELISA proved to be useful for monitoring antibody responses following vaccination of leghorn chicks. The titers obtained with the ELISA and SNIB showed an overall correlation, but results suggest the involvement of different antibody classes.