六株鸽源新城疫病毒的分离、鉴定及生物信息学分析

摘要

新城疫是严重危害世界养禽业的一种禽类传染病。近年来，鸽新城疫的流行越来越严重，引起了养殖户和兽医工作者的广泛关注。本研究在4批发病鸽组织病料中分离到了4株鸽源新城疫强毒株和2株鸽源弱毒株。病毒分离株经鸡胚有限稀释法纯化5次后，新鲜尿囊液用于提取病毒RNA。经RT-PCR扩增、测序得到分离株的F基因和HN基因序列。通过对F基因的遗传进化分析发现，4株鸽源强毒株属于ClassⅡ基因Ⅵb亚型，2株鸽源弱毒株与Class II基因II型疫苗株La Sota高度同源。进一步遗传进化分析结果显示，4株鸽源新城疫病毒强毒株与目前国内流行株属于同一遗传分支，这些毒株与欧洲流行株亲缘关系十分相近，而与中国20世纪90年代鸽源分离株遗传距离稍远，因此推测当前鸽源流行强毒株可能源自欧洲。生物信息学分析显示鸽源毒株HN蛋白345~353位的一个线性抗原表位发生了改变，而血清学实验显示La Sota疫苗株和鸽源毒株的交叉血凝抑制存在明显差异，暗示鸽源毒株已经发生了抗原性变异。因此，使用鸡疫苗株La Sota免疫鸽存在免疫失败的风险，需要研发鸽专用新城疫病毒疫苗用于鸽新城疫的防疫工作。%Newcastle disease is a severe infectious disease in birds, threatening poultry industry worldwide. Pigeons are very susceptible to Newcastle disease virus (NDV) infection, which causes high morbidity and mortality. In this study, six NDV strains were isolated from the pigeon tissue samples, four of which were highly virulent and two were low virulent. These pigeon viruses were cloned 5 times in embroynated SPF chicken eggs via limited dilution method. The fresh allantoic fluids were collected for RNA extraction. The resulting RNA samples were converted to cDNAs followed by PCR amplification and sequencing of F and HN genes. Phylogenetic analysis based on F gene sequences showed that four highly virulent strains belonged to ClassⅡⅥb subgenotype and two low virulent strains were similar to La Sota and belonged to ClassⅡgenotypeⅡ. In addition, four highly virulent strains were more closely related to the strain isolated in Italy a few years ago than those strains obtained in China in 1990’s. Therefore, these virulent pigeon strains currently prevailing in China might originate from Europe. Bioinformatics analysis revealed that a linear epitope consisting of amino acids 345-353 on HN protein of these pigeon strains changed as compared with the classic vaccine strain La Sota. In addition, hemagglutination inhibition revealed a noticeable difference between these pigeon strains and La Sota. The results support that chicken vaccine strains do not effectively protect pigeons from NDV infection thus the vaccines designed for pigeon use are much needed.