IBDV超强毒GX8/99株的细胞适应毒和鸡体传代毒的致病性和VP5基因的比较

Comparisons of pathogenicity and VP5 gene of the very virulent infectious bursal disease virus strain GX8/99 and its cell-adapted virus and chicken backpassaged viruses

传染性法氏囊病病毒（Infectious Bursal Diseas Virus，IBDV）的超强毒株GX8／99经在鸡胚成纤维细胞（CEF）上连续盲传23代后，细胞适应毒株GXC23对鸡的致病性显著减弱，对4～6周龄SPF鸡的致死率从85％～90％减为0～5％。GXC23在96孔细胞培养板上经2次无限稀释法后得到3个克隆病毒。经SPF鸡回传20代后，相应的毒株GX-2820、GX-4820、GX-5820对SPF鸡的致死率仍维持在0～5％。序列比较表明，GX8／99在传代过程中，其细胞适应毒GXC23有8个碱基突变，其中5个碱基突变导致了氨基酸改变。bp#2T→c突变，导致传代毒ORF中的起始编码子后移了4个氨基酸。而3个克隆化病毒回鸡20代后致病性不变，这几个位点也保持不变，且与3个疫苗株完全一致。进一步分析表明，有4个超强毒株和4个强毒株的VP5基因的5个位点与GX8／99原始毒相同，而另3个超强毒及6个强毒株与细胞适应毒GXC23一致。这些结果表明，超强毒GX8／99在细胞传代过程中VP5基因上5个氨基酸的变异主要与适应细胞培养相关而与致病性无关。

A very virulent infectious brusal virus（vvlBDV） strain GX8/99 was passaged blindly in cell cuhrue for 23 passages, pathogenicity of the cell adapted virus GXC23 signifcantly decreased from 85%-90% to 0-5 % of mortality when 4 6 weeks SPF chickens were challenged with them. The GXC23 was cloned twice by unlimited dilutions in 96-well plates. 3 clones of which were backpassaged. The mortality in chickens challeged with 3 baekpassaged viruses GX 2B20,GX-4B20,GX 5B20 was still as low as GXC23 in the range of 0-5%. The sequence comparison of VP5 genes of GX8/99 and GXC23 demonstrated mutations of 8 base sites and 5 of them made the amino acid changes. Especially,the mutation from T to C at bp# 2 of VP5 gene made GXC23 to lose its first ＂ATG＂and 4 aa at the N-ter minal of VP5 protein. The mutations were very similar to 3 vaccine strains. Furthermore, such mutations did not change in the 3 backpassaged viruses after passed for 20 generations in chickens as their pathogenicity was still low as GXC23. However,further comparisons indicated that the bases at the sites of VP5 genes in 4 vvlBDV reference strains and 4 vlBDV reference strains were exactly same as primary GX8/99, while another 3 vvIBDV reference strains and 6 vIBDV strains were same as cell-adapted GXC23. The results suggested that the 5 amino acids mutations of VP5 gene from the primary GX8/99 to its cell-adapted GXC23 and backpassaged viruses may not be associated with pathogenicity but more related to the adaptation processes in cell cultures.