一株鸭源H4N8亚型禽流感病毒的全基因测序及遗传进化分析

doi:10.3864/j.issn.0578-1752.2015.15.014

摘要/Abstract

摘要： 【目的】禽流感病毒(avian influenza virus, AIV)根据其表面糖蛋白血凝素(hemagglutinin, HA)和神经氨酸(neuraminidase, NA)的不同，可分为16种HA和9种NA亚型。根据其致病力的差异可分为高致病性禽流感病毒(highly pathogenic avian influenza virus, HPAIV)和低致病性禽流感病毒(low pathogenic avian influenza virus, LPAIV)。虽然H4亚型禽流感病毒为低致病性AIV，感染家禽表现为无症状感染，但其对禽类甚至是哺乳动物是一个潜在的威胁，因此必须要加强对H4亚型禽流感病毒的调查监控。【方法】为了探讨H4亚型禽流感病毒的分子特征及遗传演化规律，对2010年在中国华东地区某活禽市场进行流行病学监测时分离到的一株H4N8亚型禽流感病毒A/duck/Nanjing/1102/2010（简称DK/NJ /1102）进行了全基因组序列测定及遗传进化分析。通过常规的血清学试验确定其HA亚型，提取病毒总RNA，并通过RT-PCR方法分别扩增出其各基因片段，连接 pGEM-Teasy载体上后进行序列测定。利用GenBank中的BLAST工具进行核苷酸序列的同源性分析，并与GeneBank 中的H4亚型流感病毒及其它相关序列进行遗传进化分析。【结果】DK/NJ/1102的HA基因与Mongolia 分离株A/duck/Mongolia/274/2007(H4N3)的核苷酸同源性最高，为98.9%。推导的氨基酸剪切位点序列为“P-E-K-A-S-R-G”，符合典型的低致病性禽流感病毒特征；NA基因与华东地区分离的鸭源毒株A/Duck/Eastern China/n91/2009(H3N8)核苷酸同源性最高，达99.4%；PB1、PA和NP基因均与H1亚型禽流感病毒亲缘关系最近；M基因与A/wild duck/Korea/CSM4-12/2009(H5N1)核苷酸同源性最高，高达99.9%；NS基因与韩国2009年分离的H7N7亚型流感病毒遗传距离最近。NS1蛋白的80-84处氨基酸没有发生氨基酸缺失。【结论】该H4N8亚型禽流感病毒基因组构成比较复杂，可能是一株多基因重组病毒。

关键词: 禽流感病毒, H4N8亚型, 遗传进化分析, 重组

Abstract: 【Objective】 Based on the difference of hemagglutinin (HA) and neuraminidase (NA), avian influenza viruses (AIVs) are classified into 16 hemagglutinin (HA) and 9 neuraminidase (NA) subtypes. According to the differences in pathogenicity, AIVs can be divided into highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV). H4 AIVs are low pathogenic influenza viruses which are generally produced asymptomatic infections in poultry. But H4 AIVs also has potential threats to both poultry and mammals. Strengthening investigation on H4 subtype avian influenza viruses is important for the study of evolution of AIVs. The objective of this experiment is to investigate the molecule characteristics and genetic evolution of H4 subtype avian influenza virus. 【Method】One H4N8 subtype avian influenza virus, designated as A/duck/Nanjing/1102/2010 (H4N8) (DK/NJ/1102), was isolated from a live poultry market in eastern China during epidemiological surveillance in 2010. The complete genome sequences of the strain was sequenced and analyzed. The virus was identified by HA/HI test and RT-PCR test. The gene was cloned into pGEM-Teasy vector for sequencing, respectively. BLAST the nucleotide identity in GeneBank. The genome sequences of H4 subtype influenza viruses available in GeneBank and some other reference sequences were downloaded for genetic analysis .【Result】The results showed that the HA gene of DK/NJ/1102 had the highest nucleotide sequence identity of 98.9% with A/duck/Mongolia/274/2007(H4N3), and the amino acid sequence at the cleavage region of the HA gene was “P-E-K-A-S-R-G”, which is typical for low pathogenicity AIVs. NA gene had the highest nucleotide sequence identity of 98.8% with a duck-origin virus A/Duck/Eastern China/n91/2009 (H3N8) isolated from eastern China, whereas PB1, PA and NP genes were all mostly related with H1 subtype avian influenza viruses. The M gene shared the greatest nucleotide sequence identities (over 99.9%) with A/wild duck/Korea/CSM4-12/2009(H5N1) while the NS gene was most closely related to H7N7 subtype AIV isolated from Korea in 2009 and there was no amino acids missing in NS1 protein at 80-84 sites.【Conclusion】These data indicated that the genome composition of the strain A/duck/ Nanjing/1102/2010(H4N8) was complicated, it may be a reassortant whose genes originated from different subtypes of AIV.

Key words: avian influenza virus, H4N8 subtype, phylogenetic analysis, reassortment

赵晴晴，李群辉，朱杰，钟蕾，刘晶晶，顾敏，王晓泉，刘文博，刘秀梵. 一株鸭源H4N8亚型禽流感病毒的全基因测序及遗传进化分析[J]. 中国农业科学, 2015, 48(15): 3040-3049.

ZHAO Qing-qing, LI Qun-hui, ZHU Jie, ZHONG Lei, LIU Jing-jing, GU Min, WANG Xiao-quan, LIU Wen-bo, LIU Xiu-fan. Genome Sequencing and Genetic Analysis of H4N8 Subtype Avian Influenza Virus Isolated from Duck[J]. Scientia Agricultura Sinica, 2015, 48(15): 3040-3049.

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