台灣禽類流感病毒H5N2的源生、演化及其與禽流感病毒H6N1之關係：公共衛生啟示

Abstract

2003年底台灣養雞場爆發「低」致病性禽流感H5N2疫情，全島撲殺超過30萬隻雞。然2008年再度爆發雞的禽流感H5N2疫情。研究顯示此波病毒已演化成「高」致病性之潛能；但此病毒來源、流行幅度仍未知。不幸在2012年爆發一連串雞的禽流感疫情，政府首度對外宣布其致病原已演變為「高」致病性禽流感H5N2病毒。因此，科學上極關注的課題是：不同年代的台灣雞H5N2病毒之間的關係為何？是否來自台灣的鴨H5N2病毒？又其與鄰國及美洲的H5N2病毒之關係為何？更重要的是台灣長年流行的雞H6N1病毒在台灣雞H5N2病毒扮演何種角色？有鑒於此，本研究目的有四：（一）探討台灣雞流感H5N2病毒之源起、引入、演化及生態衝擊；（二）台灣雞與鴨H5N2病毒的流行病學特徵及其與1994年墨西哥H5N2病毒之演化關係；（三）檢視台灣雞和鴨的流感H5N2病毒之分子標記，評估其公共衛生的潛在風險；及（四）基於上述研究成果，提供防治建議。 研究設計上，禽流感偵測分兩階段，第一階段在2012年12月之前（2005年10月至2006年10月及2008年8月至2012年11月）；第二階段自2012年12月(官方宣布「高」致病性禽流感後的當年冬季)至2013年7月進行強化偵測。研究地主擇選北部一大活禽市場，每月取雞、鴨糞便及雞血，分別進行病毒與血清偵測。實驗上，糞便檢體以特殊無菌雞胚蛋培養病毒後，萃取核酸，進行分子檢測(reverse transcriptase-polymerase chain reaction, RT-PCR)、八段基因定序及使用最大似然法(Maximum-likelihood method, ML)與貝式統計馬可夫鏈-蒙特卡羅法[Bayesian Markov Chain Monte Carlo (BMCMC)]分別分析病毒親緣關係及病毒核酸演化速率。血清偵測第一階段是用禽流感病毒之酵素連結免疫吸附法(enzyme-linked immunosorbent assay, ELISA)，檢測雞血清中是否有抗流感病毒NP蛋白的抗體。第二階段以血球凝集抑制試驗[hemagglutination inhibition (HI) test]檢測雞血清抗流感病毒NP蛋白(+)的抗體對五禽流感病毒[TW853/12 (H3N8)、TW1030/12 (H6N8)、TW2267/12 (H6N1)、TW2593/12 (H5N2)、HK-NT155/08 (H9N2)]的抗體力價。 偵測結果，第一階段鴨與雞糞的禽流感病毒總分離率各為2.6%(124/4,798)與0.3%(9/3,363)；且各有五亞型(H1、H3、H4、H5、H12)與一亞型(H6)。而第二階段強化偵測結果中，鴨與雞糞的禽流感病毒總分離率已提高而各為5.1%(132/2,58)與0.8%(67/8,659)；另各有五亞型(H3、H4、H5、H6及H7)與三亞型(H3、H5及H6)。但此兩階段均未分離得H9N2病毒。 禽流感病毒八段基因的核酸親源樹分析結果，發現所有16株雞的H5N2病毒有兩來源:(1)表面基因：兩群均源於2003年流行於台灣雞的低致病性禽流感H5N2病毒株，而非來自台灣家鴨的禽流感H5病毒，且可溯源自製備1994年墨西哥禽流感H5N2之疫苗株；此兩表面基因的演化速率相當低，甚發現2003年台灣雞H5N2病毒株和1994-96年墨西哥H5N2病毒株兩者的表面基因之核酸總累積變異度相對於共同始祖是等同；(2)六段內部基因：全來自台灣禽流感H6N1病毒而呈基因重組現象；且除PB1之外的五段基因也和表面基因有兩群，即來自2012及2013年B群的雞H5N2病毒之內部基因和台灣2004-05年的H6N1病毒親緣相近，而2012-13年A群的雞H5N2病毒卻和台灣2012-13年的H6N1病毒親緣相近；再檢視其HA蛋白的切割位，發現鹼性胺基酸數從3演變成4個，又從4演變成3個的反覆逆轉現象，且近年台灣H5N2病毒株HA蛋白切割位的鹼性胺基酸有三類型(REKR, RKKR, RRKR)，甚而同次流行中出現多元化。種種跡象顯示此雞H5N2病毒至少兩次引入而分別在養雞場出現重組，有違病毒學及自然演化的常理。 血清偵測發現活禽市場雞對2003年H5N2禽流感病毒有中至低度的HI抗體 血清盛行率。有趣的是台灣過去從未偵測到禽流感H9N2病毒，卻在雞血有8成以上的抗體陽性率，有些甚達高力價(1280)。綜言之，台灣雞可能有用禽流感疫苗。 檢視雞H5N2及鴨H5病毒對人的風險[病毒致病能力、病毒傳播及辨認哺乳類受體(α2,6 鍵結之醣類)的病毒接合位(receptor binding site)及抗病毒藥神經胺酸酶抑制劑(neuraminidase inhibitor, NAI)的相關胺基酸]，發現這些病毒對NAI均有感受性；且幾乎全保有源自禽的分子標記，但出現一些特殊位點的胺基酸可能對鼠有致病力，如在雞H5N2病毒有八位點變異，各為PB2-L89V、PB2-G309D、PB2-T339K、PB2-R477G、PB2-I495V、M1-N30D、M1-T215A及NS1-I106M；在鴨H5病毒除此八位點變異之外，另有三位點變異(PB2-A676T、NS1-P42S及NS1-L103F)與NS1蛋白C端出現ESEV。 台灣政府雖嚴禁禽類施打禽流感疫苗，但本研究發現台灣雞打禽流感疫苗已為不爭的事實。在去年五月出現H6N1禽流感病毒感染人肺炎病例之際，發現此病毒與2013年2月台灣雞H6N1病毒8段基因中已有4段基因(NS1, NP, PB1, HA)的核酸序列相同度達97.6- 99.5%。因此本研究建議：(1)嚴格依法取締動物用禽流感疫苗；(2)必須在高風險區強化禽流感病毒偵測，並探索禽流感H9N2病毒是否已大量進入台灣禽場；(3)禽畜產業相關人員及家庭成員應參與年度禽流感血清學調查；(4)家禽載具宜徹底消毒與路線管理，防禽流感H5N2病毒擴散至其他縣市。在未來研究方面：(1)須持續監測禽流感H6N1及H5N2病毒，尤其是目前H6N1病毒感染人的相關機制還未明朗前，更需注意H6N1病毒或H5N2從H6N1病毒得感染人相關的決定因子；(2)研發醣晶片，以快速大規模篩選出具接合哺乳類受器能力的禽流感病毒，期達及早預警疫情之效；(3)探查台灣雞流感H5N2-Ck/TW1680/13病毒株為何能在降低病毒毒力的醣化作用位出現下卻仍可造成高致病性的分子機轉。最重要的是推動整合禽流感病毒跨領域研究，以科學探究積極面對禽流感病毒對公共衛生的挑戰。

English Abstract The outbreaks of avian influenza (AI) caused by low pathogenic AI (LPAI) H5N2 in chickens occurred island-wide in Taiwan in 2003 and reoccurred in 2008 attributed to the H5N2 virus with elevated potential of high pathogenicity (HP). However, the origin, prevalence and distribution of these H5N2 viruses were unclear. In 2012, government firstly announced that HPAI H5N2 viruses resulted in several severe AI outbreaks. Therefore, the scientific issues were: What were the relationships among the H5N2 viruses emerged from different years? Were they derived from Taiwan duck H5N2 viruses? What were their relationships with the H5N2 viruses from the neighboring countries and North America? Most importantly, what were the roles of Taiwan enzootic chicken H6N1 viruses playing in the genesis and evolution of Taiwan chicken H5N2 viruses? Therefore, this study involved the four specific aims: (1) to investigate the genesis, introduction, evolution, and impacts of Taiwan chicken H5N2 viruses; (2) to elucidate the epidemiological characteristics of Taiwan chicken/duck H5N2 viruses and their evolutionary relationships with the 1994 Mexican-like H5N2 viruses; (3) to evaluate the molecular markers of Taiwan chicken and duck H5N2 viruses with clinical and public health importance; and (4) to address recommendations for future prevention/control. AI surveillance involved two stages (I and II) was conducted mainly at a large-scale live-bird market (LBM) in northern Taiwan. The stage I was before Dec. 2012 (Oct. 2005 to Oct. 2006, and Aug. 2008 to Nov. 2012). An enhanced surveillance was implemented in stage II from Dec. 2012 (the first winter after Government officially announced the HPAI H5N2 outbreaks) to Jul. 2013. Monthly chickens’/ducks’ fecal samples and chickens’ bloods were collected for virological and serological surveillance, respectively. AI viruses (AIVs) were isolated from chicken embryonated eggs, and then identified for their subtypes after RNA extraction, reverse transcriptase-polymerase chain reaction (RT-PCR). After alignment of full-length viral sequencing, phylogenetic analyses and evolutionary rates of each virus gene segment were conducted using Maximum-likelihood phylogenies and Bayesian Markov Chain Monte Carlo (MCMC), respectively. For serological surveillance, anti-influenza-NP antibody was firstly screened in chickens’ blood specimens by enzyme-linked immunosorbent assay (ELISA). During the stage II, the seropositive ones were then tested their serotiters for 5 AIVs [TW/12 H3N8, TW/12 H6N8, TW/12 H6N1, TW/12 H5N2, and also HK/08 (H9N2)]. Results of virological surveillance revealed that the isolation rates of AIVs were higher in ducks than those in chickens in both stages [stage I: 2.6% (124/4,798) vs. 0.3% (9/3,363) p < 0.001; stage II: 5.1% (132/2,580) vs. 0.8% (67/8,659), p < 0.001]. Furthermore, subtypes of AIVs isolated from ducks (stage I: H1, H3, H4, H5, H12), stage II: H3, H4, H5, H6, H7) were more diversified than those from chickens (stage I: H6 only, stage II: H3, H5, H6). However, no H9N2 viruses were isolated. The phylogenetic analyses of the 8 viral gene segments of all the isolated 16 chicken H5N2 viruses showed two sources. (1) Surface Genes: Both of the two groups of HA/NA were all closely related with the 2003 LPAI chicken H5N2 viruses that can be traced back to the 1994-Mexican H5N2-like vaccine strain, but away from the domestic duck H5N2 viruses. The evolutionary rates of HA/NA were extremely low, and the accumulated genetic substitutions of the 2003 Taiwan H5N2-like viruses and those of the 1994-96 Mexican-like H5N2 strains to their the most recent common ancestor (MRCA) were almost equal. (b) Internal Genes: All the internal genes of Taiwan chicken H5N2 viruses were derived from Taiwan enzootic chicken H6N1 viruses, different from the sources of their HA/NA, resulted in novel reassortants, unlike the six internal genes of the 1994-Mexican- like H5N2 viruses. Similarly, the five internal (except PB1) genes of the Taiwan chicken H5N2 viruses involved two groups. Group A viruses were clustered with the 2012-13 Taiwan H6N1 viruses whereas group B viruses were clustered with the 2004-05 Taiwan H6N1 viruses. Moreover, the increasing number of the basic amino acids at the connecting peptide of HA from 3 to 4 and repeatedly back to 3 plus the three patterns (REKR, RKKR, RRKR) identified from the recent chicken H5N2 viruses, even two patterns detected in the same sampling, support the unusual diversity of these chicken H5N2 viruses. All together reveal that Taiwan chicken H5N2 viruses had multiple introductions, at least twice in the field, violated against virological and evolutionary behaviors. Serological surveillance verified the presence of low to middle level of anti-Ck/TW2593/12(H5N2) antibody in chickens. Unexpectedly, more than 80% sero-positive rate of H9N2 was detected for the first time and occasionally with very high HI serotiter (1280), although no H9N2 virus has been isolated in Taiwan. It is very likely vaccination was implemented in chickens in Taiwan. To evaluate possible risk of the Taiwan chicken H5N2 and duck H5 viruses to human, viral amino acid (a.a.) associated with pathogenicity, aerosol transmissibility, receptor binding site recognizing mammalian receptors (α2,6 linkage sialosides), and resistance to anti-viral drug [neuraminidase inhibitor (NAI)] were examined. The results revealed that all these AIVs almost were susceptible to NAI and kept the molecular markers belonged to avian type viruses. However, several molecular markers might be related to increasing pathogenicity in mice (chicken H5N2 viruses had 8 substitutions: PB2-L89V, PB2-G309D, PB2-T339K, PB2-R477G, PB2-I495V, M1-N30D, M1-T215A and NS1-I106M; duck H5 viruses had the same 8 substitutions plus additional PB2-A676T, NS1-P42S, NS1-L103F substitutions and ESEV pattern in NS1 C-terminus). This study provides evidence supporting the vaccination of AIV in chickens in Taiwan, even though it is legally prohibited. As increasing AI subtypes infecting humans are identified recently, particularly the world’s human H6N1 pneumonia case in May of 2013 in Taiwan (with nucleotide sequence identities of NS1, NP, PB1, HA to the 2013 Taiwan chicken H6N1 viruses isolated in February from this study ranging 97.6-99.5%), this study recommends three prevention measures: (1) strictly taking legal action on illegal usage of AI vaccine in poultry, (2) enhancing surveillance of AIVs in high risk areas, and investigating whether H9N2 viruses entering Taiwan poultry in a large-scale, (3) comprehensively disinfecting vehicles/cages, evaluation, and traffic route control can prevent further spreading. In future research directions, it is necessary to implement continuous surveillance of AIVs and monitor changes in molecular markers of H5N2 and H6N1 viruses and their other determinants to infect humans. Furthermore, developing a glycan-array applicable to rapidly large-scale screening the AIVs recognizing mammalian receptor can provide early warming and prevent possibly inter-species transmission as well. The mechanism involved in the elevating pathogenicity of A/Ck/TW1680/13(H5N2) strain even under the presence of the potential glycosylation site is worthwhile investigating. Most importantly, integrating inter-disciplinary research on AIVs will better equip us to face future public health challenges.