3株H3N2亚型禽流感病毒的基因组特征与演化分析

doi:10.11843/j.issn.0366-6964.2022.11.038

摘要/Abstract

摘要： 旨在了解浙江地区家禽H3N2亚型禽流感病毒（AIV）的流行变异情况，采用RT-PCR技术对2021年浙江923份样品进行检测，对AIV分离株进行分子特征及遗传演化分析。结果表明，AIV样品阳性率为7.69%（71/923）；共分离到2株鸡源和1株鸭源H3N2亚型AIVs，其HA和NA基因相似性分别为93.4%～100%和94.0%～99.9%，分离株内部基因片段来源复杂，与H1N2、H1N4、H10N7等亚型亲缘关系密切；遗传进化分析显示，H3N2亚型AIV主要流行于华东地区，鸭是其主要宿主，3株H3N2亚型分离株 HA和NA基因均属于禽源进化分支；分离株HA蛋白裂解位点均为PEKQTR↓GLF，符合低致病性禽流感病毒特征，HA蛋白与受体结合相关位点为226Q和228G，PB2蛋白与哺乳动物适应性相关的氨基酸位点为627E，均不同于人流感病毒对应蛋白的相关位点（226L、228S和627K），推测其跨种传播至人的潜力较低；分离株PB1蛋白的66位氨基酸突变为S，提示其对哺乳动物的致病性可能增强。综上所述，本研究分离的H3N2亚型AIV符合低致病性禽流感病毒特征，基因片段来源复杂，跨种传播至人的潜力较低，但是否影响对宿主的致病性仍需进一步探究。

关键词: H3N2亚型禽流感病毒, 浙江地区, 遗传进化, 氨基酸变异

Abstract: To understand the prevalence and variation of the H3N2 subtype avian influenza virus (AIV) in poultry in Zhejiang province, RT-PCR technology was utilized to detect a total of 923 samples from Zhejiang province in 2021, the isolated AIVs were subjected to molecular characteristics and genetic evolution analysis. The results showed that the positive rate of AIV in Zhejiang province in 2021 was 7.69% (71/923), 2 chicken-derived AIVs and 1 duck-derived H3N2 subtype AIVs were isolated, and their HA and NA genes homology were 93.4%-100% and 94.0%-99.9%, respectively. The origins of the internal gene fragments of the isolated AIVs were complex, and they were closely related to subtypes such as H1N2, H1N4, and H10N7. The genetic evolution analysis showed that H3N2 subtype AIV was mainly prevalent in East China, and the duck was its main host. The HA and NA genes of the three H3N2 subtype isolates belonged to avian-derived evolutionary branches. Amino acid analysis revealed that the cleavage site of HA protein was PEKQTR↓GLF, which was in line with the characteristics of the low pathogenic AIV. The 226Q and 228G of HA protein as well as the 627E of PB2 protein, which was related to receptor binding and mammalian adaptability respectively, were consistent with those of AIV, suggesting that the cross-species transmission to mammals appeared unlikely. However, amino acid 66 was mutated to S in the PB1 protein might increase the pathogenicity in mammals and need to be further investigated. In summary, the H3N2 subtype AIVs isolated in this study were consistent with the characteristics of low pathogenic avian influenza virus, and the origins of the gene fragments were complex. The potential of the isolates for cross-species transmission to humans was low, but whether they affect the pathogenicity of the host remains to be further explored in the near future.

Key words: H3N2-subtype avian influenza virus, Zhejiang province, genetic evolution, amino acid variation

中图分类号:

S852.659.5

崔明仙, 王星博, 黄彦铭, 卞希一, 冯梦珂, 颜焰, 董伟仁, 周继勇. 3株H3N2亚型禽流感病毒的基因组特征与演化分析[J]. 畜牧兽医学报, 2022, 53(11): 4116-4122.

CUI Mingxian, WANG Xingbo, HUANG Yanming, BIAN Xiyi, FENG Mengke, YAN Yan, DONG Weiren, ZHOU Jiyong. Genetic Characterization and Evolution of Three Strains of H3N2 Avian Influenza Viruses[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(11): 4116-4122.

参考文献 25

[1]	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)
[2]	赵国, 赵坤坤, 鹿欣伦, 等.华东地区家禽低致病性禽流感的病原学检测与带毒情况的分析[J].畜牧兽医学报, 2010, 41(9):1133-1137.ZHAO G, ZHAO K K, LU X L, et al.Infection of low pathogenic avian influenza in poultry in eastern China[J].Acta Veterinaria et Zootechnica Sinica, 2010, 41(9):1133-1137.(in Chinese)
[3]	PENG Y, XIE Z X, LIU J B, et al.Visual detection of H3 subtype avian influenza viruses by reverse transcription loop-mediated isothermal amplification assay[J].Virol J, 2011, 8:337.
[4]	LIU M, HE S Q, WALKER D, et al.The influenza virus gene pool in a poultry market in South Central China[J].Virology, 2003, 305(2):267-275.
[5]	SONG M S, OH T K, MOON H J, et al.Ecology of H3 avian influenza viruses in Korea and assessment of their pathogenic potentials[J].J Gen Virol, 2008, 89(4):949-957.
[6]	谭小艳, 顾大勇, 何建安, 等.2016-2019年深圳口岸入境人群感染甲型流感的流行病学特征分析[J].中国人兽共患病学报, 2020, 36(11):922-927.TAN X Y, GU D Y, HE J A, et al.Epidemiological characteristics of influenza A infection among the inbound population at Shenzhen ports, 2016-2019[J].Chinese Journal of Zoonoses, 2020, 36 (11):922-927.(in Chinese)
[7]	赵婉宸, 李扬, 才天宇, 等.2019-2021年华东地区6株禽源H3亚型流感病毒的遗传进化分析[J/OL].中国动物传染病学报, 2022.[2022-03-05].https://doi.org/10.19958/j.cnki.cn31-2031/s.20220302.005.ZHAO W C, LI Y, CAI T Y, et al.Genetic evolution analysis of six avian H3 subtype influenza viruses in Eastern China from 2019 to 2021[J/OL].Chinese Journal of Animal Infectious Diseases, 2022.[2022-03-05].https://doi.org/10.19958/j.cnki.cn31-2031/s.20220302.005.(in Chinese)
[8]	刘婷婷, 谢芝勋, 宋德贵, 等.11株H3N2亚型禽流感病毒广西分离株全基因序列测定与分析[J].中国兽医学报, 2017, 37(5):859-865.LIU T T, XIE Z X, SONG D G, et al.Whole genome sequencing and analysis of eleven strains of avian influenza virus subtype H3N2 isolated in Guangxi province[J].Chinese Journal of Veterinary Science, 2017, 37(5):859-865.(in Chinese)
[9]	仇保丰, 刘武杰, 胡顺林, 等.混合感染的多种亚型禽流感病毒的纯化与鉴定[J].微生物学报, 2010, 50(1):107-112.CHOU B F, LIU W J, HU S L, et al.Purification and identification of avian influenza viruses causing mixed multi-infection[J].Acta Microbiologica Sinica, 2010, 50(1):107-112.(in Chinese)
[10]	张锐.禽流感病毒的流行株序列分析及PA单克隆抗体的制备[D].杭州:浙江大学, 2020.ZHANG R.Sequence analysis of avian influenza virus epidemic strains and preparation of a monoclonal antibody against PA protein[D].Hangzhou:Zhejiang University, 2020.(in Chinese)
[11]	LI H, DURBIN R.Fast and accurate short read alignment with Burrows-Wheeler transform[J].Bioinformatics, 2009, 25(14):1754-1760.
[12]	LI H, HANDSAKER B, WYSOKER A, et al.The sequence alignment/map format and SAMtools[J].Bioinformatics, 2009, 25(16):2078-2079.
[13]	STAMATAKIS A.RAxML version 8:a tool for phylogenetic analysis and post-analysis of large phylogenies[J].Bioinformatics, 2014, 30(9):1312-1313.
[14]	LETUNIC I, BORK P.Interactive tree of life (iTOL) v3:an online tool for the display and annotation of phylogenetic and other trees[J].Nucleic Acids Res, 2016, 44(W1):W242-W245.
[15]	何世成, 彭志, 王卫国, 等.2011-2015年环洞庭湖区H3亚型禽流感病毒的分离鉴定与遗传演化[J].畜牧兽医学报, 2019, 50(2):382-389.HE S C, PENG Z, WANG W G, et al.Isolation, identification and genetic evolution of H3 subtype avian influenza virus in Dongting lake region from 2011 to 2015[J].Acta Veterinaria et Zootechnica Sinica, 2019, 50(2):382-389.(in Chinese)
[16]	CHEN X Y, GUO F C, PAN J B, et al.Rare homologous recombination in H3N2 avian influenza A viruses[J].J Infect, 2020, 80(3):350-371.
[17]	LIN Y P, XIONG X L, WHARTON S A, et al.Evolution of the receptor binding properties of the influenza A(H3N2) hemagglutinin[J].Proc Natl Acad Sci U S A, 2012, 109(52):21474-21479.
[18]	SHINYA K, HAMM S, HATTA M, et al.PB2 amino acid at position 627 affects replicative efficiency, but not cell tropism, of Hong Kong H5 N1 influenza A viruses in mice[J].Virology, 2004, 320(2):258-266.
[19]	CONENELLO G M, ZAMARIN D, PERRONE L A, et al.A single mutation in the PB1-F2 of H5N1 (HK/97) and 1918 influenza A viruses contributes to increased virulence[J].PLoS Pathog, 2007, 3 (10):e141.
[20]	KATZ J M, LU X H, TUMPEY T M, et al.Molecular correlates of influenza A H5N1 virus pathogenesis in mice[J].J Virol, 2000, 74(22):10807-10810.
[21]	SONG M S, PASCUA P N Q, LEE J H, et al.The polymerase acidic protein gene of influenza a virus contributes to pathogenicity in a mouse model[J].J Virol, 2009, 83(23):12325-12335.
[22]	FAN S F, DENG G H, SONG J S, et al.Two amino acid residues in the matrix protein M1 contribute to the virulence difference of H5N1 avian influenza viruses in mice[J].Virology, 2009, 384(1):28-32.
[23]	TATE M D, JOB E R, DENG Y M, et al.Playing hide and seek:how glycosylation of the influenza virus hemagglutinin can modulate the immune response to infection[J].Viruses, 2014, 6 (3):1294-1316.
[24]	BROWN L E, FFRENCH R A, GAWLER J M, et al.Distinct epitopes recognized by I-Ad-restricted T-cell clones within antigenic site E on influenza virus hemagglutinin[J].J Virol, 1988, 62(1):305-312.
[25]	WU H B, WU N P, PENG X R, et al.Molecular characterization and phylogenetic analysis of H3 subtype avian influenza viruses isolated from domestic ducks in Zhejiang Province in China[J].Virus Genes, 2014, 49(1):80-88.