鸡传染性支气管炎分离株S1基因的序列分析

传染性支气管炎病毒(IBV)为冠状病毒科冠状病毒属成员,基因组为单股正链RNA,长27.6 kb,带有poly ( A)尾.病毒粒子基因组编码6种蛋白,其中位于病毒囊膜表面的纤突蛋白(spike protein , S )是病毒主要的免疫原蛋白基因,IBV纤突蛋白是由2～3个拷贝的S1, S2亚单位组成.由于IBV的特殊的转录合成机制使得S1基因易发生点突变、插入、缺失和基因重组,使得IBV极易产生变异株.S1蛋白具有许多重要的生物学功能,是IBV主要的免疫原蛋白,它能刺激机体产生中和及血凝抑制抗体.因此,根据不同地区的分离毒株S1基因的差异,来确定流行IBV的血清型,研究其分子生物学特性,深入分析IBV的变异原因,进一步揭示IBV分离株的变异机制,对从根本上控制IB,具有重要意义.     

鸡肾型传染性支气管炎病毒分离株S1基因的序列分析

对鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)肾型毒株BJQ、BJS、BJY、SDW和HBN的S1全基因进行了扩增、克隆和序列测定,并将所分离毒株的S1基因序列与5个参考毒株进行了比较.结果表明,IBV分离株S1基因间核苷酸同源性在76.7%～92.1%之间,氨基酸同源性在73.9%～89.5%之间.氨基酸序列特征分析表明,S1基因多处存在突变、缺失和插入现象,其中在氨基酸69～81和142～150位点区出现较高的变异.系统进化树分析表明,除SDW株与Gray属于相同的进化分支外,其他国内肾型分离株属于同一个进化分支,而韩国、日本等亚洲分离株和美洲分离株分别属于其他不同的进化分支,说明IBV病毒的发生和流行与地域及所致病型有一定的相关性.     

鸡传染性支气管炎病毒分离株核衣壳基因克隆、序列分析和真核表达

根据GenBank公开序列自行设计一对引物，采用RT—PCR扩增出鸡传染性支气管炎病毒（Infectious bronchitis virus，IBV）W和C9001分离株完整的核衣壳（N）基因，并将其克隆至pMD18-T载体进行核苷酸序列测定和分析。结果表明，扩增的2个IBV分离株核衣壳基因片段长度均为1230bp，编码409个氨基酸，彼此间核苷酸和氨基酸同源性分别为88．0％和89．5％，与GenBank中有代表性的参考毒株相应基因核苷酸和氨基酸序列比较显示，w株核苷酸序列与GenBank中的广东分离株（AY646283）同源性最高，为94．1％，氨基酸序列同源性为94．6％；与国内部分毒株核苷酸序列同源性在86．1％～88．0％之间，氨基酸序列同源性在88．0％～90．7％之间；C9001株与国内部分毒株核苷酸序列同源性在86．4％～99．8％之间，氨基酸序列同源性在88．0％～99．8％之间。从核衣壳基因编码的氨基酸序列的系统进化树可见，W株与C9001株处于不同的进化分枝，亲缘关系较远。同时将核衣壳基因构建于真核表达质粒pVAX1中，用脂质体法将重组质粒转染入COS-7细胞中，间接免疫荧光检测出核衣壳蛋白的体外表达。研究结果为进一步研究IBV核衣壳蛋白的结构与功能以及基因工程疫苗的研制奠定了基础。     

Molecular epidemiology of infectious bronchitis virus isolates from China and Southeast Asia

In order to trace the origin and evolution of avian infectious bronchitis virus (IBV) isolates in China and Southeast Asia, genomic sequencing was used for molecular characterization of 24 IBV isolates and two reference strains in comparison with the published sequences. The 5' region of the S1 genes, containing hypervariable regions I and II, and 3' region of the nucleocapsid genes, containing cytotoxic T lymphocyte epitopes, were used to construct phylogenetic trees for analysis. The results showed that the 24 isolates could be divided into three distinct groups, that is, American, Asian, and European. Some isolates formed a distinct Asian phylogenetic group, suggesting that IBV has existed for some time in Asia. Our results also showed that in vivo recombination of IBV may have occurred at a rather high frequency, contributing to the diversity of these IBV isolates. Importantly, recombination events have probably occurred between vaccine strains and field strains in the natural condition.     

表达传染性支气管炎病毒S1基因重组鸡痘病毒的构建

将鸡传染性支气管炎病毒S1基因插入到鸡痘病毒转移载体pSY681中，获得重组转移载体pSY681。将pSY681-IBVS1转染已感染亲本鸡痘病毒S-FPV-017株的鸡胚成纤维细胞，使其在鸡胚成纤维细胞内与鸡痘病毒基因组发生同源重组，产生表达鸡IBVS1蛋白的重组鸡痘病毒rFPV-IBVS1。在含有X-gal的营养琼脂培养基上进行蓝斑筛选且进一步纯化14代。S1基因的PCR检测表明，获得的含传染性支气管炎病毒S1基因的重组鸡痘病毒能够稳定遗传，间接免疫荧光和Western blot等试验证实该重组病毒在CEF内真实地表达了分子量约为90Ku的具有免疫学活性的IBV S1糖蛋白。     

河南不同临床型鸡支气管炎病毒流行毒株的分离鉴定及其S1基因变异分析

经鸡胚接种、RT-PCR、气管环培养等方法从河南地区发病鸡群中分离、鉴定出2株鸡传染性支气管炎病毒(Infectious bronchitic virus,IBV),分别命名为HN/HL株和HN/SG株。应用RT-PCR方法对尿囊液中HN/HL株和HN/SG株以及本室保存的H120株的S1基因全序列进行了扩增,并对其进行克隆测序。结果显示,3株IBV目的片段全长分别为1828、1825、1819bp,3个序列相互之间存在多位点的变异,同时存在插入和缺失现象;对推导的氨基酸序列进行分析表明,HN/HL株S蛋白裂解识别位点序列为HRRRR,而HN/SG株和H120株S蛋白裂解识别位点同为RRFRR。将测序结果同GenBank上登录的其他IBVS1基因相比较,发现本试验的HN/HL株与疫苗H120株的同源性仅为78.1%,HN/SG株与H120株的同源性仅为81.1%,而HN/HL株与HN/SG株的同源性为87.9%。同源性及遗传进化分析还发现,已报道的腺胃型ZJ971株和QX株在分子水平上应该分别属于呼吸型和肾型IBV。     

Preparing hemagglutinating antigen from isolates of infectious bronchitis virus

The hemagglutinating (HA) activity of 14 strains of infectious bronchitis virus (IBV) was investigated. The optimal conditions for IBV antigen preparation include inoculation of 10- or 11-day-old specific pathogen-free embryonated eggs and incubation for 30 hours at 37 C. Embryos were inoculated via the allantoic cavity with 0.1 ml of a low embryonic passage of the virus (10(7) to 10(8) EID50/ml). Allantoic fluid was harvested and pooled, and a 100-fold concentration of virus particles was achieved by centrifugation for 3 hours at 30,000 x g. Virus pellets were resuspended in Tris-hydrochloride buffer containing 3 units of phospholipase-C (type-1) enzyme/ml and incubated for 2 hours at 37 C. All IBV strains tested demonstrated positive HA activity with chicken red blood cells. The antigen was stored in liquid state or lyophilized at 4 C.     

Pathogenicity of infectious bronchitis virus isolates from Ontario chickens

Infectious bronchitis (IB) is one of the important viral diseases of chickens, and in spite of regular vaccination, IB is a continuous problem in Canadian poultry operations. In an earlier study using sentinel chickens we determined the incidence of infectious bronchitis virus (IBV) in Ontario commercial layer flocks. The objective of this study was to determine the pathogenicity of 5 nonvaccine-related IBV isolates recovered from the sentinel birds. The clinical signs, gross, and histological lesions in specific pathogen-free chickens indicated that all 5 isolates caused mild lesions in the respiratory tract. An important finding of this study was the significantly lower average daily weight gain among virus-inoculated groups of chickens during the acute phase of infection. Based on sequences of part of the S1 gene IBV-ON2, IBV-ON3, and IBV-ON5 formed a cluster and they were closely related to strain CU-82792. IBV-ON4 had 98.7% identity with the strain PA/1220/9, a nephropathogenic variant.     

传染性支气管炎病毒TY1株结构蛋白基因部分片段的表达

根据已报道的传染性支气管炎病毒S基因序列设计了1对引物，利用从传染性支气管炎病毒TY1株中提取的RNA，经PCR扩增获得了300bp的产物；序列测定与分析表明，所扩增产物是传染性支气管炎病毒结构蛋白基因的部分片段。将扩增片段插入pET32a构建表达载体，并于大肠埃希氏菌BL21中进行表达；结果表明，该片段在大肠埃希氏菌中成功表达，产物为30ku、以可溶性形式存在的蛋白。Western—blotting分析表明，该蛋白可与传染性支气管炎病毒TY1株阳性血清发生反应。     

禽传染性支气管炎病毒S1基因在毕赤酵母中的表达

根据GenBank已公布的传染性支气管炎病毒(Infectious bronchitis virus,IBV)株S1基因序列及pPIC9K表达载体序列,设计1对IBV S1基因表达片段的PCR引物,用RT-PCR方法扩增出长度为1 566 bp IBV S1基因表达片段,5′端不含信号肽序列,3′端添加了终止密码子。用限制性内切酶SnaB和Not将S1基因和载体pPIC9K酶切回收后连接,构建了重组表达载体pPIC9K-S1。用限制性内切酶Bgl将表达质粒pPIC9K-S1线性化,然后用电转化的方法导入毕赤酵母GS115,在MD平板上生长的转化子经过PCR鉴定和表型筛选后,获得了整合型阳性重组菌株GS115/pPIC9K-S1 His Muts。将重组菌株在1%甲醇中进行诱导分泌表达,并对表达产物进行SDS-PAGE、Western blot分析。结果显示,IBV S1基因在毕赤酵母中成功获得了表达,表达蛋白的分子量约为76 000,能与IBV阳性血清特异性结合,表达的蛋白占上清中总蛋白量的12.5%。     

鸡传染性支气管炎病毒广西分离株血清型的分析

应用病毒感染的鸡胚材料免疫新西兰兔的方法制备抗鸡传染性支气管炎病毒(IBV)单因子血清,然后在鸡胚气管环培养(Tracheal organ cultures,TOC)上对广西分离的7个IBV代表性毒株和3个常用疫苗株进行交叉病毒中和试验。结果显示,10个毒株被分为6个血清型。根据试验所得的R值,应用聚类分析法分析了各血清型毒株之间的亲缘关系,显示目前在广西流行的IBV野毒株之间以及其与疫苗株间的抗原性存在很大程度的差异,分属不同的血清型。同时还对IBV基因分型和血清分型之间的关系进行了探讨。     

鸡传染性支气管炎病毒东北分离株S1基因的序列分析

鸡传染性支气管炎(IB)是由冠状病毒科冠状病毒属的传染性支气管炎病毒(AIBV)引起的一种急性高度接触性传染性疾病.其呈世界性流行,我国为高发区.该病毒基因因点突变和重组易发生变异,故该病毒血清型较多.     

鸡传染性支气管炎病毒分离鉴定及S1基因遗传演化分析

为调查辽宁地区鸡传染性支气管炎病毒(IBV)的流行及遗传演化情况,从辽宁某鸡场疑似鸡传染性支气管炎病料中分离得到1株病毒,经分子生物学检测、鸡胚矮小化试验、新城疫病毒血凝特性干扰试验和动物回归试验,确定该毒株为IBV,并命名为CH/LN/2019.扩增该毒株S1基因并进行序列分析发现,分离株S1基因全长1 620 nt...     

鸡传染性支气管炎病毒S1和N基因遗传变异的相关性

对国内1992—2005年分离的IBV毒株的纤突蛋白(S1)和核衣壳蛋白(N)基因分别进行克隆测序,结合在GenBank中发表的IBV S1和N基因的序列,对其不同毒株的S1或N基因片段和全长、S1和N基因全长分别进行遗传变异的研究,利用统计学软件SPSS11.5进行同源性相关分析。结果表明:不同IBV毒株N或S1基因片段与其全长之间遗传变异高度相关,核苷酸r≥0.892,氨基酸0.854≤r≤0.968;但S1与N基因全长之间核苷酸的遗传变异相关性相对较低一些,而且有些毒株间S1基因同源性很低(r=0.645),但N基因同源性仍很高,显示每个基因变异的独立性。国内IBV疫苗株之间S1和N基因核苷酸高度同源(S1≥97.3%;N≥90.7%),而野毒株与疫苗株相比S1和N基因同源性均较低(S1≤84.3%;N≤87.1%),这也可能与常规疫苗对IBV野毒株不能有效保护有关。     

S1 and N gene analysis of avian infectious bronchitis viruses in Taiwan

The disease caused by infectious bronchitis virus (IBV) produces great economic for the poultry industry. The purpose of this study is to investigate the molecular epidemiology of IBV in Taiwan. An old IBV strain isolated in 1964 and another 31 strains isolated from 1991 to 2003 were selected for N-terminal S1 gene analysis. Based on their phylogenetic tree, 13 strains were selected for sequencing the entire S1 and partial nucleocapsid (N) genes. The results indicated that Taiwanese IBV strains could be divided into two distinct lineages, Taiwan Group I and Taiwan Group II, with one Massachusetts strain and one Chinese strain. No recombination was found between H120 and the Taiwanese strains in the S1 gene. However, the S1 gene showed a noticeably higher divergence than the N gene. The phylogenetic trees constructed from the S1 and N genes indicate that intergenic recombination has occurred. Since most local strains are in Taiwanese clusters, developing vaccines from local strains is necessary for IBV control in Taiwan.     

4株IBV中国地方分离株部分结构基因变异的研究

本研究通过RT-PCR分别获得了4个国内IBV分离株的S1、M和N基因，并进行了克隆及测序。序列分析结果表明：HaN2-95株的S1、M和N基因核苷酸序列均与IBV H120疫苗株的同源性最高；尽管HaN1-95株的S1基因与IBV H52疫苗株的亲缘关系最近，但是该毒株的M基因和N基因却与IBV Gray株的同源性最高；GX1-98株的S1和M基因均与IBV H52疫苗株的亲缘关系最近，但其N基因却与IBV Gray株和Ark99株有高度的同源性；GX2-98株的S1基因却与IBV Holte株的亲缘关系最近。上述结果提示国内有些IBV分离株的出现可能与疫苗株的使用有关。     

Isolation and identification of four infectious bronchitis virus strains in China and analyses of their S1 glycoprotein gene

Between 2003 and 2005, four strains of infectious bronchitis virus (IBV) were isolated from the vaccinated chicken flocks in China. The results from chicken embryo cross-neutralization assays showed that all the four isolates were relative to strain A2 of IBV, which was isolated in 1996 in Beijing and related to strain 4/91. The S1 gene of the spike protein was amplified and sequenced. The nucleotide and amino acid sequence of the S1 gene had a similar degree of identity (88.98-99.28%) among the four Chinese IBV isolates. The identity of the S1 protein gene between the four Chinese IBV isolates and 14 strains of other IBVs varied from 70.06 to 81.59%. Phylogenetic analysis suggested that there are at least four groups of IBVs circulating in China and the disease outbreaks might have been caused by infection of multiple strains of IBV.