孔子榮 李三木 何奇松 曾詠芳 楊可妍 馮淑萍 孫翔翔 熊毅 顏健華
摘要:【目的】明確廣西豬源H9N2亞型流感病毒的遺傳特征及分子生物學(xué)特性(抗原性、耐藥性和致病性),為廣西豬源H9N2亞型流感病毒的防控提供科學(xué)依據(jù)?!痉椒ā恳苑蛛x自廣西百色地區(qū)的2株豬源H9N2亞型流感病毒(SW/GX/P2/2011株和SW/GX/P3/2011株)為研究對(duì)象,運(yùn)用RT-PCR擴(kuò)增其全基因組的8個(gè)基因片段(HA、NA、NP、M、NS、PB1、PB2和PA基因),經(jīng)克隆測序后進(jìn)行核苷酸序列及氨基酸位點(diǎn)分析?!窘Y(jié)果】2株廣西豬源H9N2亞型流感病毒的核苷酸序列開放閱讀框(ORF)分別是PB2:2280 bp、PB1:2274 bp、PA:2151 bp、HA:1683 bp、NP:1497 bp、NA:1410 bp、M:982 bp和NS:838 bp。2株廣西豬源H9N2亞型流感病毒全基因組僅M基因核苷酸序列與豬源H9N2亞型流感病毒的相似性較高,而HA、NA、NP、NS、PA、PB2和PB1基因核苷酸序列均與禽源H9N2亞型流感病毒的相似性較高,在基因型分類上屬于G57基因型,為我國廣泛流行的H9N2亞型基因型。2株廣西豬源H9N2亞型流感病毒的HA蛋白發(fā)生R180Q、T213A、D216E、M224L、N285S和V287T突變,NA蛋白抗原決定簇S331V、W403S和Q431K也發(fā)生突變;NA蛋白在N2亞型的耐藥性關(guān)鍵位點(diǎn)119E、151D、292R、276E和294N位點(diǎn)未發(fā)生突變,但在NA蛋白抗原區(qū)存在S331V、K367E和Q432K突變,且M2氨基酸位點(diǎn)發(fā)生S31N突變。2株廣西豬源H9N2亞型流感病毒HA蛋白連接HA1和HA2的氨基酸均為RSSR↓GLF;HA蛋白存在1個(gè)因P315S突變而新增的潛在糖基化位點(diǎn)(NCS);NA蛋白未缺失NA潛在糖基化位點(diǎn),也未出現(xiàn)NA蛋白頸部桿狀結(jié)構(gòu)63~65 aa缺失現(xiàn)象,在NA潛在糖基化位點(diǎn)中69、86、146、200和234 aa處非常保守,但發(fā)生W402S突變。【結(jié)論】從廣西百色分離獲得的2株豬源H9N2亞型流感病毒(SW/GX/P2/2011株和SW/GX/P3/2011株)雖為低致病力毒株,但在流感病毒基因重組過程中發(fā)揮重要作用,且其致病性有增強(qiáng)趨勢,已對(duì)金剛烷胺類藥物產(chǎn)生耐藥性。因此,應(yīng)加強(qiáng)廣西地區(qū)哺乳動(dòng)物H9N2亞型流感病毒的監(jiān)控,并警惕其跨種間傳播。
關(guān)鍵詞: 豬流感病毒;H9N2亞型;基因重組;抗原性;耐藥性;致病性
中圖分類號(hào): S852.659.5? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 文獻(xiàn)標(biāo)志碼: A 文章編號(hào):2095-1191(2020)09-2304-07
Genetic evolution and biological characteristics analysis of two strains of H9N2 subtype influenza virus from swine in Guangxi
KONG Zi-rong1, LI San-mu2,HE Qi-song3, ZENG Yong-fang1, YANG Ke-yan1,
FENG Shu-ping3, SUN Xiang-xiang1, XIONG Yi3, YAN Jian-hua2*
(1College of Animal Science and Technology, Guangxi University, Nanning? 530004, China; 2Medical College,Guangxi University, Nanning? 530004, China; 3Guangxi Center for Animal Disease Prevention and Control, Nanning? 530001,China)
Abstract:【Objective】To understand the genetic and molecular biological characteristics(antigenicity, drug resistance and pathogenicity) of pig H9N2 subtype influenza virus from swine in Guangxi and to provide scientific basis for the prevention and control of swine H9N2 influenza virus in Guangxi. 【Method】 Two strains of pig H9N2 subtype influenza virus from swine isolated from Baise in Guangxi(SW/GX/P2/2011 and SW/GX/P3/2011) were used as materials, eight segments(HA, NA, NP, M, NS, PB1, PB2 and PA) of both virus strains? whole genome were amplified by RT-PCR. After cloning and sequencing, the nucleotide sequence and amino acid sites were analyzed. 【Result】Nucleotide sequence open reading frame(ORF) of two strains of H9N2 influenza virus isolated from pigs were PB2:2280 bp, PB1:2274 bp, PA:2151 bp, HA:1683 bp, NP:1497 bp, NA:1410 bp, M:982 bp and NS:838 bp, respectively. In genomes of the two isolated strains,only the nucleotide sequence of M gene of the two isolates had high similarity with H9N2 subtype influenza virus of swine origin, while the nucleotide sequences of HA,NA,NP,NS,PA,PB2 and PB1 genes had high similarity with H9N2 subtype influenza virus of avian origin, and belonged to G57 genotype in genotype classification, which was the H9N2 subtype genotype widely prevalent in China. Mutations of R180Q, T213A, D216E, M224I, N285S and V287T occurred in HA protein of the two isolated strains, and the NA protein antigenic determinants S331V, W403S and Q431K were also mutated. In the drug resistance locus analysis, the isolated virus strains did not mutate at the 119E, 151D, 292R, 276E and 294N sites of the NA protein in the N2 subtype resistance,but S331V, K367E and Q432K mutations were found in the antigen region of NA protein,and the M2 amino acid site produced S31N mutations.The amino acids connec-ting HA1 and HA2 of the two isolates were RSSR↓GLF. A new potential glycosylation site(NCS) existed in HA protein due to P315S mutation. There was no deletion of NA potential glycosylation site and 63-65 aa deletion in the neck rod structure of NA protein. Among the potential glycosylation sites of NA,69,86,146,200 and 234 aa were very conservative, but W402S mutation occurred. 【Conclusion】The two H9N2 influenza viruses(SW/GX/P2/2011 and SW/ GX/P3/2011) isolated from Baise, Guangxi are low virulence strains, but they play an important role in the gene recombination of influenza viruses, and their pathogenicity tends to increase. They have developed amantadine resistance. Therefore, it is necessary to strengthen the surveillance of H9N2 subtype influenza virus in mammals in Guangxi, and to guard against its cross species transmission.
Key words: swine influenza virus; H9N2 subtype; genetic recombination; antigenicity; drug resistance; pathogenicity
Foundation item: National Natural Science Foundation of China(31660713); Guangxi Natural Science Foundation(2015GXNSFEA139002)
0 引言
【研究意義】豬流感病毒(Swine influenza virus,SIV)隸屬于正黏病毒科(Orthomyxoviridae)A型流感病毒屬,主要引發(fā)豬群發(fā)生呼吸道疾病(汪琪等,2017;李鴿等,2019)。豬是流感病毒進(jìn)化過程中的混合器,能將禽源、豬源和人源的不同亞型流感病毒基因進(jìn)行重組(李海燕等,2004;蘭德松等,2018;汪琪等,2018)。H9N2亞型是一種極易跨越種間屏障的流感病毒亞型(Butt et al.,2005),若在豬群中與其他亞型流感病毒不斷重組或重配,極有可能產(chǎn)生感染人類的新型流感病毒。因此,加強(qiáng)豬源H9N2亞型流感病毒流行監(jiān)測對(duì)整個(gè)流感病毒防控體系的建設(shè)和完善具有重要意義?!厩叭搜芯窟M(jìn)展】李海燕等(2004)研究發(fā)現(xiàn),我國豬群中存在H9N2亞型SIV,且證實(shí)豬只充當(dāng)禽源、豬源和人源流感病毒重組的混合器。H9N2亞型流感病毒能為H5N1亞型、H7N9亞型和H5N6亞型等感染人類的流感病毒提供內(nèi)部基因,是一個(gè)極其危險(xiǎn)的信號(hào)(Gu et al.,2014)。陳建新等(2011)研究表明,H9N2亞型SIV不僅能感染小鼠,還在小鼠體內(nèi)表現(xiàn)出較強(qiáng)的致病性,與HA蛋白裂解位點(diǎn)顯示的低致病性不一致,其原因可能是流感病毒在哺乳動(dòng)物體內(nèi)與在家禽體內(nèi)的致病機(jī)理不一致。伍和明等(2012)對(duì)廣西豬源H9N2亞型流感病毒全基因組序列進(jìn)行分析,結(jié)果發(fā)現(xiàn)分離病毒株具備較強(qiáng)的跨種屬傳播能力。殷斌等(2015)對(duì)1株山東H9N2亞型SIV進(jìn)行遺傳進(jìn)化分析,發(fā)現(xiàn)該毒株是禽流感病毒(Avian influenza virus,AIV)向豬體適應(yīng)的毒株。劉曉敏等(2017)成功建立了禽源H9N2亞型SIV反向遺傳操作技術(shù)平臺(tái),為后續(xù)開展SIV的基因功能研究打下基礎(chǔ)。孫王楊吉等(2019)對(duì)1株浙江H9N2亞型SIV進(jìn)行遺傳進(jìn)化和致病性分析,結(jié)果表明豬可能是AIV獲得感染哺乳動(dòng)物能力的過渡宿主。Sun等(2019)于2017—2018年從華南地區(qū)的家禽中分離獲得8株H9N2亞型流感病毒,通過系統(tǒng)發(fā)育進(jìn)化樹及生物學(xué)特性分析,發(fā)現(xiàn)H9N2亞型流感病毒向哺乳動(dòng)物傳播的能力不斷增強(qiáng)。此外,Samir等(2019)通過對(duì)埃及的家養(yǎng)雞群進(jìn)行樣品采集,并選取5種亞型毒株進(jìn)行測序及HA基因進(jìn)化分析,結(jié)果發(fā)現(xiàn)HA基因與亞洲G1系的親緣關(guān)系較近,且各基因片段在不斷進(jìn)化或重組?!颈狙芯壳腥朦c(diǎn)】至今,有關(guān)廣西地區(qū)豬源H9N2亞型流感病毒的研究報(bào)道較少,尤其在遺傳特征研究及生物學(xué)特性分析方面更少?!緮M解決的關(guān)鍵問題】對(duì)分離自廣西百色地區(qū)的2株豬源H9N2亞型流感病毒進(jìn)行遺傳進(jìn)化分析,并通過關(guān)鍵位點(diǎn)分析了解其抗原性、耐藥性及致病性,為廣西豬源H9N2亞型流感病毒的防控提供科學(xué)依據(jù)。
1 材料與方法
1. 1 試驗(yàn)材料
豬源H9N2亞型流感病毒SW/GX/P2/2011株和SW/GX/P3/2011株均分離自廣西百色市樂業(yè)縣送檢的病豬組織樣品,由廣西動(dòng)物疫病預(yù)防控制中心分離鑒定并保存。SPF雞胚購自北京梅里亞維通實(shí)驗(yàn)動(dòng)物技術(shù)有限公司;病毒核酸抽提、膠回收和質(zhì)粒提取試劑盒及DL2000 DNA Marker等購自天根生化科技(北京)有限公司;M-MLV反轉(zhuǎn)錄酶、抑制降解酶(RRI)、Taq DNA聚合酶、pMD18-T載體及大腸桿菌DH5α感受態(tài)細(xì)胞等購自寶生物工程(大連)有限公司。
1. 2 引物設(shè)計(jì)與合成
參考Hoffmann等(2001)擴(kuò)增A型流感病毒基因組的引物設(shè)計(jì)原則,利用Primer 5.0設(shè)計(jì)8對(duì)引物用于擴(kuò)增廣西豬源H9N2亞型流感病毒基因組各片段(表1)。所有引物均委托寶生物工程(大連)有限公司合成。
1. 3 RT-PCR擴(kuò)增
以接種SPF雞胚增殖獲得的病毒尿囊液抽提病毒總RNA,并以此為模板進(jìn)行RT-PCR擴(kuò)增。RT-PCR反應(yīng)體系25.0 μL:2×Step Bufffer 10.0 μL,PrimeScript 1 Step Enzyme Mix 1.0 μL,上、下游引物各0.5 μL,RNA模板5.0 μL, RNase Free dH2O補(bǔ)足至25.0 μL。擴(kuò)增程序:42 ℃反轉(zhuǎn)錄1 h;94 ℃預(yù)變性5 min;94 ℃ 40 s,56 ℃ 40 s,72 ℃ 60 s,進(jìn)行30個(gè)循環(huán);72 ℃延伸10 min。擴(kuò)增產(chǎn)物采用1.5%瓊脂糖凝膠電泳進(jìn)行檢測。
1. 4 目的條帶回收與克隆
按瓊脂糖凝膠DNA回收試劑盒說明進(jìn)行DNA目的片段回收純化,將回收純化獲得的DNA目的片段與pMD18-T載體連接,經(jīng)轉(zhuǎn)化和涂板后挑斑進(jìn)行重組質(zhì)粒鑒定,陽性重組質(zhì)粒送至寶生物工程(大連)有限公司測序。
1. 5 病毒基因組測序分析
利用SeqMan進(jìn)行序列拼接以獲得完整的病毒基因組序列,再使用MEGA 5.0繪制毒株遺傳進(jìn)化樹并分析相關(guān)功能位點(diǎn)。
2 結(jié)果與分析
2. 1 豬源H9N2亞型流感病毒各基因片段擴(kuò)增結(jié)果
利用設(shè)計(jì)的特異性引物進(jìn)行RT-PCR擴(kuò)增,電泳檢測獲得的目的條帶分別為HA、NA、NP、M、NS、PB1、PB2和PA基因(圖1),與預(yù)期結(jié)果一致,說明廣西豬源H9N2亞型流感病毒SW/GX/P2/2011株和SW/GX/P3/2011株全基因組各片段均擴(kuò)增成功。
2. 2 豬源H9N2亞型流感病毒基因核苷酸序列比對(duì)及遺傳進(jìn)化分析結(jié)果
廣西豬源H9N2亞型流感病毒SW/GX/P2/2011株和SW/GX/P3/2011株經(jīng)基因克隆測序得到的核苷酸序列開放閱讀框(ORF)分別是PB2:2280 bp、PB1:2274 bp、PA:2151 bp、HA:1683 bp、NP:1497 bp、NA:1410 bp、M:982 bp和NS:838 bp。使用NCBI中的BLAST進(jìn)行比對(duì)分析,獲得與2株廣西豬源H9N2亞型流感病毒各基因片段核苷酸序列相似性最高的參考毒株(表2)。利用MEGA 5.0對(duì)SW/GX/P2/2011株和SW/GX/P3/2011株的各基因片段進(jìn)行遺傳進(jìn)化分析,其中,基于HA基因核苷酸序列的遺傳進(jìn)化分析顯示這2株豬源H9N2亞型流感病毒與A/duck/Hong Kong/Y280/97株屬于同一分支(圖2),為BJ/94系中的Y280亞系;基于NA基因核苷酸序列的遺傳進(jìn)化分析顯示這2株豬源H9N2亞型流感病毒與A/chicken/Hong Kong/G9/97株屬于同一分支,為BJ/94系中的G9/97亞系;基于NS基因核苷酸序列的遺傳進(jìn)化分析顯示這2株豬源H9N2亞型流感病毒與A/chicken/Shanghai/F/98株屬于同一分支,為BJ/94系中的F/98亞系;基于PB1、PA和NP基因核苷酸序列的遺傳進(jìn)化分析均顯示這2株豬源H9N2亞型流感病毒屬于F/98亞系;基于PB2基因核苷酸序列的遺傳進(jìn)化分析顯示這2株豬源H9N2亞型流感病毒與A/duck/Shantou/2102/2000株屬于同一分支,屬于DK1系;基于M基因核苷酸序列的遺傳進(jìn)化分析顯示這2株豬源H9N2亞型流感病毒與A/quail/Hong Kong/G1/97株屬同一分支,屬G1/97系。由于SW/GX/P2/2011株和SW/GX/P3/2011株的基因組成與目前廣泛流行的G57基因型(圖3)相一致,故確定廣西豬源H9N2亞型流感病毒為G57基因型。
2. 3 豬源H9N2亞型流感病毒表面蛋白抗原性分析結(jié)果
SW/GX/P2/2011株和SW/GX/P3/2011株與參考株DK/HK/Y280/97、DK/HK/Y439/97和QA/HK/G1/97的HA抗原表位比對(duì)分析結(jié)果顯示,7個(gè)抗原區(qū)在147~153 aa、162~167 aa和267~273 aa等3個(gè)抗原位區(qū)上保守,除此之外有7個(gè)抗原位點(diǎn)發(fā)生變異,分別是117~181 aa區(qū)的R180Q、195~205 aa區(qū)的T/E198A、211~227 aa區(qū)的T213A、D216E和M/V224L及284~289 aa區(qū)的N285S和V287T。以疫苗株SH/F/98、CK/HK/G9/97和QA/HK/G1/97為參考,進(jìn)行豬源H9N2亞型流感病毒NA抗原性位點(diǎn)比對(duì)分析,結(jié)果發(fā)現(xiàn)在抗原決定簇發(fā)生S331V、W403S和Q431K突變,且在紅細(xì)胞結(jié)合位點(diǎn)發(fā)現(xiàn)K367E、D399G、W403S和Q432K存在明顯變異。NA酶活性位點(diǎn)分析結(jié)果表明,2株豬源H9N2亞型流感病毒在NA蛋白基質(zhì)結(jié)合位點(diǎn)(118R、224R、243D、277E、350K和425E)上與參考毒株無差異。由此推測,SW/GX/P2/2011株和SW/GX/P3/2011株的表面蛋白抗原性已發(fā)生改變。
2. 4 豬源H9N2亞型流感病毒耐藥性分析結(jié)果
通過對(duì)比N2亞型抗神經(jīng)氨酸酶抑制劑NA蛋白酶活性位點(diǎn)氨基酸序列,結(jié)果顯示SW/GX/P2/2011株和SW/GX/P3/2011株在119E、151D、292R、276E和294N位點(diǎn)均未發(fā)生突變,說明廣西豬源H9N2亞型流感病毒對(duì)神經(jīng)氨酸酶抑制劑不具耐藥性;但在NA蛋白抗原區(qū)發(fā)生S331V、K367E和Q432K突變,而這些突變可能會(huì)影響病毒的耐藥性;M2氨基酸位點(diǎn)分析發(fā)現(xiàn)其氨基酸序列發(fā)生S31N突變,說明廣西豬源H9N2亞型流感病毒對(duì)金剛烷胺類藥物耐藥。綜上所述,SW/GX/P2/2011株和SW/GX/P3/2011株已對(duì)金剛烷胺類藥物產(chǎn)生耐藥性,但對(duì)神經(jīng)氨酸酶抑制劑依然敏感。
2. 5 豬源H9N2亞型流感病毒致病性關(guān)鍵位點(diǎn)分析結(jié)果
SW/GX/P2/2011株和SW/GX/P3/2011株HA蛋白連接HA1和HA2的氨基酸序列均為RSSR↓GLF,僅存在2個(gè)堿性氨基酸殘基,說明這2株豬源H9N2亞型流感病毒為低致病性毒株;HA蛋白有8個(gè)保守的潛在糖基化位點(diǎn),但發(fā)生P315S突變導(dǎo)致在313 aa處新增1個(gè)糖基化位點(diǎn)(NCS);NA蛋白共有8個(gè)保守的潛在糖基化位點(diǎn),未發(fā)生因NA潛在糖基化位點(diǎn)缺失而增強(qiáng)病毒致病性的情況;與參考毒株CK/SH/F/98相比,發(fā)現(xiàn)這2株豬源H9N2亞型流感病毒NA蛋白頸部桿狀結(jié)構(gòu)在63~65 aa處并未發(fā)生缺失(表3)。在對(duì)病毒致病性產(chǎn)生影響的NA潛在糖基化位點(diǎn)中,69、86、146、200和234 aa處非常保守,但發(fā)生W402S突變,可能對(duì)神經(jīng)氨酸酶活性產(chǎn)生影響;在內(nèi)部基因中,與致病力有關(guān)的氨基酸位點(diǎn)與參考毒株相比均較保守,如PB2蛋白的627E和701D(陸家海等,2007),PB1蛋白的436Y、622G和709V,PA蛋白的315F和515T,NP蛋白的184K和319N。可見,SW/GX/P2/2011株和SW/GX/P3/2011株為低致病性H9N2亞型流感病毒,但存在毒力變強(qiáng)的趨勢。
3 討論
H9N2亞型SIV致死率較低因而在實(shí)際生產(chǎn)中常被忽視,但從整個(gè)養(yǎng)殖業(yè)的發(fā)展及公共衛(wèi)生意義角度出發(fā),SIV尤其是H9N2亞型SIV的監(jiān)測和防控意義重大。本研究結(jié)果表明,SW/GX/P2/2011株和SW/GX/P3/2011株全基因組僅M基因核苷酸序列與豬源H9N2亞型流感病毒的相似性較高,而HA、NA、NP、NS、PA、PB2和PB1基因核苷酸序列均與禽源H9N2亞型流感病毒的相似性較高,故推測這2株豬源H9N2亞流感病毒應(yīng)是由禽源H9N2亞型流感病毒感染豬群進(jìn)化產(chǎn)生的宿主適應(yīng)性變異毒株,但以現(xiàn)有數(shù)據(jù)不能推斷其產(chǎn)生來源。這2株豬源H9N2亞型流感病毒在基因型分類上屬于G57基因型,為我國廣泛流行的H9N2亞型基因型,且與我國已報(bào)道的H7N9、H5N2和H5N1亞型流感病毒具有相似內(nèi)部基因組成,說明SW/GX/P2/2011株和SW/GX/P3/2011株具備為多種亞型流感病毒提供內(nèi)部基因的可能,與孫王楊吉等(2019)認(rèn)為禽源H9N2亞型流感病毒未發(fā)生突變即能感染豬群的觀點(diǎn)一致,進(jìn)一步佐證豬作為流感病毒進(jìn)化混合器的觀點(diǎn)。
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(責(zé)任編輯 蘭宗寶)