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该研究分析了监测发现的H10N3亚型禽流感病毒的遗传特征，并与人源H10亚型禽流感病毒进行比较，分析禽源与人源H10病毒的遗传相关性。2020年6月至2022年6月在浙江某地采集外表健康家禽的喉头与泄殖腔拭子，接种鸡胚后取尿囊液进行血凝效价和流感病毒荧光定量PCR检测，对流感病毒核酸阳性的样品进行全基因测序和遗传变异分析。在采集的1 462份样本中，共分离到14株H10N3禽流感病毒。HA和NA基因分析显示，分离毒株与人源H10N3毒株位于同一进化分支，相似性分别在95.1%～98.5%和95.2%～99.4%；分离毒株的HA基因与人源H10N8毒株的相似性为91.8%～93.2%；在分离鉴定的禽源H10N3毒株中，A/Dk/ZJ/4-23HZBX0004-O/2021毒株的HA和NA基因与人源H10N3毒株相似性最高。内部基因分析显示，人源H10N3和H10N8毒株的内部基因均源自H9N2禽流感病毒，而分离毒株的6个内部基因片段来自H9N2或其他亚型低致病性禽流感病毒。基因位点分析显示，13株H10N3病毒HA基因均为禽源受体（α-2,3-SA）结合特征（226Q和228G）；发现1株H10N3(A/Dk/ZJ/4-23HZBX0004-O/2021)与人源H10N3毒株的226和228位具有相同的氨基酸残基Q和S，其中228S可能会促进病毒与人源受体（α-2,6-SA）的结合。同时，分离毒株与人源毒株的多个基因存在哺乳动物适应性突变，如HA蛋白S138A、Q220R和S212N突变，可能影响病毒的受体结合能力；PA蛋白K356R、S409N突变，PB1蛋白I368V、S375N突变，可能会增强病毒在哺乳动物细胞中的聚合酶活性或对小鼠等哺乳动物的致病力；此外，M2携带S31N突变，提示分离毒株对金刚烷胺类药物具有耐药性。本研究分离鉴定的H10N3禽流感病毒与人源H10N3毒株的HA和NA基因具有一定遗传相关性，但病毒内部基因片段来源有明显差异。提示：H10N3病毒在家禽中经过基因重配和变异后，突破种间屏障进而造成人类的感染。虽然本研究鉴定的禽源H10N3病毒和人源毒株具有一定遗传差异，但禽源毒株携带人源受体结合能力及哺乳动物适应性增强的基因位点突变，进一步说明了H10N3禽流感病毒的公共卫生风险，需要加强关注。
Abstract：Objective to study the genetic characteristics of H10N3 subtype avian influenza virus(AIV), and analyze the genetic relationship between avian and human H10 viruses. From June 2020 to June 2022, throat and cloacal swabs were collected from apparently healthy poultry in Zhejiang Province,China. The samples were inoculated into specific pathogen-free(SPF) chicken embryos for virus isolation. The allantoic fluid was collected for influenza virus testing using hemagglutination assay and quantitative reverse transcription-PCR(RT-qPCR). The AIV positive samples were used for whole genome sequencing and genetic variation analysis.In the 1,462 samples, a total of 14 H10N3 AIV strains were isolated. Phylogenic analysis of the HA and NA genes showed that these AIV isolates and the human-derived H10N3 strain were located in the same evolutionary branch, with nucleotide identities ranging from 95.1%-98.5% and 95.2%-99.4%, respectively.The nucleotide identity of HA gene between these isolates and the human H10N8 strain was 91.8%-93.2%.Among the 14 H10N3 isolates, the HA and NA genes of A/Dk/ZJ/4-23HZBX0004-O/2021 shared the highest identities with the human H10N3. All the six internal genes of human H10N3 and H10N8 strains were derived from H9N2 AIVs, while those of the AIV isolates were derived from the H9N2 or other subtypes of low pathogenic avian influenza virus(LPAIV). The HA protein of 13 H10N3 strains was characterized by 226Q and 228G(H3 numbering), indicating that they preferred avian receptor(α-2,3-SA). However, one strain H10N3(A/Dk/ZJ/4-23HZBX0004-O/2021) was found to share the same amino acid residues 226Q and 228S as human H10N3 strain. The mutation G228S might promote the virus bind to human receptor(α-2,6-SA). We further described other key mammalian adapted mutations in the avian isolates and human strains. S138A, Q220R, and S212N were observed in HA protein, which might affect the viral receptor binding properties.K356R and S409N in PA protein, I368V and S375N in PB1 protein were found, indicating an increased polymerase activity in mammalian cells or the viral pathogenicity to mice or other mammals. In addition, the M2protein had S31N mutation, suggesting their drug-resistance to amantadine. The H10N3 AIV isolates exhibited close genetic similarity with human H10N3 strain in HA and NA genes, while their internal genes were derived from different LPAIVs. It indicated that the human H10N3 virus might have been generated through reassortment among different AIVs in poultry and broke through the species barrier to cause human infections to cause human infections. Furthermore, we summarized the molecular variations associated with the increased ability to bind to human receptors and enhanced adaptation to mammalian observed in the avian isolates in this study and the human H10N3 viruses, which suggested that more attention should be paid to the public health risk of H10N3 AIVs.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004369
中图分类号:R373.13
引用信息:
[1]袁悦,田甜,杨静茹,等.14株H10N3亚型禽流感病毒的遗传变异研究[J].病毒学报,2023,39(04):949-961.DOI:10.13242/j.cnki.bingduxuebao.004369.
基金信息:
国家重点研发计划（项目号：2022YFC2601602）,题目：重要跨境野生动物病原传播机制及发生风险; 中美疾控中心合作项目（项目号：5 U01IP001106-04-00）,题目：China-U.S.Collaborative Program on Emerging and Re-emerging Infectious Diseases; 广州国家实验室自立项目（项目号：SRPG22-001）,题目：重要呼吸道病原遗传变异特征和跨种传播机制研究; 中国科学院国际合作局—海教中心（项目号：151C53KYSB20210023）,题目：禽流感病毒的遗传多样性与流行风险预警;中国科学院青年创新促进会（项目号：Y2021034）; 泰山学者青年专家计划项目（项目号：tsqn202211217）~~
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