超强毒IBDV细胞克隆化毒回归鸡后的致病性与VP_2基因高变区的分子变化

Pathogenicity and the Sequence Analysis of High Variable Region of VP_2 Gene of the Very Virulent Infectious Bursal Disease Virus During the Process of Inoculating the Cell-cloned Strains Back to SPF Chickens

为了研究超强毒鸡传染性法氏囊病病毒(vvIBDV)的致病性及其VP2基因高变区的分子变化,以vvIBDVGX8/99株囊毒为研究对象,将该毒在SPF鸡胚连传10代后,再在鸡胚成纤维细胞(CEF)上连续盲传。该病毒在CEF上传至22代时开始引发CEF细胞病变,随之在96孔细胞培养板上用无限稀释法连续克隆2次后获得了4个病毒克隆,再将该4个病毒克隆分别连续回传3～5周龄SPF鸡10代。分别比较4个克隆毒及其回传SPF鸡后不同传代毒,对4～6周龄SPF鸡的致病性及VP2高变区氨基酸分子的变化,结果表明,4个克隆化毒的细胞培养毒对SPF鸡只有0～6 7%的致死率,不同克隆毒的SPF鸡传代毒对SPF鸡的致病性却都逐渐增强,但程度差异很大,其中克隆#5在回鸡1、5和10代后的致死率从0分别增加到10%、20%和27%;克隆#4从6 7%增加至13%、17%和23%;但克隆#1和#3的致病性变化相对较小。相对于原始囊毒及鸡胚毒,4个克隆化毒在测序的VP2高变区的约145个氨基酸中,有10个位点发生了相同的变异,变得与适应细胞的疫苗毒D78株基本一致,在回鸡传代导致对鸡毒力增强的过程中,这10个位点中大多数氨基酸不再变化,只有第253位和256位氨基酸从囊毒的Q和I变为细胞适应毒的H和V后,有些病毒克隆回鸡至第10代时又变为原囊毒的Q和I。

In order to study the pathogenicity and the sequence analysis of high variable region of VP_2 gene of the very virulent infectious bursal disease virus(vvIBDV),we extracted RNA from different passages of the initial bursa virus,chicken embryo-adapted virus,CEF cell-adapted virus,cell-cloned virus,and SPF-chicken-passaged virus separately.After reverse transcription polymerase chain reaction(RT-PCR),gene cloning,nucleotide sequence examination and analysis,we grasped the sequence of VP_2 region during the passage process and deduced the evolution rules of amino acid sequence.Compared with the initial bursa virus and the embryo virus,10 sites in 145 amino acids of high variable VP_2 region from four cell-adapted viruses changed the same,identical to that of the vaccine D78 virus strain.In the process of inoculation back the virus to chickens,the virulence increased,but the amino acids in the 10 sites no longer changed.Only the 253th and the 256th amino acids changed from Q and I of the initial bursa virus to H and V of the cell-adapted virus.The 253th and the 256th amino acids of some strains of cell-cloned virus changed back to Q and I when the virus was inoculated back to chikens at the 10th generation.The results suggested that the variation of VP_2 region did not associate with pathogenicity,but had something to do with cell culture or tissue appetency.The research is of high significance.It establishes multiple clones from cell-adapted virus and a series of corresponding continuous chicken-passaged virus strains,which offers a new idea and necessary materials for research on other gene's variance and pathogenicity and also other biological characteristics of the virus.