一株H9N2亚型猪流感病毒的遗传进化和致病性分析
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摘要
本研究从有流感症状的病猪中分离到一株H9N2亚型猪流感病毒(SIV),命名为A/swine/Jiangsu/1/2015 (SW/JS/1/15)。为探究其遗传特征和生物学特性,本研究采用RT-PCR技术扩增其全部基因节段后测序并进行遗传分析,并研究了其对鸡和豚鼠的致病特性。遗传进化分析显示,分离病毒SW/JS/1/15株是由BJ/94系、DK1系、G1系和F/98系4个分支病毒重组而成,8个基因节段均属于G57基因型。分离株HA蛋白裂解位点为PSRSSR*GL,符合低致病性流感病毒的特征。HA蛋白有9个潜在糖基化位点,其中218位糖基化位点缺失,145位与313位各新增一个糖基化位点。与疫苗株SH/F/98、SD/6/96、GD/SS/94相比,分离病毒HA抗原位点发生了G~(90)E、S~(127)R、S~(145)N、D~(153)G、N~(167)S、A~(168)N、A~(198)T、T~(200)R、N~(201)D、和Q~(235)M (H9 numbering)突变;NA蛋白发生6个氨基酸突变:K~(367)R、K/E~(368)N、D~(369)N、D~(401)E、K~(143)N和T~(434)P。同时NA蛋白颈部缺失aa63～aa65。分离病毒的8个基因节段与2株禽源H9N2病毒的相应基因高度同源,其6个内部基因与两株人源H7N9病毒的内部基因高度同源。致病性试验结果显示分离病毒可以感染鸡和豚鼠,但不能在豚鼠群内水平传播,且可能作为H7N9等新型流感病毒内部基因供体,同时表明猪可以感染禽流感病毒(AIV),且可能是AIV获得感染哺乳动物能力的过渡宿主。本研究为H9N2亚型SIV的致病性以及遗传特征的研究提供科学依据。
In this study, an H9 N2 subtype swine influenza virus(SIV) was isolated from a pig with flu symptoms and named of A/swine/Jiangsu/1/2015(SW/JS/1/15). In order to investigate its genetic characteristics, the 8 gene fragments were amplified by RT-PCR, sequenced and analyzed. The pathogenicity of the isolate was evaluated in chickens and guinea pigs. Genetic analysis results showed that this isolate belonged to G57 genotype and was a quadruple reassortant virus consisting of genes from BJ/94-like lineage, DK1-like lineage, F/98-like lineage and G1-like lineage. The HA cleavage sites of the isolate were PSRSSR*GL, which is the molecular characteristic of low pathogenic avian influenza virus. This isolate contained 9 potential glycosylation sites in HA gene with the deletion of one glycosylation site at position 218, and the addition of two glycosylation sites at position 145 and 313 of HA. G~(90)E、S~(127)R、S~(145)N、D~(153)G、N~(167)S、A~(168)N、A~(198)T、T~(200)R、N~(201)D、Q~(235)M mutations were found in the antigenic sites of HA and K~(367)R、K/E~(368)N、D~(369)N、D~(401)E、K~(143)N and T~(434)P mutations occurred in NA. The deletion of amino acid residues at positions 63-65 was observed in the stalk of NA gene. This isolate shared high homology across all 8 genes with the two avian H9N2 strains, and the 6 internal genes of the isolate were highly homologous to the two human H7N9 strains.Pathogenicity experiments showed that the isolate was low pathogenicity virus and exhibited limited replication in chickens and guinea pigs, and could not horizontally transmit in guinea pigs. The results indicated that the H9N2 virus isolated in pig could easily infected chickens and guinea pigs and shared the internal genes with H7N9 viruses, and demonstrated that pigs could be infected with the H9N2 influenza virus and considered as the potential intermediate host for avian influenza viruses to acquire the ability to infect mammals. The present study provided a scientific basis for exploring the pathogenicity and genetic characteristics of the H9N2 SIV.
In this study, an H9 N2 subtype swine influenza virus(SIV) was isolated from a pig with flu symptoms and named of A/swine/Jiangsu/1/2015(SW/JS/1/15). In order to investigate its genetic characteristics, the 8 gene fragments were amplified by RT-PCR, sequenced and analyzed. The pathogenicity of the isolate was evaluated in chickens and guinea pigs. Genetic analysis results showed that this isolate belonged to G57 genotype and was a quadruple reassortant virus consisting of genes from BJ/94-like lineage, DK1-like lineage, F/98-like lineage and G1-like lineage. The HA cleavage sites of the isolate were PSRSSR*GL, which is the molecular characteristic of low pathogenic avian influenza virus. This isolate contained 9 potential glycosylation sites in HA gene with the deletion of one glycosylation site at position 218, and the addition of two glycosylation sites at position 145 and 313 of HA. G~(90)E、S~(127)R、S~(145)N、D~(153)G、N~(167)S、A~(168)N、A~(198)T、T~(200)R、N~(201)D、Q~(235)M mutations were found in the antigenic sites of HA and K~(367)R、K/E~(368)N、D~(369)N、D~(401)E、K~(143)N and T~(434)P mutations occurred in NA. The deletion of amino acid residues at positions 63-65 was observed in the stalk of NA gene. This isolate shared high homology across all 8 genes with the two avian H9N2 strains, and the 6 internal genes of the isolate were highly homologous to the two human H7N9 strains.Pathogenicity experiments showed that the isolate was low pathogenicity virus and exhibited limited replication in chickens and guinea pigs, and could not horizontally transmit in guinea pigs. The results indicated that the H9N2 virus isolated in pig could easily infected chickens and guinea pigs and shared the internal genes with H7N9 viruses, and demonstrated that pigs could be infected with the H9N2 influenza virus and considered as the potential intermediate host for avian influenza viruses to acquire the ability to infect mammals. The present study provided a scientific basis for exploring the pathogenicity and genetic characteristics of the H9N2 SIV.
引文
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[12] Wan Hong-quan, Sorrell E M, Song Hai-chen, et al. Replication and transmission of H9N2 influenza viruses in ferrets:evaluation of pandemic potential[J]. PLoS One, 2008, 3(8):e2923.
[13] Matrosovich M N, Matrosovich T Y, Gray T, et al. Human and avian influenza viruses target different cell types in cultures of human airway epithelium[J]. PNAS, 2004, 101(13):4620-4624.
[14] Lin Y P, Shaw M, Gregory V, et al. Avian-to-human transmission of H9N2 subtype influenza A viruses:relationship between H9N2 and H5N1 human isolates[J]. PNAS, 2000, 97(17):9654-9658.
[15] Matrosovich M N,Krauss S,Webster R G. H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity[J]. Virology, 2001, 281(2):156-162.
[16] Okamatsu M, Sakoda Y, Kishida N, et al. Antigenic structure of the hemagglutinin of H9N2 influenza viruses[J]. Arch Virol,2008, 153(12):2189-2195.
[17] Peacock T,Reddy K,James J,et al. Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape[J]. Sci Rep, 2016,6:18745.
[18] Kaverin N V, Rudneva I A, Ilyushina N A, et al. Structural dif-ferences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture:analysis of H9 escape mutants[J]. J Virol,2004, 78(1):240.
[19]王新卫,赵军,陈陆,等.H9N2AIVHA蛋白S145N变异毒株的抗原性和免疫原性[J].中国兽医学报,2012,32(02):182-188.
[2] Ma Weng-jun, Kahn R E, Richt J A. The pig as a mixing vessel for influenza viruses:Human and veterinary implications[J]. J Mol Genet Med, 2008, 3(1):158-166.
[3] Cong Yan L, Pu Juan, Liu Qin F, et al. Antigenic and genetic characterization of H9N2 swine influenza viruses in China[J]. J Gen Virol, 2007,(7):2035-2041.
[4]李海燕,于康震,杨焕良,等.中国猪源H5N1和H9N2亚型流感病毒的分离鉴定[J].中国预防兽医学报,2004,26(1):1-6.
[5] Gu Min, Chen Hong-zhi, Li Qun-hui, et al. Enzootic genotype S of H9N2 avian influenza viruses donates internal genes to emerging zoonotic influenza viruses in China[J]. Vet Microbiol,2014, 174(3-4):309-315.
[6] OIE Biological Standards Commission. Manual of diagnostic tests and vaccines for terrestrial animals(Mammals, Birds and Bees). 5th ed[M]. Paris:Office International Des Epizooties,2004.
[7] Hoffmann E,Stech J, Guan Y,et al. Universal primer set for the full-length amplification of all influenza A viruses[J]. Arch Virol, 2001, 146(12):2275-2289.
[8]刘金华,史为民,吴清民,郭玉璞.鸡源H9N2亚型流行性感冒病毒神经氨酸酶基因序列分析[J].病毒学报,2004,(03):237-241.
[9]彭欠欠.H9N2亚型流感病毒HA蛋白糖基化位点变化影响病毒生物学特性的研究[D].扬州:扬州大学,2017.
[10] Pu Juan, Wang Shuo-guo, Yin Yan-bo, et al. Evolution of the H9N2 influenza genotype that facilitated the genesis of the novel H7N9 virus[J]. PNAS USA, 2015, 112(2):548-553.
[11] Dong Guo-ying, Luo Jing, Zhang Hong, et al. Phylogenetic Diversity and Genotypical Complexity of H9N2 Influenza A Viruses Revealed by Genomic Sequence Analysis[J]. PLoS One,2011, 6(2):e17212.
[12] Wan Hong-quan, Sorrell E M, Song Hai-chen, et al. Replication and transmission of H9N2 influenza viruses in ferrets:evaluation of pandemic potential[J]. PLoS One, 2008, 3(8):e2923.
[13] Matrosovich M N, Matrosovich T Y, Gray T, et al. Human and avian influenza viruses target different cell types in cultures of human airway epithelium[J]. PNAS, 2004, 101(13):4620-4624.
[14] Lin Y P, Shaw M, Gregory V, et al. Avian-to-human transmission of H9N2 subtype influenza A viruses:relationship between H9N2 and H5N1 human isolates[J]. PNAS, 2000, 97(17):9654-9658.
[15] Matrosovich M N,Krauss S,Webster R G. H9N2 influenza A viruses from poultry in Asia have human virus-like receptor specificity[J]. Virology, 2001, 281(2):156-162.
[16] Okamatsu M, Sakoda Y, Kishida N, et al. Antigenic structure of the hemagglutinin of H9N2 influenza viruses[J]. Arch Virol,2008, 153(12):2189-2195.
[17] Peacock T,Reddy K,James J,et al. Antigenic mapping of an H9N2 avian influenza virus reveals two discrete antigenic sites and a novel mechanism of immune escape[J]. Sci Rep, 2016,6:18745.
[18] Kaverin N V, Rudneva I A, Ilyushina N A, et al. Structural dif-ferences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture:analysis of H9 escape mutants[J]. J Virol,2004, 78(1):240.
[19]王新卫,赵军,陈陆,等.H9N2AIVHA蛋白S145N变异毒株的抗原性和免疫原性[J].中国兽医学报,2012,32(02):182-188.