超强毒IBDV GX8/99株不同传代毒致病性与VP5基因的关系分析  

A relationship between pathogenicity of different passage virus of vvIBDV GX8/99 strain and VP5 gene

作　　者：赵昌亮[1] 朱瑞良[1] 王尊民[1] 王军委[1] 赵庆友[1] 杨世发[1] 左雪梅[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2012年第8期1121-1125,共5页Chinese Journal of Veterinary Science

基　　金：山东省科技攻关资助项目(2008GG10009023);农业部公益性行业科研专项资助项目(#200803019)

摘　　要：将vvIBDV GX8/99株原代毒、鸡胚毒、克隆化毒、回传SPF鸡10代次毒及克隆化细胞传20代次毒分别测定ELD50,以相同的ELD50病毒量分别接种SPF鸡,从而观察vvIBDV GX8/99株在传代过程中致病性的变化规律。同时分别提取病毒RNA,通过RT-PCR、PCR扩增、基因克隆、核苷酸序列测定和分析,对IBDV GX8/99株的24株不同传代毒的VP5基因进行比较,发现其同源性在94%～100%,并且有15个易发生变异的位点,其中位点bp#2、#8、#52、#145、#232、#272、#310、#364、#385、#409的碱基变异引起了相应氨基酸的改变,而位点bp#18、#285、#331、#354、#397的碱基变异没有引起相应氨基酸的变化。通过比较分析,可以看到在致死率由原代毒的86.7%降为GXE10的43.0%时VP5基因的核苷酸有4个位点发生了变异,致死率由GXE10的43.0%降为GXC23的3.3%时VP5基因的核苷酸有10个位点发生了变异;而将GXC23克隆化后的4株毒株致病性变化不大,并且仅在GXCL1-1的#8位点,GXCL2-1和GXCL4-1的#364位点发生变异;将4株克隆化毒株回鸡传10代后的致死率有一定程度的回升,且有2个位点发生变异;4株克隆化毒株在细胞上连续传20代后其致死率都降为0.0%,并且克隆株5没有发生核苷酸变异而克隆株1,2,4也仅有1个核苷酸位点发生变异。由此推论vvIBDVGX 8/99株的致病性与VP5基因某些氨基酸的变异有一定的关系,更可能与病毒对细胞的亲嗜性关系密切;这为探明vvIBDV毒力改变的分子基础,从而解释自然界中vvIBDV出现和致弱的原因提供了佐证。ELD50 of the original virus（chicken virus）, embryo virus, virus of adapted to cells, cloned virus in cells, the 10th passage virus returned SPF chickens（chicken virus） ,and the 20th passage virus of cloned cells of vvIBDV GX8/ 99 strains were detected. By inoculating SPF chicken with virus led the same ELDs0 ,we observed pathogenic changes of vvIBDV GX8/99 strains during the course of passage. RNA was extracted from the certain passage strains of the above virus. By RT PCR,PCR amplification, gene cloning, nucleotide sequence analysis, we compared the VP5 gene of the virus IBDV of the 32 different passages,and we obtained their homology were between 94 % and 100 %. How- ever,there are 15 sites prone to mutation,and the base variation in the sites bp 2,8,52,145,232,272,310,364,385 and 409 caused the changes of corresponding amino acid,but the base variation in the site bp 18,285,331,354 and 397 did not cause changes of corresponding amino acid. By comparison, when the death rate reduced from 86.7% of the original virus to 43.0% of GXE10, four sites of the VP5 nucleotide mutated, and when the death rate reduced from 43.0% of GXC23 to 3.3% of GXE10,10 sites of the VP5 nucleotide mutated. But the pathogenicity have little change in the four strains of the cloned GXC23,and only the sites bp 8 of GXCI.1 1,364 of GXCL2- 1 and GXCL4 1 mutated;the mortality have some degree of recovery after the four strains of cloned virus returned SPF chickens passage 10 generations,and there were two sites mutated;in four cloned virus strains in the cells after 20 generations of continuous passage,its death rate dropped to 0,and cloned 5 strains did not change nucleotide, and cloned 1,2,4 strains has only one nucleotide site mutated. Therefore,we can ascertain that the majority variation of amino acid of VP5 may have a certain relationship with the pathogenicity of the virus, especially with the cell culture and tissue affinity.

关 键 词：VVIBDV 细胞适应毒 传代毒 致病性 VP5基因 

分 类 号：S852.65[农业科学—基础兽医学]