H5N1亚型禽流感病毒鸡胚分离株蚀斑克隆及生物学特性比较

建立了H5N1亚型禽流感病毒（AIV）蚀斑克隆方法，用不加中性红的营养琼脂为覆盖层，在倒置显微镜下挑斑，对3株H5N1亚型AIV鸡胚分离毒株进行了蚀斑克隆纯化，从同一鸡胚分离毒株中纯化得到了蚀斑大小和形态均存在显著差异的毒株，共获得13株蚀斑克隆纯化株。研究结果表明，当小牛血清浓度为2%时，加入终浓度50 μg/ml的胰酶可明显促进和刺激蚀斑的形成，蚀斑的直径和PFU/ml均有显著的增加。通过TCID₅₀、EID₅₀和LD₅₀测定发现，来自同一鸡胚分离株的不同蚀斑克隆纯化株中，蚀斑大、毒力强的克隆毒株占优势，而且蚀斑较大的，其毒力也较强。对得到的13株蚀斑克隆毒株的全基因组各片段序列分析表明，蚀斑克隆纯化株在HA蛋白裂解位点附近的氨基酸序列均符合高致病性通报性禽流感病毒的分子特征。

H5N1 Subtype AIV Strains Plaque Clone and the Biological Characteristics Comparison

With the method developed by our research, using agar without neutral red, plaques were picked under an inverted microscope, we plaque-purified 3 strains H5N1 subtype AIV chicken embryo isolates and obtained thirteen plaque-purified strains with marked difference in size and shape. Our research indicate addition of suitable amount of trypsin could facilitate plaque formation with marked increase in plaque diameter and plaque-forming units, its optimal concentration being 50 μg/ml, when the concentration of calf serum is 2%. TCID₅₀, EID₅₀ and LD₅₀ testing were done on embryo isolates and the plaque-purified strains, when It was found in the study that for the size and shape of purified strains which came from the same embryo isolate exist notable difference, the larger the plaque the higher the virulence, and that the virulence of the large plaque strains approached that of the embryo isolates, which indicate that virus particles producing large plaques were dominant in embryo isolates. Analysis of the nucleotide sequences and deduced amino acid sequences of HA genes of the 13 plaque-purified strains revealed that the cleavage site amino acid sequences of the HA protein were all conforming to structures assigned to the molecular characteristics of highly pathogenic notifiable avian influenza virus.