H5N1亚型禽流感病毒A/duck/Shandong/093/2004株的全基因克隆及序列分析

应用流感病毒通用引物［4］和H5N1亚型禽流感（Avian influenza virus, AIV）的型特异性引物，成功的扩增出H5N1亚型禽流感病毒A/duck/Shandong/093/2004株（简称A/D/SD/04）的全基因序列（包括5′和3′端的非编码区序列）。A/D/SD/04的基因组核苷酸全序列与18株网上公布的禽流感基因序列进行比较和分析，结果与4株鸭源H5N1的5～7个基因具99%以上的同源性；与14株H5N1有至少一个以上内部基因同源性在95%以上。与H9亚型AIV代表株A/Quail/Hongkong/G1/97（简称G1株）和A/Chicken/Beijing/1/94（简称BJ94）比较，除了非结构基因（Nonstructural gene, NS）与G1株的同源性为95.3%外，其余基因均在36.6%～92.1%之间。说明A/D/SD/04没有H9N2基因的直接整合，是H5N1毒株在自然界的重组株。推导的HA氨基酸序列分析，A/D/SD/04 的血凝素（Heamgglutinin，HA）裂解位点与比较的16株AIV的序列一致，是高致病性禽流感的分子特征（PQRERRRKKR/G），第226位氨基酸是对禽类和哺乳细胞均具有亲嗜性的蛋氨酸（Met）。神经氨酸酶（Neuraminidase, NA）在第48位氨基酸（颈部）后有20个氨基酸的缺失，但非结构蛋白（NS）没有在79～84氨基酸发生缺失。碱性聚合酶2（PB2）的627位氨基酸是亲禽类细胞的谷氨酸（Glu, E）。结合生物学特性和分子特征，A/D/SD/04对小鼠的致病力是由多种因素决定，其可能是一株对鸡高度致病，并逐渐获得对哺乳动物致病能力的中间重组病毒。

Cloning of full-length genes of H5N1 subtype Avian influenza virus strain A/duck/Shandong/093/2004 and analysis of the sequences

The eight full-length genes, including the 5' and 3' ends of H5N1 subtype Avian influenza virus (A/duck/ Shandong/093/2004) were amplified by using the universal primers and H5 specific primers. The method used for the amplification of Avian influenza virus's full-length sequence was more easily and rapidly than that of rapid amplification of cDNA ends assay (RACE). The amplified segments were cloned into the T vector PCR 2.1, respectively. Three to five positive clones of each gene were sequenced and the same two sequencing results of the full-length genes were obtained. The phylogenetic analysis results showed that all the eight segments of the A/duck/Shandong/093/2004 were different from the A/Quail/Hongkong/G1/97 and A/Chicken/Beijing/1/94, but showed highly similarity (99% and above) to that of four H5N1 strains, which were isolated in 2002 in duck. It revealed that this strain was resulted from re-assortment of H5N1 rather than H9N2. The NA sequence of A/D/SD/04 was analyzed and the result demonstrated that there are 20 amino acids missing in 48 - 68 sites, however, there was no residue lost in NS gene in 263 to 277 sites. The motif of HA cleavage site is PQRERRRKKR/G, which is the characteristic of HPAIV. The 226 amino acid residue was Met (M), which can react with both Aalpha-2, 3Gal and SAalpha-2, 6Gal receptor. And the 627 residue of PB2 was Glutamic acid (E). The result mentioned above confirmed that H5N1 subtype AIV has multiple determinants in its virulence. A/D/SD/04 is the mid-strain evolving from HPAIV to a virulent strain of mammal.