Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence
Passaging of a Newcastle disease virus pigeon variant in chickens results in selection of viruses with mutations in the polymerase complex enhancing virus replication and virulence
复制标题
DOI：
10.1099/vir.0.026344-0
复制
发表时间：
2011-02-01
影响因子：
3.8
通讯作者：
Peeters, B. P. H.
中科院分区：
文献类型：
--
作者：
Dortmans, J. C. F. M.;Rottier, P. J. M.;Peeters, B. P. H.
从鸽子中分离到的一些新城疫病毒(NDV)变异株(鸽子副粘病毒1型；ppmv-1)对家鸡没有显示出完全的毒力潜力，但一旦在这些动物中传播就可能变得强大。在这项研究中，我们通过在鸡身上传代一种低致病性的ppmv-1分离株，检查了导致这种毒力增强的分子变化。经过1、3和5代病毒的全基因组测序表明，毒力的增加并不伴随着融合蛋白的变化，而是伴随着L和P复制蛋白的突变。融合蛋白是新城疫病毒的一个众所周知的毒力决定因素。这些突变对毒力的影响是通过使用感染性克隆的反向遗传学的方法来证实的。获得三个氨基酸突变，两个在L蛋白中，一个在P蛋白中，通过对1日龄鸡的脑内致病指数试验确定，显著增加了毒力。这些突变增强了病毒在体外和体内的复制，并增加了感染细胞培养单层中的空斑大小。此外，他们增加了病毒复制复合体的活性，这是通过体外微型基因组复制试验确定的。我们的数据表明，ppmv-1在鸡体内的复制会导致聚合酶复合体而不是病毒融合蛋白的突变，并且鸽子副粘病毒的毒力水平与病毒复制复合体的活性直接相关。
Some Newcastle disease virus (NDV) variants isolated from pigeons (pigeon paramyxovirus type 1; PPMV-1) do not show their full virulence potential for domestic chickens but may become virulent upon spread in these animals. In this study we examined the molecular changes responsible for this gain of virulence by passaging a low-pathogenic PPMV-1 isolate in chickens. Complete genome sequencing of virus obtained after 1, 3 and 5 passages showed the increase in virulence was not accompanied by changes in the fusion protein - a well known virulence determinant of NDV - but by mutations in the L and P replication proteins. The effect of these mutations on virulence was confirmed by means of reverse genetics using an infectious cDNA clone. Acquisition of three amino acid mutations, two in the L protein and one in the P protein, significantly increased virulence as determined by intracerebral pathogenicity index tests in day-old chickens. The mutations enhanced virus replication in vitro and in vivo and increased the plaque size in infected cell culture monolayers. Furthermore, they increased the activity of the viral replication complex as determined by an in vitro minigenome replication assay. Our data demonstrate that PPMV-1 replication in chickens results in mutations in the polymerase complex rather than the viral fusion protein, and that the virulence level of pigeon paramyxoviruses is directly related to the activity of the viral replication complex.