一株人感染高致病性禽流感(H5N1)病毒基因组分子特征分析

Genomic molecular characterization of a highly pathogenic avian influenza A (H5N1) virus

【背景】H5N1禽流感病毒可以感染人类导致重症呼吸道感染,致死率高。【目的】研究我中心确认的一例人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015的可能起源及基因组分子特征。【方法】对病人痰液样本中的H5N1病毒进行全基因组测序,使用CLC Genomics Workbench 9.0对序列进行拼接,使用BLAST和MEGA 5.22软件进行同源性比对和各片段分子特征分析。【结果】该株禽流感病毒属于H5亚型的2.3.2.1c家系,其8个片段均与江浙地区禽类中分离的病毒高度同源,未发现有明显的重配。分子特征显示,该病毒血凝素(Hemagglutinin,HA)蛋白裂解位点为PQRERRRR/G,受体结合位点呈现禽类受体特点,但出现D94N、S133A和T188I氨基酸置换增强了病毒对人类受体的亲和性。神经氨酸酶(Neuraminidase,NA)蛋白颈部在49-68位缺失20个氨基酸,非结构蛋白1 (Non-structure protein,NS1)存在P42S置换和80-84位氨基酸的缺失。其他蛋白中也存在多个增强病毒致病力和对人类细胞亲和力的氨基酸突变。对耐药位点分析发现存在对奥司他韦的耐药突变H274Y,病毒对金刚烷胺仍旧敏感。【结论】人感染高致病性禽流感H5N1病毒A/Nanjing/1/2015属于2.3.2.1c家系,禽类来源,关键位点较保守,但仍出现了多个氨基酸的进化与变异使其更利于感染人类。H5N1禽流感病毒进化活跃,持续动态监测不能放松。

[Background] H5 N1 influenza virus can cause severe respiratory infection in human with high fatality. [Objective] To analyze the virus A/Nanjing/1/2015, a human infected highly pathogenic avian influenza virus H5 N1 confirmed in our laboratory, and to investigate its possible origins and genomic molecular characterization. [Methods] Whole genome sequencing was performed on the samples of patient’s sputum. CLC Genomics Workbench 9.0 was used for sequence assembly. Genome homology and genetic molecular characterization were analyzed by BLAST and MEGA 5.22 software. [Results] The virus belonged to H5 subtype clade 2.3.2.1 c. The 8 segments were highly homologous with those viruses isolated from domestic poultry in Jiangsu and Zhejiang area and no re-assortment was observed. Molecular characterization analysis revealed that the amino acid sequences of hemagglutinin(HA) protein cleavage site were PQRERRRR/G. The Receptor Binding Sites(RBS) was in avian type, however, D94 N, S133 A and T188 I substitutions enhanced the affinity to human receptors. A 20 amino acid deletion at position 49-68 in neuraminidase(NA) stalk, and a deletion at position 80-84 together with a P42 S mutation in the non-structural protein 1(NS1) were observed. Several amino acid mutations enhancing virulence and affinity to human cells in other proteins were also found in the present strain. Analysis on drug resistance sites revealed the presence of oseltamivir resistant mutation H274 Y, and the virus remained sensitive to amantadine. [Conclusion] The human infected highly pathogenic avian influenza A(H5 N1) virus A/Nanjing/1/2015 belonged to clade 2.3.2.1 c and was avian in origin. The key sites in the present strain were relatively conservative;however, there were still several amino acid evolution and mutations to make it more favorable for infecting human. H5 N1 avian influenza virus is evolving actively and continued surveillance is warranted.