Guo Yuan-ji, Wen Le-ying, Wang Min, Li Zi, Zhang Ye, Guo Jun-feng
Department of Influenza, National Institute of Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2004 Mar;18(1):7-11.
To understand the origin of HA and NA genes of swine influenza A (H9N2) viruses isolated from pigs in the mainland of China and on basis of these to reveal the pathogenicity of them in pigs.
The target gene was amplified by PCR, the PCR product was ligated with PGEM-T Easy Vector (Promega company, USA) at 4 degrees, the recombined plasmid was transferred into DH-10-beta bacteria; positive colonies were selected and identified then digested with restriction enzyme. Afterwards,the nucleotide sequence was determined. Finally,phylogenetic analysis of the sequencing data was performed with MegAlign (Version 1.03) and Editseg (Version 3.69) softwares.
Two strains of swine influenza A(H9N2) virus isolated in the mainland had an amino acid residue, leucine (L) at position 226 (H3 numbering) on HA protein molecule found in H9N2 viruses isolated either in pigs or humans previously; the amino acid sequence at HA connecting peptide of isolates possessed R-L-S-R, whereas the other H9N2 viruses with virulence in poultry had R-S-S-R at HA connecting peptide. The two pig H9N2 isolates shared the same three-amino-acids deletion in the NA stalk at 62.64 position found in A/Shaoguan/408/98 and A/Swine/Hong Kong/9/98, as well as A/Duck/Hong Kong /Y280/97(H9N2) viruses. The analysis of the phylogenetic tree indicated that the HA and NA genes of new isolates were closely related to those of A/Chicken/Hong Kong/G23/97 and A/Chicken/Hong Kong/G9/97 and A/Shaoguan/408/98 viruses, respectively.
The HA and NA genes of swine influenza A(H9N2) viruses isolated in the mainland of China probably were derived from those of avian influenza A(H9N2) virus. The occurrence of substitution of amino acid sequence at HA connecting peptide, could result in the H9N2 virus from non pathogenic to pathogenic in pigs. However, avian influenza A(H9N2) virus had deletion in the stalk of the NA that resulted in host range transmission. Therefore they could infect pigs directly.
了解中国大陆猪源甲型流感病毒（H9N2）HA和NA基因的来源，并以此揭示其对猪的致病性。
采用PCR扩增目的基因，PCR产物于4℃与PGEM-T Easy载体（美国Promega公司）连接，重组质粒转入DH-10-β细菌；挑选阳性菌落进行鉴定，然后用限制性内切酶消化。之后，测定核苷酸序列。最后，用MegAlign（1.03版）和Editseg（3.69版）软件对测序数据进行系统发育分析。
中国大陆分离的两株甲型流感病毒（H9N2）在HA蛋白分子226位（H3编号）有一个氨基酸残基亮氨酸（L），该位点在之前猪或人分离的H9N2病毒中也有发现；分离株HA连接肽的氨基酸序列为R-L-S-R，而其他对家禽有致病性的H9N2病毒在HA连接肽处为R-S-S-R。这两株猪H9N2分离株在NA柄部62、64位有相同的三个氨基酸缺失，该缺失在A/韶关/408/98、A/猪/香港/9/98以及A/鸭/香港/Y280/97（H9N2）病毒中也有发现。系统发育树分析表明，新分离株的HA和NA基因分别与A/鸡/香港/G23/97、A/鸡/香港/G9/97和A/韶关/408/98病毒的基因密切相关。
中国大陆分离的猪源甲型流感病毒（H9N2）的HA和NA基因可能来源于甲型禽流感病毒（H9N2）。HA连接肽氨基酸序列的替换可能导致H9N2病毒由无致病性变为对猪有致病性。然而，甲型禽流感病毒（H9N2）的NA柄部有缺失，导致宿主范围传播。因此它们可直接感染猪。
