2017—2018年华东地区H9亚型禽流感病毒分离毒株的抗原差异分析
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摘要
为掌握华东地区H9亚型禽流感病毒(avian influenza virus, AIV)的流行情况及变异程度,采集临床疑似病料和活禽交易市场家禽的棉拭子样品,通过鸡胚接种、HA和HI试验及RT-PCR等方法分离鉴定H9 AIV,对其中部分毒株进行测序和序列分析,筛选4个代表毒株免疫SPF鸡制备抗血清,利用交叉血凝抑制试验测定抗原差异性。结果如下:共分离鉴定出62株H9亚型禽流感病毒,分离率为2.02%(62/3 074)。对25株H9亚型禽流感病毒株序列分析发现,分离株属于h9.4.2.5谱系,并进一步细分为A和B亚系。HA基因裂解位点氨基酸为PSRSSR↓G,符合低致病性禽流感病毒的特征。与Y280代表株HA基因的推导氨基酸相比,分离株受体结合位点左侧臂全部变为NGLMGR。在抗原位点92位氨基酸出现R(64%)/K(36%)的变化。交叉血凝抑制试验结果表明分离株间的HI抗体滴度相差2～8 log2,可分为2类抗原型。因此,目前流行的H9亚型AIV发生了抗原变异,至少同时存在两种抗原型。
This experiment was conducted to understand the prevalence and variation of H9 subtype avian influenza virus(AIV) isolated in East China. Diseased samples from clinical suspected cases and swab samples from poultry in live poultry markets were collected and subjected to inoculate SPF chicken embryos. H9 AIVs were identified by HA and HI test. HA genes of partial H9 AIVs were amplified by RT-PCR, and PCR productions were subjected to sequence. Antisera from SPF chickens vaccinated with four selected strains were performed cross-reactivity by HI test to analyze the antigen variation. Results were as follows: 62 isolates of H9 subtype AIV were identified, and the isolation rate was 2.02%(62/3 074). Sequence analysis of 25 H9 subtype AIVs revealed that the HA genes were belonged to the h9.4.2.5 lineage, and further were divided into Group A and Group B. The amino acid at the HA gene cleavage site was PSRSSR↓G, which is consistent with the characteristics of low pathogenic avian influenza virus. Compared with the amino acid of the HA gene of the Y280-like strain, the left arm of the receptor binding site(RBS) was changed to NGLMGR. There was a change of R(64%)/K(36%) at antigenic residue 92. The cross-reactivity of antisera showed that the HI antibody titers were differed by 2-8 log2 between the isolates and the isolates could be categorized to two antigenic phenotypes. Therefore, antigen drift is appeared in the prevalent H9N2 AIVs, and at least two antigenic phenotypes are existed simultaneously.
This experiment was conducted to understand the prevalence and variation of H9 subtype avian influenza virus(AIV) isolated in East China. Diseased samples from clinical suspected cases and swab samples from poultry in live poultry markets were collected and subjected to inoculate SPF chicken embryos. H9 AIVs were identified by HA and HI test. HA genes of partial H9 AIVs were amplified by RT-PCR, and PCR productions were subjected to sequence. Antisera from SPF chickens vaccinated with four selected strains were performed cross-reactivity by HI test to analyze the antigen variation. Results were as follows: 62 isolates of H9 subtype AIV were identified, and the isolation rate was 2.02%(62/3 074). Sequence analysis of 25 H9 subtype AIVs revealed that the HA genes were belonged to the h9.4.2.5 lineage, and further were divided into Group A and Group B. The amino acid at the HA gene cleavage site was PSRSSR↓G, which is consistent with the characteristics of low pathogenic avian influenza virus. Compared with the amino acid of the HA gene of the Y280-like strain, the left arm of the receptor binding site(RBS) was changed to NGLMGR. There was a change of R(64%)/K(36%) at antigenic residue 92. The cross-reactivity of antisera showed that the HI antibody titers were differed by 2-8 log2 between the isolates and the isolates could be categorized to two antigenic phenotypes. Therefore, antigen drift is appeared in the prevalent H9N2 AIVs, and at least two antigenic phenotypes are existed simultaneously.
引文
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[17] 陈伯伦,张泽纪,陈伟斌.禽流感研究:I.鸡A型禽流感病毒的分离与血清学初步鉴定[J].中国兽医杂志,1994,20(10):3-5.CHEN B L,ZHANG Z J,CHEN W B.Isolation and serological identification of chicken Avian influenza A virus[J].Chinese Journal of Veterinary Medicine,1994,20(10):3-5.(in Chinese)
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[19] 黄艳艳,李悦,张琳,等.6株H9N2亚型禽流感病毒的分子演化和抗原变异分析[J].畜牧兽医学报,2018,49(10):2205-2214.HUANG Y Y,LI Y,ZHANG L,et al.Molecular and antigenic analyses of six H9N2-subtype avian influenza viruses isolated from broiler chicken farms of Shandong province in 2017[J].Acta Veterinaria et Zootechnica Sinica,2018,49(10):2205-2214.(in Chinese)
[20] XIA J,CUI J Q,HE X,et al.Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China,2013-2016[J].PLoS One,2017,12(2):e0171564.
[2] SUN Y P,LIU J H.H9N2 influenza virus in China:a cause of concern[J].Protein Cell,2015,6(1):18-25.
[3] UMAR S,YOUNUS M,REHMAN M U,et al.Role of aflatoxin toxicity on transmissibility and pathogenicity of H9N2 avian influenza virus in turkeys[J].Avian Pathol,2015,44(4):305-310.
[4] KANG N,CHEN M,BI F Y,et al.First positive detection of H9 subtype of avian influenza virus nucleic acid in aerosol samples from live poultry markets in Guangxi,South of China[J].Chin Med J,2016,129(11):1371-1373.
[5] WANG M W,FU C X,ZHENG B J.Antibodies against H5 and H9 avian influenza among poultry workers in China[J].N Engl J Med,2009,360(24):2583-2584.
[6] GAO R B,CAO B,HU Y W,et al.Human infection with a novel avian-origin influenza A (H7N9) virus[J].N Engl J Med,2013,368(20):1888-1897.
[7] LI K S,XU K M,PEIRIS J S M,et al.Characterization of H9 subtype influenza viruses from the ducks of southern China:a candidate for the next influenza pandemic in humans?[J].J Virol,2003,77(12):6988-6994.
[8] GUAN Y,SHORTRIDGE K F,KRAUSS S,et al.Molecular characterization of H9N2 influenza viruses:were they the donors of the “internal” genes of H5N1 viruses in Hong Kong?[J].Proc Natl Acad Sci U S A,1999,96(16):9363-9367.
[9] 孟芳,徐怀英,张伟,等.近20年中国部分地区鸡源H9N2亚型禽流感病毒HA基因遗传演化及其变异频率[J].微生物学报,2016,56(1):35-43.MENG F,XU H Y,ZHANG W,et al.Genetic evolution and substitution frequency of avian influenza virus HA gene in chicken H9N2 subtype in China in the last 20 years[J].Acta Microbiologica Sinica,2016,56(1):35-43.(in Chinese)
[10] 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.
[11] JIANG W M,LIU S,HOU G Y,et al.Chinese and global distribution of H9 subtype avian influenza viruses[J].PLoS One,2012,7(12):e52671.
[12] ARCHETTI I,HORSFALL F L.Persistent antigenic variation of influenza a viruses after incomplete neutralization in ovo with heterologous immune serum[J].J Exp Med,1950,92(5):441-462.
[13] LIU Y F,LAI H Z,LI L,et al.Endemic variation of H9N2 avian influenza virus in China[J].Avian Dis,2016,60(4):817-825.
[14] KAVERIN N V,RUDNEVA I A,ILYUSHINA N A,et al.Structural differences among hemagglutinins of influenza A virus subtypes are reflected in their antigenic architecture:analysis of H9 escape mutants[J].J Virol,2004,78(1):240-249.
[15] WAN Z M,YE J Q,XU L L,et al.Antigenic mapping of the hemagglutinin of an H9N2 avian influenza virus reveals novel critical amino acid positions in antigenic sites[J].J Virol,2014,88(7):3898-3901.
[16] ZHU Y B,YANG D,REN Q,et al.Identification and characterization of a novel antigenic epitope in the hemagglutinin of the escape mutants of H9N2 avian influenza viruses[J].Vet Microbiol,2015,178(1-2):144-149.
[17] 陈伯伦,张泽纪,陈伟斌.禽流感研究:I.鸡A型禽流感病毒的分离与血清学初步鉴定[J].中国兽医杂志,1994,20(10):3-5.CHEN B L,ZHANG Z J,CHEN W B.Isolation and serological identification of chicken Avian influenza A virus[J].Chinese Journal of Veterinary Medicine,1994,20(10):3-5.(in Chinese)
[18] ZHANG Y,YIN Y B,BI Y H,et al.Molecular and antigenic characterization of H9N2 avian influenza virus isolates from chicken flocks between 1998 and 2007 in China[J].Vet Microbiol,2012,156(3-4):285-293.
[19] 黄艳艳,李悦,张琳,等.6株H9N2亚型禽流感病毒的分子演化和抗原变异分析[J].畜牧兽医学报,2018,49(10):2205-2214.HUANG Y Y,LI Y,ZHANG L,et al.Molecular and antigenic analyses of six H9N2-subtype avian influenza viruses isolated from broiler chicken farms of Shandong province in 2017[J].Acta Veterinaria et Zootechnica Sinica,2018,49(10):2205-2214.(in Chinese)
[20] XIA J,CUI J Q,HE X,et al.Genetic and antigenic evolution of H9N2 subtype avian influenza virus in domestic chickens in southwestern China,2013-2016[J].PLoS One,2017,12(2):e0171564.
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