2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境H9N2亞型禽流感病毒分子流行特征分析

張如勝，姚 棟，葉 文，陳靜芳，黃 政，劉曉蕾，陳田木，歐新華，孫邊成

2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境H9N2亞型禽流感病毒分子流行特征分析

張如勝，姚 棟，葉 文，陳靜芳，黃 政，劉曉蕾，陳田木，歐新華，孫邊成

目的 了解2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H9N2亞型禽流感病毒(Avian influenza virus，AIV)血凝素(Hemagglutinin, HA)、神經(jīng)氨酸酶(Neuraminidase, NA)和非結(jié)構(gòu)蛋白(ron-structural, NS)基因的分子進(jìn)化特征，為人感染H9N2亞型AIV防控提供實(shí)驗(yàn)室科學(xué)證據(jù)支持。方法 對(duì)2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所中采集的501份環(huán)境標(biāo)本(禽類(lèi)飲水263份，污水221份，其他標(biāo)本17份)利用real-time PCR方法進(jìn)行A型、H5、H7和H9亞型流感病毒核酸檢測(cè)，然后對(duì)單獨(dú)H9陽(yáng)性標(biāo)本利用HA和NA基因通用引物進(jìn)行RT-PCR擴(kuò)增和核苷酸測(cè)序，病毒HA、NA及NS基因測(cè)序結(jié)果在線BLAST分析，利用Bioedit和Mega5軟件進(jìn)行氨基酸比對(duì)和進(jìn)化樹(shù)構(gòu)建。結(jié)果 從501份環(huán)境標(biāo)本中檢出A型AIV核酸陽(yáng)性標(biāo)本350份，H9亞型核酸陽(yáng)性標(biāo)本191份，占總標(biāo)本數(shù)量的38.12%，H5亞型核酸陽(yáng)性標(biāo)本177份(35.33%)，H7亞型核酸陽(yáng)性11份(2.20%)，H5和H9亞型混合核酸陽(yáng)性標(biāo)本68份(13.57%)。部分單獨(dú)H9亞型核酸陽(yáng)性標(biāo)本經(jīng)RT-PCR和核苷酸測(cè)序鑒定出陽(yáng)性H9N2亞型病毒核酸標(biāo)本23份，分子進(jìn)化分析表明2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中的絕大部分H9N2亞型AIV HA、NA、NS基因來(lái)源于A/Chicken/Shanghai/F/98 (F98)類(lèi)似株病毒，屬于新的S基因型，HA蛋白受體結(jié)合位點(diǎn)(Receptor binding site，RBS)第235位(對(duì)應(yīng)H3流感第226位)氨基酸為L(zhǎng)eu(L)，表現(xiàn)為特異性結(jié)合人流感病毒受體特征，病毒HA、NA及NS蛋白關(guān)鍵分子位點(diǎn)表現(xiàn)為低致病性的分子特征。結(jié)論 2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H9亞型AIV核酸陽(yáng)性率最高，H9N2亞型AIV的HA、NA和NS基因表現(xiàn)為低致病性的分子特征，但具有容易感染人類(lèi)的特征，需要進(jìn)一步加強(qiáng)監(jiān)測(cè)。

禽類(lèi)場(chǎng)所；環(huán)境；禽流感病毒；H9N2亞型；基因；進(jìn)化分析

禽流感病毒(Avian influenza virus，AIV)屬于正粘病毒科A型流感病毒屬。根據(jù)流感病毒表面糖蛋白血凝素(Hemagglutinin, HA)和神經(jīng)氨酸酶(Neuraminidase, NA)抗原性的不同，A型流感病毒可分為18個(gè)HA亞型(H1-H18)和11個(gè)NA亞型(N1-N11)[1-2]。AIV除感染禽外，還可感染人、豬、馬、水貂和海洋哺乳動(dòng)物。近年來(lái)，部分AIV亞型突破種間屏障，造成人類(lèi)感染，如H5N1、H5N2、H6N1、H9N2、H7N7、H7N2、H7N3、H10N7、H7N9、H10N8和H5N6亞型AIV[3-13]。截至2017年2月22日，全球共報(bào)告22例人感染H9N2亞型AIV病例[14]，其中21例發(fā)生在中國(guó)大陸和香港地區(qū)(廣東12例，香港5例，湖南2例，安徽2例)，1例發(fā)生在孟加拉國(guó)，病例年齡7月-86歲不等，13例為女性，9例為男性，以?xún)和癁橹?，其?歲以下兒童占68.1%(15例)，病例臨床表現(xiàn)以呼吸道癥狀為主，大多表現(xiàn)為輕癥，無(wú)死亡病例；流行病學(xué)調(diào)查顯示病例大多有禽類(lèi)接觸史。研究[12,15]表明中國(guó)大陸首次報(bào)道的人感染新型重配H7N9和H10N8亞型AIV病毒，其內(nèi)部基因均來(lái)自于H9N2亞型AIV。

AIV的致病性與病毒的基因特征相關(guān)。AIV基因組由8個(gè)基因節(jié)段(PB2: RNA polymerase basic subunit 2；PB1: RNA polymerase basic subunit 1；PA: RNA polymerase acidic subunit；HA: haemagglutinin；NP: nucleoprotein；NA: neuraminidase；M: matrix gene；NS: non-structural gene)組成，其中節(jié)段4編碼的HA蛋白，具有抗原性，除了能對(duì)A型流感病毒HA亞型進(jìn)行區(qū)分外，還在感染宿主和增加致病力方面起重要作用，其HA1和HA2連接肽處編碼堿性氨基酸的多少與病毒的致病性相關(guān)[16]，且HA蛋白編碼氨基酸第226-228位點(diǎn)(H3亞型流感病毒氨基酸位點(diǎn))為特異性受體結(jié)合區(qū)域，如為Gln-Ser-Gly(QSG)則表現(xiàn)為特異性結(jié)合AIV受體-唾液酸A-2,3半乳糖(SA-2,3-Gal)，Leu-Ser-Ser(LSS)則表現(xiàn)為對(duì)人流感病毒受體-唾液酸A-2,6半乳糖(SA-2,6-Gal)親和[17-18]。節(jié)段6編碼的NA蛋白氨基酸[19]出現(xiàn)69-73位點(diǎn)缺失則表明該病毒在小鼠體內(nèi)的毒力明顯增加。節(jié)段8編碼NS1和NS2蛋白，其中NS1蛋白編碼氨基酸的第42位出現(xiàn)P42S突變及其末端結(jié)構(gòu)編碼氨基酸的第218-230位出現(xiàn)缺失，分別與病毒在小鼠體內(nèi)的毒力增強(qiáng)和減輕相關(guān)[20-21]。

黃一偉等[22]報(bào)道了1例H9N2亞型AIV輕癥病例，該病例AIV病毒分離株基因特征為禽源，與禽類(lèi)市場(chǎng)環(huán)境中分離到的H9N2亞型AIV病毒高度相似(98.5～99.8%)，其感染來(lái)源為與病例有流行病學(xué)關(guān)聯(lián)的活禽市場(chǎng)。近年來(lái)研究[23-25]也表明活禽市場(chǎng)為人感染H5N1、H7N9和H10N8亞型AIV的重要感染來(lái)源，研究[26]顯示長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H5N1亞型禽流感病毒核酸陽(yáng)性率較高，存在傳播AIV的風(fēng)險(xiǎn)。為此，本研究對(duì)2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中監(jiān)測(cè)到的H9N2亞型AIV HA、NA及NS基因進(jìn)行測(cè)序和基因特性分析，對(duì)陽(yáng)性基因中與致病性相關(guān)的氨基酸變異和分子遺傳(Phylogenetic)等分子特征進(jìn)行探討，為人感染H9N2亞型AIV疫情防控提供科學(xué)數(shù)據(jù)支持。

1 材料與方法

1.1 儀器及試劑 7300 Real-time RT-PCR System購(gòu)于美國(guó)AB Applied Biosystems公司，MycyclerTMthermal cycler購(gòu)于美國(guó)BIO-RAD公司，Centrifuge 5430R購(gòu)于德國(guó)eppendorf公司，PowerpacTM Universal購(gòu)于美國(guó)BIO-RAD公司，SuperScript○RIII One-Step RT-PCR System with Platinum○RTaq購(gòu)于美國(guó)Life Technologies公司，SuperScript○RIII Platinum○ROne-Step Quantitative RT-PCR System購(gòu)于美國(guó)Life Technologies公司，Rneasy○RMini Kit購(gòu)于德國(guó)QIAGEN公司。

1.2 禽類(lèi)場(chǎng)所環(huán)境標(biāo)本H9亞型AIV核酸檢測(cè) 按照中國(guó)疾病預(yù)防控制中心下發(fā)的《職業(yè)暴露人群血清學(xué)和環(huán)境高致病性禽流感監(jiān)測(cè)方案》(2011年版)要求，采集2014年長(zhǎng)沙市轄區(qū)禽類(lèi)場(chǎng)所環(huán)境標(biāo)本501份(禽類(lèi)飲水263份、禽類(lèi)污水221份、其他標(biāo)本17份)，利用real-time RT-PCR方法進(jìn)行A型及H5、H7、H9亞型流感病毒核酸檢測(cè)，核酸提取、檢測(cè)步驟及結(jié)果判斷按照《全國(guó)流感監(jiān)測(cè)方案(2010年版)》進(jìn)行[27-28]，檢測(cè)用real-time RT-PCR引物和探針序列來(lái)源于中國(guó)國(guó)家流感中心(Chinese National Influenza Center，CNIC)[29-30]，引物及探針由上海Invitrogen公司合成。

1.3 H9N2亞型AIV HA、NA及NS基因擴(kuò)增及核苷酸序列測(cè)定 將在2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中監(jiān)測(cè)到的H9亞型AIV陽(yáng)性標(biāo)本(禽類(lèi)污水、禽類(lèi)飲水共23份，排除H5和H7亞型AIV核酸陽(yáng)性)利用Rneasy○RMini Kit (德國(guó)QIAGEN 公司)進(jìn)行RNA核酸提取(提取步驟參照試劑盒說(shuō)明書(shū)進(jìn)行)，提取的RNA利用SuperScript○RIII One-Step RT-PCR System with Platinum○RTaq RT-PCR(Life Technologies 公司)對(duì)HA和NA基因片段分別進(jìn)行RT-PCR核酸擴(kuò)增，反應(yīng)體積(25 μL)：RNase Free Water 6.0 μL；2×PCR反應(yīng)緩沖液12.5 μL；Platinum@Taq酶混合液1．0 μL；20 μmol/L引物(Bm-HA-1、Bm-NS-890R; Ba-NA-1、Ba-NA-1413R，引物序列來(lái)源于文獻(xiàn)[31]，見(jiàn)表1)各0.25 μL；RNA模板5.0 μL。HA和NA基因預(yù)期擴(kuò)增片段長(zhǎng)度分別為1 778 bp和1 413 bp，其中HA基因擴(kuò)增能同時(shí)擴(kuò)增出NS片段，預(yù)期片段長(zhǎng)度為890 bp。擴(kuò)增反應(yīng)條件：60 ℃ 60 min；94 ℃ 2 min；再按94 ℃ 15 s，58 ℃ 30 s，68 ℃ 7 min 循環(huán)40 次，68 ℃ 7 min。分別取擴(kuò)增后的HA和NA基因片段RT-PCR產(chǎn)物10 μL，在瓊脂糖凝膠中90 v 40 min電泳后成像，觀察有無(wú)預(yù)期目的片段。如有預(yù)期目的片段，將PCR產(chǎn)物TA克隆后進(jìn)行核苷酸序列測(cè)定，TA克隆測(cè)序由上海Life Technologies 公司利用ABI PRISMTM 3730XL DNA Analyzer測(cè)序儀和BigDye○RTerminator v3.1 Cycle Sequencing Kit測(cè)序試劑完成核苷酸序列測(cè)定，正、反兩方向測(cè)序，測(cè)通。

1.4 環(huán)境中H9N2亞型AIV HA、NA及NS基因進(jìn)化分析 長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H9N2亞型AIV病毒HA、NA及NS基因測(cè)序結(jié)果利用BioEdit軟件拼接后提交至美國(guó)國(guó)立生物信息中心(National Center for Biotechnology Information，NCBI)，利用在線基本局部相似性搜索工具(Basic local alignment search tool, BLAST)進(jìn)行同源性分析(http：//BLAST.ncbi.nlm.nih.gov/BLAST.cgi)。從NCBI 流感病毒資源庫(kù)(Influenza Virus Resource，http：//www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi)下載長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H9N2亞型AIV、國(guó)內(nèi)H9N2亞型AIV及G1(A/Quail/Hong Kong/G1/97)、Y280(A/duck/Hong Kong/Y280/97)、Y439(A/duck/Hong Kong/Y439/1997)、F98(A/Chicken/Shanghai/F/98)和BJ/1/94(A/Chicken/Beijing/1/94)等譜系代表株HA、NA及NS基因片段核苷酸序列，利用MEGA 5軟件的Neighbor-joining方法和Tamura-Nei 模式構(gòu)建進(jìn)化樹(shù)。

表1 H9N2亞型禽流感病毒測(cè)序引物序列

Tab.1 Sequences of sequencing primers for H9N2 virus

PrimerSeguence(5'→3')Bm-HA-1TATTCGTCTCAGGGAGCAAAAG-CAGGGGBm-NS-890RATATCGTCTCGTATTAGTAGAAA-CAAGGGTGTTTTBa-NA-1TATTGGTCTCAGGGAGCAAAAG-CAGGAGTBa-NA-1413RATATGGTCTCGTATTAGTAGAAA-CAAGGAGTTTTTT

1.5 環(huán)境中H9N2亞型AIV HA、NA及NS基因關(guān)鍵位點(diǎn)分析 選擇并從NCBI流感病毒資源庫(kù)中下載人感染H9N2亞型病毒湖南株A/Lengshuitan/11197/2013 (LST/11197)、H9N2亞型AIV各譜系代表株(G1、Y280、Y439、F98、BJ/1/94)和H5N1亞型AIV A/duck/Guangxi/xa/2001(GX/xa)HA、NA及NS基因片段編碼氨基酸序列，利用BioEdit軟件將上述AIV和本研究中的環(huán)境H9N2亞型AIV進(jìn)行多重比對(duì)、分析各基因關(guān)鍵位點(diǎn)分子特征。

2 結(jié) 果

2.1 禽類(lèi)場(chǎng)所環(huán)境標(biāo)本各基因相關(guān)檢測(cè)結(jié)果 對(duì)2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中采集到的501份禽類(lèi)飲水(263份)、污水(221份)和其他(17份)標(biāo)本進(jìn)行AIV病毒核酸檢測(cè)，共檢出A型病毒核酸陽(yáng)性標(biāo)本350份，其中主要A型病毒核酸亞型為H9和H5， H9亞型核酸陽(yáng)性標(biāo)本191份，占總標(biāo)本的38.12%， H5亞型核酸陽(yáng)性標(biāo)本177份(35.33%)，H7亞型核酸陽(yáng)性11份(2.20%)，H5和H9亞型混合核酸陽(yáng)性標(biāo)本68份(13.57%)；263份禽類(lèi)飲水標(biāo)本中H9和H5亞型核酸檢出率分別為50.19%和32.70%，221份污水標(biāo)本中H9和H5亞型核酸檢出率分別為25.34%和39.37%，具體結(jié)果見(jiàn)表2。

表2 長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境標(biāo)本AIV real-time RT-PCR檢測(cè)結(jié)果分析

Tab.2 Prevalence of AIV in specimens collected from poultry markets in Changsha City, Hunan Province, China, determined by real-time RT-PCR

No.ofpositivesamplesanddetectionrate(%)fortypeandsubtypesSampleNo.ofsamplesAH9H5H7H5,H9H5,H7H7,H9H5,H7,H9Othersubtypesdrinkingwater263216(82.13%)132(50.19%)86(32.70%)8(3.04%)33(12.55%)5(1.90%)2(0.76%)1(0.38%)8(3.04%)sewage221126(57.01%)56(25.34%)87(39.37%)3(1.36%)34(15.38%)1(0.45%)1(0.45%)0(0%)20(9.05%)others178(47.06%)3(17.65%)4(23.53%)0(0%)1(5.88%)0(0%)0(0%)0(0%)0(0%)Total501350(69.86%)191(38.12%)177(35.33%)11(2.20%)68(13.57%)6(1.20%)3(0.60%)1(0.20%)28(5.59%)

2.2 H9N2亞型AIV核苷酸序列測(cè)定結(jié)果 從H9亞型核酸陽(yáng)性標(biāo)本中篩選出23份(H5、H7亞型AIV核酸均為陰性)，通過(guò)HA和NA基因通用引物(其中HA基因擴(kuò)增引物能夠同時(shí)擴(kuò)增出NS基因片段)RT-PCR擴(kuò)增和TA克隆核苷酸序列測(cè)定，成功獲得58條基因序列。其中HA和NA基因序列分別為23條，NS序列為12條，各基因序列已上傳至NCBI，GenBank登錄號(hào)為：KX247857-KX247914。其中H9N2亞型AIV HA基因核苷酸序列全長(zhǎng)1 683 bp，NA基因核苷酸序列全長(zhǎng)1 401 bp，NS基因核苷酸序列全長(zhǎng)883 bp。在線BLAST相似性比對(duì)分析表明：23條HA基因序列分別與來(lái)源于湖南、河南、江西、江蘇、北京和青島等省市環(huán)境及雞H9N2、H9亞型AIV分離株HA基因高度相似(≥98.63%)；22條H9N2亞型AIV NA基因序列分別與來(lái)源于湖南、江蘇、江西、北京、東莞和深圳等省市環(huán)境及雞H9N2亞型AIV分離株NA基因高度相似(≥98.07%), 1條H9N2亞型AIV(A/environment/Changsha/369/2014(H9N2))NA基因序列與來(lái)源于蒙古麻鴨H11N2亞型AIV分離株(A/ruddy shelduck/Mongolia/590C2/2009(H11N2))NA基因高度相似(97.94%)；7條NS基因序列分別與武漢和溫州等地雞源H9N2亞型AIV分離株NS基因高度相似(≥98.98%)，5條NS基因序列分別與安徽、無(wú)錫、溫州等省市人、雞和鴨H7N9亞型AIV分離株NS基因高度相似(≥99.09%)；HA和NA基因BLAST結(jié)果表明上述23份環(huán)境標(biāo)本中存在的病毒為H9N2亞型AIV，具體病毒名稱(chēng)及其BLAST比對(duì)結(jié)果見(jiàn)表3。

2.3 H9N2亞型AIV進(jìn)化樹(shù)構(gòu)建 H9N2 亞型AIV在世界范圍內(nèi)廣泛分布，在遺傳進(jìn)化上可以分為北美和歐亞譜系兩大分支，其中歐亞系又進(jìn)一步衍生出以Y280、G1、Y439、F98以及BJ/1/94等為代表的病毒系進(jìn)化分支[32-33]。HA基因進(jìn)化樹(shù)構(gòu)建出Y280、G1和Y439三個(gè)譜系進(jìn)化分支，其中本研究中的23株H9N2亞型AIV、國(guó)內(nèi)H9N2亞型AIV HA基因聚集在一個(gè)亞分支內(nèi)，Y280、F98和CK/BJ/1譜系代表株HA基因聚集在另一個(gè)亞分支內(nèi)，兩個(gè)亞分支共同構(gòu)成Y280譜系進(jìn)化分支[22]，本研究中的23株H9N2病毒與Y280譜系代表株進(jìn)化距離近；NA進(jìn)化樹(shù)顯示本研究中的23株環(huán)境H9N2亞型AIV有22株病毒聚集在一個(gè)單獨(dú)進(jìn)化分支內(nèi)，與Y280、F98譜系代表株及H9N2亞型人源湖南株LST/11197進(jìn)化距離近，而本研究中的另外1株病毒CS/369位于Y439譜系；NS基因進(jìn)化樹(shù)顯示，本研究中的12株H9N2亞型AIV NS基因全部聚集在F98譜系，但分別形成3個(gè)亞分支，見(jiàn)圖1。

表3 長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境23株H9N2亞型病毒BLAST相似性分析

Tab.3 Comparisons of twenty-three H9N2 viruses from Changsha City with isolates in GenBank of highest nucleotide identity (%) (The date of this BLAST search was Feb 27, 2016)

VirusHANucleotideidentityofH9N2virus(%)NANucleotideidentityofH9N2virus(%)NSNucleotideidentityofH9N2virus(%)CS/92A/chicken/Beijing/0331/2013(99.17%)A/chicken/Jiangsu/JS86/2013(99.13%)A/chicken/Wenzhou/YHQL04/2014(98.98%)CS/94A/chicken/Jiangxi/29075/2013(99.58%)A/chicken/Jiangxi/29075/2013(99.71%)A/chicken/Wuhan/JXQL01/2015(99.66%)CS/219A/environment/Hunan/27420/2014(99.94%)A/chicken/Dongguan/1674/2014(99.93%)A/chicken/Wuhan/JXQL01/2015(99.09%)CS/250A/environment/Hunan/28028/2014(99.23%)A/environment/Hunan/28034/2014(99.79%)A/Anhui/DEWH72-09/2013b(99.55%)CS/279A/environment/Hunan/28034/2014(99.58%)A/environment/Hunan/28034/2014(99.64%)A/duck/Wuxi/0405006/2013b(99.09%)CS/334A/environment/Hunan/27420/2014(98.87%)A/chicken/Jiangsu/YZLH3/2013(99.07%)NosequenceCS/341A/chicken/Beijing/1115/2013(99.23%)A/chicken/Jiangxi/20482/2013(99.14%)A/chicken/Wuhan/JXQL01/2015(99.66%)CS/347A/environment/Hunan/28034/2014(99.47%)A/environment/Hunan/28034/2014(99.71%)NosequenceCS/360A/environment/Hunan/27420/2014(99.52%)A/chicken/Beijing/0512/2013(99.00%)NosequenceCS/366A/environment/Hunan/27420/2014(99.47%)A/environment/Hunan/28034/2014(99.57%)NosequenceCS/368A/environment/Hunan/27420/2014(99.58%)A/environment/Hunan/28034/2014(99.50%)NosequenceCS/369A/environment/Hunan/27420/2014(99.47%)A/ruddyshelduck/Mongolia/590C2/2009a(97.94%)NosequenceCS/372A/chicken/Qingdao/013/2014(98.63%)A/chicken/Jiangxi/20478/2013(99.29%)NosequenceCS/400A/chicken/Henan/SH01/2015(99.52%)A/chicken/Dongguan/1674/2014(99.64%)NosequenceCS/408A/environment/Hunan/27420/2014(99.23%)(A/chicken/Dongguan/1674/2014(99.50%)NosequenceCS/418A/environment/Hunan/27420/2014(99.70%)(A/chicken/Dongguan/1674/2014(99.07%)A/chicken/Wenzhou/RAQL18/2015b(99.09%)CS/450A/chicken/Jiangsu/J3156/2014(99.05%)A/chicken/Jiangxi/20482/2013(99.21%)A/chicken/Wuhan/JXQL01/2015(99.21%)CS/451A/environment/Hunan/27420/2014(99.41%)A/chicken/Dongguan/1674/2014(99.36%)NosequenceCS/462A/chicken/Henan/SH01/2015(99.34%)A/chicken/Dongguan/1674/2014(99.57%)A/chicken/Wuhan/JXQL01/2015(99.21%)CS/478A/chicken/Henan/SH01/2015(99.28%)A/chicken/Jiangxi/29117/2013(98.93%)A/chicken/Wuhan/JXQL01/2015(99.21%)CS/481A/environment/Hunan/27420/2014(99.41%)A/environment/Hunan/28034/2014(99.29%)NosequenceCS/484A/chicken/Jiangsu/J3294/2014(99.35%)A/chicken/Shenzhen/1949/2013(98.07%)A/duck/Wuxi/0405006/2013b(99.21%)CS/490A/environment/Hunan/27420/2014(99.17%)A/chicken/Jiangxi/29117/2013(98.86%)(A/duck/Wuxi/0405006/2013b(99.32%)

Note: CS/92: A/environment/Changsha/92/2014; CS/94: A/environment/Changsha/94/2014; CS/219: A/environment/Changsha/219/2014; CS/250: A/environment/Changsha/250/2014; CS/279: A/environment/Changsha/279/2014; CS/334: A/environment/Changsha/334/2014; CS/341: A/environment/Changsha/341/2014; CS/347: A/environment/Changsha/347/2014; CS/360: A/environment/Changsha/360/2014; CS/366: A/environment/Changsha/366/2014; CS/368: A/environment/Changsha/368/2014; CS/369: A/environment/Changsha/369/2014; CS/372: A/environment/Changsha/372/2014; CS/400: A/environment/Changsha/400/2014; CS/408: A/environment/Changsha/408/2014; CS/418: A/environment/Changsha/418/2014; CS/450: A/environment/Changsha/450/2014; CS/451: A/environment/Changsha/451/2014; CS/462: A/environment/Changsha/462/2014; CS/478: A/environment/Changsha/478/2014; CS/481: A/environment/Changsha/481/2014; CS/484: A/environment/Changsha/484/2014; CS/490: A/environment/Changsha/490/2014; a: H11N2; b: H7N9.

The sequence of the H9N2 virus in our study is marked by ▲, and the sequences of representative H9N2. Viruses of G1, Y280, Y439, BJ/1/94 and F98 lineages from GenBank are marked by ●.

>圖1 2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境H9N2亞型AIV HA(a)、NA(b)及NS(c)基因進(jìn)化分析

Fig.1 Phylogenetic analysis of the HA (a), NA(b) and NS(c) genes of the influenza H9N2 viruses isolated from poultry markets in Changsha, China in 2014

2.3.1 H9N2亞型AIV HA、NA及NS基因關(guān)鍵位點(diǎn)分析 本研究中的23株H9N2亞型AIV HA基因HA1和HA2 蛋白連接肽(335-341 位點(diǎn))出現(xiàn)2個(gè)堿性氨基酸(SRSSRGL，其中R為堿性氨基酸)，與其他H9N2亞型AIV譜系代表株HA基因致病特征相似，對(duì)禽表現(xiàn)為低致病性特征，與H5N1亞型高致病性AIV具有多個(gè)堿性氨基酸的高致病性分子特征不同[16]，見(jiàn)表4。

本研究中的23株H9N2亞型AIV HA 蛋白第235～237位(對(duì)應(yīng)H3型流感病毒編碼氨基酸第226-228位)氨基酸為L(zhǎng)MG，表明其受體為人源流感病毒特異性受體[17]，與人源H9N2亞型AIV湖南株LST/11197及其他不同譜系H9N2亞型AIV代表株HA基因特征相似，具有容易感染人的受體特征，見(jiàn)表4。

本研究中的1株H9N2亞型AIV CS/369 NA基因編碼氨基酸第63～64位點(diǎn)為NR氨基酸，與G1、Y439和BJ/1/94譜系代表株病毒NA基因特征相似，但本研究中的另外22株H9N2亞型AIV NA基因編碼氨基酸第63-64位點(diǎn)出現(xiàn)缺失，與Y280和F98譜系代表株病毒相似，見(jiàn)表4。

本研究中的12株H9N2亞型AIV與其他不同譜系代表株NS1蛋白全長(zhǎng)編碼217個(gè)氨基酸，相比H5N1亞型AIV GX/xa，表4中的H9N2亞型AIV第80-84位氨基酸(TIASV)均未發(fā)生缺失現(xiàn)象，但本研究中的12株H9N2亞型AIV及1株人源H9N2亞型AIV湖南株LST/11197缺失第218-230位氨基酸，導(dǎo)致了病毒致死性相關(guān)PL基序(ESEV/EPEV)的缺失，見(jiàn)表4。本研究中的12株H9N2亞型AIV NS1編碼蛋白第92位氨基酸均為D，未出現(xiàn)D92E突變，不同于G1譜系代表株NS1蛋白出現(xiàn)D92E氨基酸突變，見(jiàn)表4。

表4 長(zhǎng)沙市H9N2亞型AIV與參考株病毒HA、NA及NS1蛋白關(guān)鍵位點(diǎn)特征分析

Tab.4 Analysis of the important amino acids of the HA, NA and NS1 proteins of H9N2 AIV strain from Changsha City with reference strains

VirusHANANS1Cleavagesite335-341Receptorbindingsite235-23763-64Totalofno.ofaaDeletionofaa80-8492CS/92SRSSRGLLMGDeletion217NODCS/94SRSSRGLLMGDeletion217NODCS/219SRSSRGLLMGDeletion217NODCS/250SRSSRGLLMGDeletion217NODCS/279SRSSRGLLMGDeletion217NODCS/334SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/341SRSSRGLLMGDeletion217NODCS/347SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/360SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/366SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/368SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/369SRSSRGLLMGNRNosequenceNosequenceNosequenceCS/372SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/400SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/408SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/418SRSSRGLLMGDeletion217NODCS/450SRSSRGLLMGDeletion217NODCS/451SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/462SRSSRGLLMGDeletion217NODCS/478SRSSRGLLMGDeletion217NODCS/481SRSSRGLLMGDeletionNosequenceNosequenceNosequenceCS/484SRSSRGLLMGDeletion217NODCS/490SRSSRGLLMGDeletion217NODLST/11197SRSSRGLLMGDeletion217NODG1ARSSRGLLQGNR218NOEY280ARSSRGLLQGDeletion218NODY439AASNRGLQQGNR218NODF98ARSSRGLQQGDeletion217NODBJ/1/94ARSSRGLQQGNR217NODGX/xaRERRRKRGLQSGIR225YESD

Note: Substitutions of particular concern are shown in bold.

3 討 論

H9N2亞型AIV在中國(guó)及東亞地區(qū)雞群中廣泛流行，其中Y280譜系病毒在中國(guó)中南部流行最為廣泛[34-35]，H9N2病毒同時(shí)也是活禽市場(chǎng)中最常見(jiàn)的AIV，研究[22]顯示與人感染H9N2亞型AIV病例有流行病學(xué)關(guān)聯(lián)的湖南省永州市活禽市場(chǎng)中H9亞型AIV核酸陽(yáng)性率最高(18%)，其他研究[36]顯示廣西地區(qū)活禽市場(chǎng)低致病性AIV具有相似的陽(yáng)性率10.8%(336/3 121)。同時(shí)人感染H7N9、H10N8、H5N6、H9N2等[22-25]亞型AIV疫情流行病學(xué)調(diào)查已證實(shí)禽類(lèi)場(chǎng)所，特別是活禽市場(chǎng)已經(jīng)成為人感染AIV的重要來(lái)源，CNIC每年都在開(kāi)展禽類(lèi)場(chǎng)所AIV日常監(jiān)測(cè)工作，監(jiān)測(cè)禽類(lèi)場(chǎng)所環(huán)境中的不同亞型AIV污染情況，本研究結(jié)果顯示2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H9亞型AIV核酸陽(yáng)性率最高(38.12%)，高于H5亞型AIV核酸陽(yáng)性率(35.33%)，并且高于其他研究報(bào)道[23,37]，原因可能與標(biāo)本種類(lèi)有關(guān)，本研究中的標(biāo)本種類(lèi)主要為禽類(lèi)飲水和污水,這兩類(lèi)標(biāo)本分別用于禽類(lèi)的飲用水和禽類(lèi)宰殺后的清洗用水，聚集了禽類(lèi)口腔和內(nèi)臟器官的AIV，造成了AIV核酸檢出率的升高。

研究對(duì)2007-2013年中國(guó)東部地區(qū)分離的H9N2病毒基因進(jìn)化分析顯示出現(xiàn)六個(gè)不同的基因型，其中的一個(gè)新基因型(S)，以F98譜系代表株病毒為骨架，通過(guò)與G1譜系代表株病毒的PB2和M基因重配而成[37]。HA及NA進(jìn)化樹(shù)分析顯示本研究中的H9N2亞型AIV與國(guó)內(nèi)2013年以后分離的部分H9N2亞型AIV HA、NA基因聚集在一個(gè)單獨(dú)的亞分支內(nèi)，但與Y280及F98譜系代表株同屬于一個(gè)大的進(jìn)化分支；由于本研究中的H9N2病毒CS/369與H11N2亞型AIV分離株(A/ruddy shelduck/Mongolia/590C2/2009)NA基因核苷酸序列高度相似(97.94%)，導(dǎo)致CS/369病毒與Y439譜系代表株進(jìn)化距離近，屬于Y439譜系；NS基因進(jìn)化樹(shù)顯示本研究中的12株H9N2亞型AIV病毒NS基因全部聚集在F98譜系，但分別形成3個(gè)亞分支，與本研究中的NS基因分別來(lái)源于H9N2和H7N9亞型AIV相關(guān)。進(jìn)化表明長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中的絕大部分H9N2亞型AIV HA、NA、NS基因來(lái)源于F98 類(lèi)似株病毒，屬于新的S基因型，并且隨著時(shí)間的推移，已經(jīng)開(kāi)始進(jìn)化形成新的分支，需要引起關(guān)注。另外HA及NA基因進(jìn)化分析還顯示本研究中的環(huán)境H9N2亞型AIV與人源H9N2亞型AIV湖南株LST/11197位于同一分支，且進(jìn)化距離近，進(jìn)一步證實(shí)了活禽市場(chǎng)是人感染H9N2亞型AIV的重要來(lái)源[22]。

流感病毒在感染宿主細(xì)胞時(shí)，HA蛋白需要蛋白酶將其裂解為HA1和HA2，然后才能吸附到宿主細(xì)胞膜上。H7N9、H10N8和H9N2等低致病性AIV在HA1和HA2連接肽區(qū)域只有一個(gè)堿性氨基酸，只能被局限在有R蛋白酶表達(dá)的粘膜表面和組織中進(jìn)行復(fù)制，感染后癥狀較輕。而H5N1，H5N6等高致病性AIV在HA1和HA2連接肽區(qū)域有4個(gè)以上堿性氨基酸，故能被廣泛分布在全身各組織和器官的蛋白酶所識(shí)別并裂解，造成全身感染，故流感病毒HA蛋白的HA1和HA2連接肽位點(diǎn)堿性氨基酸數(shù)目的多少與病毒的致病性密切相關(guān)。本研究中的2014年長(zhǎng)沙市禽類(lèi)場(chǎng)所環(huán)境中H9N2亞型AIV HA基因HA1和HA2蛋白連接肽位點(diǎn)出現(xiàn)2個(gè)堿性氨基酸，與其他H9N2亞型AIV譜系代表株HA基因致病特征相似，對(duì)禽表現(xiàn)為低致病性基因特征[16]。流感病毒HA蛋白還起識(shí)別宿主細(xì)胞受體的作用，其第226～228位氨基酸為QSG，表明其對(duì)AIV受體-SA-2,3-Gal親和，如為L(zhǎng)SS序列，則對(duì)人流感病毒受體-SA-2,6-Gal親和[38]。研究報(bào)道近年來(lái)國(guó)內(nèi)流行的H9N2病毒HA蛋白R(shí)BS區(qū)域幾乎均突變?yōu)長(zhǎng)226，并且多數(shù)具有與SA-2,6-Gal結(jié)合的能力[39]，有研究還證實(shí)含L226的H9N2亞型AIV能夠在人的氣管上皮細(xì)胞中進(jìn)行有效的復(fù)制[40]。本研究中的H9N2亞型AIV HA基因RBS區(qū)域?yàn)長(zhǎng)226，表現(xiàn)為人源流感病毒受體，具有與SA-2,6-Gal結(jié)合的能力，容易造成人類(lèi)感染。目前報(bào)道的22例人感染H9N2亞型AIV病例絕大多數(shù)表現(xiàn)為輕癥，表明更多的H9N2亞型AIV輕癥和無(wú)癥狀攜帶者尚未被發(fā)現(xiàn)，這種溫和的感染使得H9N2亞型AIV能夠進(jìn)一步在人體內(nèi)適應(yīng)并有可能與其他人流感病毒發(fā)生基因重組而具備造成流感流行的潛力。

本研究中的絕大部分H9N2亞型AIV NA基因蛋白第63-64位氨基酸出現(xiàn)缺失，僅1株H9N2亞型AIV CS/369第63-64位氨基酸未發(fā)生缺失，表現(xiàn)為NR，證實(shí)了H9N2亞型AIV NA基因來(lái)源的多樣性。研究表明H9N2亞型AIV NS1蛋白編碼氨基酸發(fā)生D92E突變，具有提高H9N2亞型AIV感染人類(lèi)的能力，本研究中的12株H9N2病毒的NS1蛋白未發(fā)生D92E突變，且缺失了與病毒致死性相關(guān)的PL基序(ESEV/EPEV), 該缺失可以減輕病毒對(duì)小鼠的致病能力[21]，與本地區(qū)環(huán)境中的其他H9N2亞型AIV NS1蛋白編碼氨基酸相似，具有低致病性流感病毒的分子特征[41]。然而，研究顯示不同譜系的H9亞型病毒的內(nèi)部基因在哺乳動(dòng)物和人體細(xì)胞模型中都具備復(fù)制能力[42]，表明H9N2亞型AIV的內(nèi)部基因仍然有潛力為產(chǎn)生新的流行株病毒提供部分甚至整套內(nèi)部基因，譬如新型H7N9和H5N1亞型病毒。

目前，國(guó)內(nèi)不斷新增人感染H9N2亞型AIV病例報(bào)告，且禽類(lèi)市場(chǎng)活禽銷(xiāo)售模式未改變、禽類(lèi)場(chǎng)所環(huán)境中H9N2亞型AIV污染狀況未進(jìn)一步減輕的情況下，需要密切監(jiān)測(cè)禽類(lèi)場(chǎng)所環(huán)境中H9N2亞型AIV病毒進(jìn)化狀況，及時(shí)評(píng)估、預(yù)警H9N2亞型AIV感染人類(lèi)的潛在風(fēng)險(xiǎn)。

[1] Tong S, Zhu X, Li Y, et al. New world bats harbor diverse influenza A viruses[J]. PLoS Pathog, 2013, 9(10): e1003657. DOI: 10.1371/journal.ppat.1003657

[2] Tong S, Li Y, Rivailler P, et al. A distinct lineage of influenza A virus from bats[J]. Proc Natl Acad Sci U S A, 2012, 109(11): 4269-4274. DOI: 10.1073/pnas.1116200109

[3] Pan M, Gao R, Lv Q, et al. Human infection with a novel, highly pathogenic avian influenza A (H5N6) virus: Virological and clinical findings[J]. J Infect, 2016, 72(1): 52-59. DOI: 10.1016/j.jinf.2015.06.009

[4] Koopmans M, Wilbrink B, Conyn M, et al. Transmission of H7N7 avian influenza A virus to human beings during a large outbreak in commercial poultry farms in the Netherlands[J]. Lancet, 2004, 363(9409): 587-593. DOI: 10.1016/S0140-6736(04)15589-X

[5] Hirst M, Astell CR, Griffith M, et al. Novel avian influenza H7N3 strain outbreak, British Columbia[J]. Emerg Infect Dis, 2004, 10(12): 2192-2195. DOI: 10.3201/eid1012.040743

[6] Ogata T, Yamazaki Y, Okabe N, et al. Human H5N2 avian influenza infection in Japan and the factors associated with high H5N2-neutralizing antibody titer[J]. J Epidemiol, 2008, 18(4): 160-166. DOI: 10.2188/jea.JE2007446

[7] Cheng VC, Chan JF, Wen X, et al. Infection of immunocompromised patients by avian H9N2 influenza A virus[J]. J Infect, 2011, 62(5): 394-399. DOI:10.1016/j.jinf.2011.02.007

[8] To KK, Ng KH, Que TL, et al. Avian influenza A H5N1 virus: a continuous threat to humans[J]. Emerg Microbes Infect, 2012, 1(9): e25. DOI: 10.1038/emi.2012.24

[9] Ostrowsky B, Huang A, Terry W, et al. Low pathogenic avian influenza A (H7N2) virus infection in immunocompromised adult, New York, USA, 2003[J]. Emerg Infect Dis, 2012, 18(7): 1128-1131. DOI: 10.3201/eid1807.111913

[10] Arzey GG, Kirkland PD, Arzey KE, et al. Influenza virus A (H10N7) in chickens and poultry abattoir workers, Australia[J]. Emerg Infect Dis, 2012, 18(5): 814-816. DOI: 10.3201/eid1805.111852

[11] Wei SH, Yang JR, Wu HS, et al. Human infection with avian influenza A H6N1 virus: an epidemiological analysis[J]. Lancet Respir Med, 2013, 1(10): 771-778. DOI: 10.1016/S2213-2600(13)70221-2.

[12] Chen H, Yuan H, Gao R, et al. Clinical and epidemiological characteristics of a fatal case of avian influenza A H10N8 virus infection: a descriptive study[J]. Lancet, 2014, 383(9918): 714-721. DOI: 10.1016/S0140-6736(14)60111-2

[13] Zhang R, Chen T, Ou X, et al. Clinical, epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus[J]. Infect Genet Evol, 2016, 40: 236-242. DOI: 10.1016/j.meegid.2016.03.010

[14] Freidl GS, Meijer A, de Bruin E, et al. Influenza at the animal-human interface: a review of the literature for virological evidence of human infection with swine or avian influenza viruses other than A(H5N1)[J]. Euro Surveill, 2014, 19(19): 8-26. DOI: 10.2807/1560-7917

[15] Gao R, Cao B, Hu Y, et al. Human infection with a novel avian-origin influenza A (H7N9) virus[J]. N Engl J Med, 2013, 368(20): 1888-1897. DOI: 10.1056/NEJMoa1304459

[16] Senne DA, Panigrahy B, Kawaoka Y, et al. Survey of the hemagglutinin (HA) cleavage site sequence of H5 and H7 avian influenza viruses: amino acid sequence at the HA cleavage site as a marker of pathogenicity potential[J]. Avian Dis, 1996, 40(2): 425-437. DOI: 10.2307/1592241

[17] Srinivasan K, Raman R, Jayaraman A, et al. Quantitative description of glycan-receptor binding of influenza A virus H7 hemagglutinin[J]. PLoS One, 2013, 8(2): e49597. DOI: 10.1371/journal.pone.0049597

[18] Shu YL,Lan Y,Wen LY, et al.Analysis of human H5N1 virus hemagglutinin gene isolated from the mainland of China[J]. Chin J Exp Clin Virol, 2006, 20(2): 8-10. DOI: 10.3760/cma.j.issn.1003-9279.2006.02.003 (in Chinese)

舒躍龍，藍(lán)雨，溫樂(lè)英，等.我國(guó)分離人H5N1禽流感病毒血凝素基因特性的研究[J]. 中華實(shí)驗(yàn)和臨床病毒學(xué)雜志,2006,20(2):8-10.

[19] Mckimm-Breschkin JL, Sahasrabudhe A, Blick TJ, et al. Mutations in a conserved residue in the influenza virus neuraminidase active site decreases sensitivity to Neu5Ac2en-derived inhibitors[J]. J Virol, 1998, 72(3): 2456-2462.

[20] Jiao P, Tian G, Li Y, et al. A single-amino-acid substitution in the NS1 protein changes the pathogenicity of H5N1 avian influenza viruses in mice[J]. J Virol, 2008, 82(3): 1146-1154. DOI: 10.1128/JVI.01698-07

[21] Jackson D, Hossain MJ, Hickman D, et al. A new influenza virus virulence determinant: the NS1 protein four C-terminal residues modulate pathogenicity[J]. Proc Natl Acad Sci U S A, 2008, 105(11): 4381-4386. DOI: 10.1073/pnas.0800482105

[22] Huang Y, Li X, Zhang H, et al. Human infection with an avian influenza A (H9N2) virus in the middle region of China[J]. J Med Virol, 2015, 87(10): 1641-1648. DOI: 10.1002/jmv.24231

[23] Wan XF, Dong L, Lan Y, et al. Indications that live poultry markets are a major source of human H5N1 influenza virus infection in China[J]. J Virol, 2011, 85(24): 13432-13438. DOI: 10.1128/JVI.05266-11

[24] Shi J, Deng G, Liu P, et al. Isolation and characterization of H7N9 viruses from live poultry markets-Implication of the source of current H7N9 infection in humans[J]. Chin Sci Bull, 2013, (16): 1857-1863. DOI: 10.1007/s11434-013-5873-4

[25] Zhang T, Bi Y, Tian H, et al. Human infection with influenza virus A(H10N8) from live poultry markets, China, 2014[J]. Emerg Infect Dis, 2014, 20(12): 2076-2079. DOI: 10.3201/eid2012.140911

[26] Zhang RS, Ou XH, Song KY, et al. Risk related to the transmission of H5N1 subtype avian influenza virus in the environment of poultry markets in Changsha, China[J]. Chin J Epidemiol, 2012, 33(8): 768-773. DOI: 10.3760/cma.j.issn.0254-6450.2012.08.003 (in Chinese)

張如勝，歐新華，宋克云, 等. 長(zhǎng)沙市家禽市場(chǎng)環(huán)境中H5N1亞型禽流感病毒傳播風(fēng)險(xiǎn)研究[J]. 中華流行病學(xué)雜志, 2012, 33(8): 768-773.

[27] Zhang RS, Sun BC, Yao D, et al. Evolution of the HA, NA and NS genes of H5N1 avian influenza viruses from sewage in live bird markets in Changsha, 2014[J]. Chin J Zoonoses, 2017, 33(1): 85-88, 80. DOI: 10.3969/j.issn.1002-2694.2017.01.016 (in Chinese)

張如勝, 孫邊成, 姚棟, 等. 2014年長(zhǎng)沙市活禽市場(chǎng)污水中H5N1亞型禽流感病毒HA、NA及NS基因進(jìn)化分析[J]. 中國(guó)人獸共患病學(xué)報(bào), 2017, 33(1): 85-88, 80.

[28] Ministry of Health of the People’s Republic of China. National influenza surveillance program (2010)[EB/OL]. (2010-09-10)[2017-02-23]. http://www.moh.gov.cn/jkj/s3577/201009/3fa356d0f4834d408fde6c12891a6482.shtml. (in Chinese)

中華人民共和國(guó)衛(wèi)生部：全國(guó)流感監(jiān)測(cè)方案(2010年版)[EB/OL]. (2010-09-10)[2017-02-23]. http://www.moh.gov.cn/jkj/s3577/201009/3fa356d0f4834d408fde6c12891a6482.shtml.

[29] WHO Global Influenza Surveillance Network. Manual for the laboratory diagnosis and virological surveillance of influenza[EB/OL]. (2013-09-15)[2017-02-23]. http://whqlibdoc.who.int/publications/2011/9789241548090_eng.pdf.

[30] World Health Organization. WHO information for molecular diagnosis of influenza virus-update[EB/OL]. (2014-03-31)[2016-06-05]. http://www.who.int/influenza/gisrs_laboratory/molecular_diagnosis_influenza_virus_humans_update_201403.pdf?ua=1.

[31] 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. DOI: 10.1007/s007050170002

[32] Xu KM, Smith GJ, Bahl J, et al. The genesis and evolution of H9N2 influenza viruses in poultry from southern China, 2000 to 2005[J]. J Virol, 2007, 81(19): 10389-10401. DOI: 10.1128/JVI.00979-07

[33] Lu JH, Liu XF, Shao WX, et al. Phylogenetic analysis of eight genes of H9N2 subtype influenza virus: a mainland China strain possessing early isolates’ genes that have been circulating[J]. Virus Genes, 2005, 31(2): 163-169. DOI: 10.1007/s11262-005-1790-1

[34] Ji K, Jiang W M, Liu S, et al. Characterization of the hemagglutinin gene of subtype H9 avian influenza viruses isolated in 2007-2009 in China[J]. J Virol Methods, 2010, 163(2): 186-189. DOI: 10.1016/j.jviromet.2009.09.01

[35] Chu YC, Cheung CL, Hung LC, et al. Continuing evolution of H9N2 influenza viruses endemic in poultry in southern China[J]. Influenza Other Respir Viruses, 2011, 5 (Suppl 1): 68-71.

[36] Peng Y, Xie ZX, Liu JB, et al. Epidemiological surveillance of low pathogenic avian influenza virus (LPAIV) from poultry in Guangxi Province, Southern China[J]. PLoS One, 2013, 8(10): e77132. DOI: 10.1371/journal.pone.0077132

[37] Gu M, Chen H, Li Q, et al. Enzootic genotype S of H9N2 avian influenza viruses donates internal genes to emerging zoonotic influenza viruses in China[J]. Vet Microbiol, 2014, 174(3/4): 309-315. DOI: 10.1016/j.vetmic.2014.09.029

[38] Stevens J, Blixt O, Tumpey TM, et al. Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus[J]. Science, 2006, 312(5772): 404-410. DOI: 10.1126/science.1124513

[39] Jiang W, Liu S, Hou G, et al. Chinese and global distribution of H9 subtype avian influenza viruses[J]. PLoS One, 2012, 7(12): e52671. DOI: 10.1371/journal.pone.0052671

[40] Vines A, Wells K, Matrosovich M, et al. The role of influenza A virus hemagglutinin residues 226 and 228 in receptor specificity and host range restriction[J]. J Virol, 1998, 72(9): 7626-7631.

[41] Zhang RS, Ou X , Song KY, et al. Genetic analysis on the NS gene of H9N2 avian influenza virus isolated from sewage in poultry market[J]. Chin Prev Med, 2013, 14(03): 205-208. (in Chinese)

張如勝，歐新華，宋克云，等. 長(zhǎng)沙市家禽市場(chǎng)污水來(lái)源H9N2亞型禽流感病毒NS基因進(jìn)化分析[J]. 中國(guó)預(yù)防醫(yī)學(xué)雜志, 2013,14(03): 205-208.

[42] The SJCEIRS H9 Working Group. Assessing the fitness of distinct clades of influenza A (H9N2) viruses[J]. Emerg Microbes Infect, 2013, 2(11): e75. DOI: 10.1038/emi.2013.75

Molecular epidemiological characteristics of avian influenza A(H9N2) viruses from environment in poultry markets in Changsha, China, 2014

ZHANG Ru-sheng, YAO Dong, YE Wen, CHEN Jing-fang, HUANG Zheng, LIU Xiao-lei, CHEN Tian-mu, OU Xin-hua, SUN Bian-cheng

(ChangshaCenterforDiseaseControlandPrevention,Changsha410004,China)

We analyzed the evolutional and molecular characteristics of Hemagglutinin(HA), Neuraminidase(NA) and non-structural(NS) genes of avian influenza A(H9n2) viruses from environment in poultry markets in Changsha, China, 2014, providing laboratory data for prevention and control of human infection with avian influenza A(H9N2) virus. Five hundred and one specimens (263 poultry drinking water specimens, 226 poultry sewage specimens and 17 others specimens) were collected from environment in poultry markets in Changsha, 2014, and real-time RT-PCR was used for influenza A typing and subtyping (H5, H7 and H9) detection. HA and NA universal primer sets for conventional RT-PCR and sequencing were used for the positivity of single H9. The sequence homology of HA, NA and NS genes of the viruses were analyzed with the online Basic Local Alignment Search Tool (BLAST). The ClustalW multiple alignments of amino acids and the phylogenetic trees for HA, NA and NS genes were constructed using the BioEdit and MEGA 5 software, respectively. Results showed that among 501 environmental samples, 350 samples were positive for influenza A virus, 191 (38.12%) for H9 subtype, 177 (35.33%) for H5 subtype, 11 (2.20%) for H7 subtype and 68 (13.57%) for H5 and H9 subtypes co-detection. Twenty-three H9N2 subtype AIV were confirmed by conventional RT-PCR and sequencing from the samples of the positivity of single H9. Phylogenetic analysis revealed that most of HA, NA and NS genes of the H9N2 subtype AIV isolated in Changsha City had gene constellations of genotype S,and these virues might have acquired their HA, NA and NS from A/Chicken/Shanghai/F/1998-like (H9N2). L235 (correspond to H3 numbering 226) of the HA protein of the receptor binding site (RBS) were found in these H9N2 viruses, and the characteristics was shown to be associated with increased affinity of HA to the glycan-receptors of human influenza virus, and the low pathogenicity molecular characteristics of HA, NA and NS genes were showed in these viruses. The positive rate of nucleic acid of the H9 subtype of avian influenza virus from environment was the highest in poultry markets in Changsha, 2014, and molecular characteristics of the HA, NA and NS of these H9N2 subtype AIV showed low pathogenicity, but that may facilitate human infection. So, the prevalence and genetic evolution of this virus should be closely monitored.

poultry markets; environment; avian influenza virus; H9N2 subtype; gene; phylogenetic analysis Supported by the Hunan Provincial Health Medicine Research Project (No. B2015-153) Corresponding author: Sun Bian-cheng, Email: sunbiancheng2013@163.com

10.3969/j.issn.1002-2694.2017.03.005

孫邊成，Email: sunbiancheng2013@163.com

長(zhǎng)沙市疾病預(yù)防控制中心，長(zhǎng)沙 410004

R373.1+3

A

1002-2694(2017)03-0212-10

2017-02-09 編輯：林丹

湖南省衛(wèi)生計(jì)生委2015年度科研計(jì)劃項(xiàng)目(No.B2015-153)