山西部分地区IBV的分离鉴定及S1基因进化分析

Isolation and identification of IBV in some areas of Shanxi Province and evolution analysis of S1 gene

为了了解山西省部分地区鸡传染性支气管炎病毒(Infectious bronchitis virus,IBV)的流行情况,试验采集山西部分地区已接种H120疫苗的7份疑似感染IBV的蛋鸡肺脏、气管、肾脏组织并进行了病毒分离,通过RT-PCR、电镜观察、血凝试验、新城疫干扰试验鉴定病毒,并对分离得到的病毒进行SPF雏鸡感染试验、鸡胚交叉中和试验和S1基因序列同源性及遗传进化分析。结果表明:将从7份疑似病料中分离纯化的病毒经尿囊腔接种SPF鸡胚,其中有3株分离毒株可导致明显的侏儒胚,经RT-PCR鉴定为IBV,分别命名为CK/CH/SX/BEI、CK/CH/SX/ZJQ和CK/CH/SX/A。3株分离毒株在电镜下均可观察到直径为80~120 nm、有囊膜、纤突呈放射状排列的病毒粒子,经Ⅰ型磷酸酯酶C处理后均可凝集1%鸡红细胞,均可干扰新城疫病毒(NDV)复制,致使SPF雏鸡发病并诱导产生IBV特异的免疫应答,3株分离毒株与H120疫苗株抗原差异较大,3株分离毒株之间S1基因核苷酸和推导氨基酸序列同源性分别为94.9%~98.0%和93.7%~96.7%,且与H120疫苗株S1基因核苷酸序列和推导氨基酸序列同源性分别为77.4%~78.6%和76.2%~78.0%,3株分离毒株与近几年我国流行的QX型IBV毒株形成了一个密切相关的系统发育分支,但与2008年分离的IBV SX8相比显示出不同程度的变异。说明山西省部分地区流行的IBV S1基因已出现不同程度的变异,应选择适宜的疫苗进行免疫。

In order to understand the prevalence of Infectious bronchitis virus(IBV) in some areas of Shanxi Province, the experiment collected seven tissue samples including lung, trachea, and kidney, from suspected IBV-infected layers that had been vaccinated with H120 vaccine in some areas of Shanxi Province, and conducted the virus isolation. The virus was identified through RT-PCR,electron microscope observation, hemagglutination test, and Newcastle disease interference test, and SPF chick infection test, and chicken embryo cross-neutralization test, were performed on the isolated virus, and S1 gene sequence homology and genetic evolution analysis were carried out. The results showed that the viruses isolated and purified from seven suspected disease materials were inoculated with SPF chicken embryos through the allantoic cavity, and among them, three disease materials caused obvious dwarf embryos, which were identified as IBV by RT-PCR and named CK/CH/SX/BEI,CK/CH/SX/ZJQ and CK/CH/SX/A respectively. All three isolated viruses could be observed under the electron microscope with virus particles of 80-120 nm in diameter, with envelope and radially arranged fiber. After treatment with type I phosphatase C,1% chicken red blood cells could be agglutinated, and the replication of Newcastle disease virus(NDV) could be interfered. The viruses caused SPF chicks to develop disease and induce IBV-specific immune response. In the cross-neutralization test, the antigens of the three isolated viruses and the H120 vaccine strain were quite different. The nucleotide and deduced amino acid sequence homologies of the S1 gene between the three isolated viruses were 94.9%-98.0% and 93.7%-96.7%,respectively;the S1 gene and deduced amino acid homologies between the three isolated viruses and the H120 vaccine strain were 77.4%-78.6% and 76.2%-78.0%,respectively. In the analysis of genetic evolution, the three isolated viruses had formed a closely related phylogenetic branch with the QX virus strains that had been circulating in China in recent years, but they had shown different degrees of variation compared with the IBVSX8 isolated in 2008. The results suggested that the IBV S1 gene that was prevalent in some areas of Shanxi Province had been mutated to varying degrees, and at the same time, appropriate vaccines should be selected for immunization.