2009年新型流感病毒A(H1N1)的基因變異、擇選、病毒性狀改變與流行病學特徵、防疫政策及其流行愈趨重要之相關性

The Genetic Variations, Selection, and Phenotypic Changes of the 2009 Pandemic Influenza A (H1N1) Viruses and their Associations with Epidemiological Characteristics, Interventions and Increasing Epidemic Significance

高全良(Chuan-Liang Kao)

指導教授 ： 金傳春

國立臺灣大學/公共衛生學院/流行病學與預防醫學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01166

摘要

2009年3-4月在美國及墨西哥等地爆發新型流感病毒(2009 pandemic influenza A H1N1, pH1N1)的流行，隨後迅速成為全球大流行。台灣地處亞熱帶且人口密度高，因此本研究主旨是探察pH1N1病毒在台灣隨著流行的變異及性狀改變，並探其病毒變異與時間、空間聚集、人口密度等流行病學特徵及重要防疫政策介入的相關性，作為未來新興流感病毒流行的防治之重要參考。本研究分三部分，其目標為: (1) 比較台灣地區台北市、高雄市的新型流感病毒pH1N1血球凝集素(hemagglutinin, HA)及 神經氨酸酶(neuraminidase, NA)的核酸及氨基酸變異，在2009-2010年流行高峰前、中、後期之差異；(2)分析台北特殊變異株(HA-E374K)的散播與時間、空間聚集、人口密度等流行病學特徵及重要防疫政策(使用抗病毒藥物、停班課與疫苗接種)介入之相關性；及 (3)探討此HA-E374K變異株擇選的可能機制，包括生物性狀改變及各段基因的共突變(co-mutations)與病毒適應性(fitness)的相關性。
針對目標(一)，以橫斷研究設計，收集自2009年6月至2010年10月人口密度高的北、高兩市各168株與28株pH1N1病毒，經狗腎細胞(Madin-Darby Canine Kidney, MDCK)兩代培養後，以反轉錄聚合酶連鎖反應(reverse transcription –polymerase chain reaction, RT-PCR)增幅196株pH1N1病毒的HA及其中40株的NA基因，進行核酸及氨基酸定序比對，並分析其在抗原位點、細胞接受器連接位點、醣化位點及對克流感敏感性。同時使用血球凝集抑制試驗(hemagglutination inhibition, HI)進行抗原分析。目標(二)是以台北市的118株pH1N1病毒，並收集患者居住區及其2009年人口密度，再進行空間流行病學研究，以空間自相關(Moran’s I) 全域型空間聚集(global spatial clustering)分析每週是否有空間聚集，且於已有E374K的時間聚集內，再進一步以區域自相關指標(local indicators of spatial association, LISA)分析何處有區域性空間聚集(local spatial clustering)；另採單變項及多變項邏輯分析(univariate and multivariate logistic regression analyses)，檢視年齡、性別、人口密度、地區聚集、使用抗病毒藥物、疫苗施打、停課、疾病嚴重性等與特殊位點氨基酸變異之相關性。最後，為探討E374K變異株持續在人群散播之機制，由美國生物訊息中心 (National Center of Biotechnology Information, NCBI)的流感病毒資料庫，得30株台灣pH1N1病毒的六段(PB2, PB1, PA, NS, NP及M)基因序列，探討此變異株與其抗原變異[HI及微量中和試驗(micro-neutralization, MNt)]、病毒在MDCK細胞的複製力等生物性狀變異及其與此6段基因的核酸、氨基酸序列是否有共突變之關聯性。再以全球觀點明瞭此E374K變異株在全球流行之趨勢。
研究結果發現流行高峰後分離pH1N1病毒的HA及NA氨基酸變異數明顯高於流行高峰前[HA：高峰前、後各為6.7% (1/15)與74.6% (47/63)，p<0.0001；NA：在高峰前、後各為36.84% (7/19)與61.9%(13/21)，p=0.205)]。進一步分析此些病毒氨基酸變異，發現共有兩變異株持續活存：一為在Ca抗原位點的S203T，最早出現在2009年第21週，34週前即佔86.84%(33/38)至35週後躍升為100% (136/136)，顯示S203T的變異與其愈趨增加可能發生在國外。另一為在HA柄上(HA2)的E374K病毒，在第34 週出現，在台灣流行高峰後愈趨增加而成為優勢株(64.65%, 64/99)。其他pH1N1抗原位點的變異均十分低[一個及二個氨基酸變異各為14.9%(26/174)及3.4%(6/174)]，而又未持續存在。此外，6株pH1N1病毒在細胞接受器結合位點上有變異 (4株在220 loop，2株在190-helix)；有2株pH1N1在HA醣化位點數減少1個醣化位點(2.56%, 2/78)，其他40株NA的醣化位點未變，也未找到抗克流感位點H275Y的變異。綜言之，大多數病毒的抗原位點、細胞接受器結合位點及HA與NA醣化位點均未變，且病毒變異(HA S203T, Q293H, D222G, N125D與R205K)與臨床嚴重性無關。
E374K變異株最早在2009年台北流行高峰前3週被分離(33.3%, 3/9)，晚6週後(第40週)在高雄出現(33.3%, 1/3)，且其頻率在北、高隨時間遞增 [高峰前、後期各為9.28% (9/97)與64.65% (64/99), p＜0.0001]，至2010-11冬更高達85.71% (96/112)[原E374株: 1.79% (2/112)，另一新變異(E374G) 株12.5% , (14/112)]。此E374K病毒與疾病嚴重性無關[輕、重症各34.8% (37/116) 與37.3% (19/52), p= 0.6]。續以週數分析，發現E374K在第41-52週較有空間聚集，且聚集處在7個區(包括板橋、萬華、中、永和等)。再以多變項邏輯分析控制年齡與人口密度後，發現流行較後週數(OR=1.53, p <0.001)與空間聚集( OR=4.565, p=0.047)兩因素與E374K分佈率有顯著相關。
三項防疫政策介入後，發現E374K病毒不但未消失，8月1日使用克流感後與其出現頻率顯著增加有相關 [0% (0/17) 與40.78% (73/179), p<0.001)]，但卻未增抗原位點病毒變異[29.41%(5/17)與17.83%(28/157), p=0.324]。此外，於停課第二波高峰(41-45週)，E374K變異株呈41-52週的時空聚集，有高檢出率(90%, 9/10)。11月16日疫苗注射後，E374K頻率更顯著增高[22.9% (32/140)與72.3%(41/56), p<0.001]；亦與HA Sa抗原位的氨基酸變異數增有相關性[2.4% (3/127)與23.4%(11/47), p<0.0001]。
細探E374K成優勢變異株的機轉，在抗原性分析上，以美疾管中心 pH1N1免疫羊、台灣動物科技中心類pH1N1免疫豬及人三血清測試HI或MNt抗體，分析7株E374K血清抗體力價與E374E無顯著差異(≦2倍)；即E374K未藉由抗原變異逃脫免疫而存活。E374K病毒在MDCK的生長曲線，在感染後4-10小時雖高於E374株約0.2-0.3 log，但無明顯差異，仍待人呼吸道細胞驗證。自2009年6月至2010年1 0月間得8株E374K變異株的PB1基因均有100%(8/8)特殊共突變(T257A)，但在2010-11年冬台灣分離的E374K病毒株，卻全為其他共突變所取代，包括PA基因 [N321K(81.82%, 9/11) , A343T (54.55%, 6/11)], M基因 [V80I(81.82%, 9/11)] 及PB1 基因[I397M(54.55%, 6/11), I435T (63.64%, 7/11)]。而E374原株全無此共突變。
綜言之，本研究發現的pH1N1- E374K病毒變異株隨流行時間愈趨增加現象，在人口密集的新加坡、英國、中國及印度亦如此，顯示此株的變異及適應性具有跨地區之共同性。可能在低免疫壓力下，經自然演化變異，在適當基因位(PB2, PB1 及PA等)生共突變，助其在人呼吸道細胞得適當繁殖力，於時空聚集及防疫介入下，仍能在人與人間快速傳播而成優勢群，此推論仍須增樣品數進一步探究。本研究為首次結合病毒學、血清學、臨床分析、時空聚集等流行病學特徵及防疫政策探討新型流感病毒之變異。未來新型流感侵襲時，應在流行上升及高峰期具高空間聚集區病毒八段基因及氨基酸的監測分析，作為疫情防治之重要參考。

關鍵字

新型流感病毒 ； 流行病學 ； 病毒變異 ； 血球凝集素 ； 公共衛生介入

並列摘要

Newly emerged triple reassortant 2009 pandemic influenza A (pH1N1) viruses were detected in the United States (US) and Mexico in March-April, 2009 and then rapidly spread worldwide. The overall objective of this study was to investigate the association between molecular and phenotypic dynamic changes of pH1N1 viruses and epidemiological characteristics and Taiwan’s public health interventions for better prevention/control of novel influenza viruses in the future. The specific aims were: 1) to compare viral sequence variations in nucleotides (NTs) and amino acids (AAs) of hemagglutinin (HA) and neuraminidase (NA) of pH1N1 isolated in Taipei and Kaohsiung metropolitans at pre-peak, on-going peak and post-peak of the 2009-2010 epidemic, 2) to analyze the association between the spreading of Taipei’s HA-E374K mutants and epidemiological characteristics and public health interventions, and 3) to explore the selection mechanisms of E374K, including viral biophenotypic changes and co-mutations in the other genes for better fitness.
A cross-sectional study was performed, using 196 pH1N1 virus strains (168 Taipei’s and 28 Kaohsiung’s) from June, 2009 to October, 2010. The viruses were passaged twice in the Madin-Darby Canine Kidney (MDCK) cells and viral nucleic acids were amplified by reverse transcription–polymerase chain reaction, (RT-PCR). The NTs and AAs of 196 HA and 40 NA genes were analyzed their viral antigenic sites, receptor binding, N-linked glycosylation sites and drug resistance genes. Strain variations in viral antigenicity used hemagglutination inhibition (HI) test. Tempo-spatial analyses of 118 pH1N1 strains of Taipei’s patients with their residential district-specific population density used the Morans’s I to measure presence of E374K cluster by global spatial clustering analysis and to further examine where were local spatial clusters by local indicators of spatial association (LISA). The association between E374K and epidemiological characteristics (age, gender, population density of the districts, and spatial clustering), or at different periods after 3 strategies of interventions (use of antiviral drug, class suspension and vaccination), or clinical severity was analyzed by univariate and multiple logistic regression analyses. Lastly, to elucidate selection mechanisms for the fitness of E374K better than E374, viral antigenicity changes, replication ability and the co-mutation of the six internal viral genes were compared, using the full-length sequences (PB2, PB1, PA, NS, M, NP) of 30 Taiwanese pH1N1 strains collected from the Influenza Virus Resource, National Center of Biotechnology Information (NCBI), USA. Global trends in increasing E374K mutants were also examined using NCBI sequence data in different countries.
The results revealed that the cumulated numbers of AA changes in HA and NA were higher in the post–peak than those in the pre-peak period of the epidemic [HA: 6.7% (1/15) vs 74.6% (47/63)，p<0.0001； NA: 36.84% (7/19) vs 61.9% (13/21)，p=0.21)]. Detail analyses identified two mutants persistently circulated with increasing percentages. One mutant, HA-S203T located at antigenic site Ca, was firstly detected at 21th week, 2009 and became dominant before week 34 (86.84%, 33/38), and totally replaced after week 35 (100%, 136/136), suggesting that the S203T mutant emerged and increased viral frequency in foreign countries in early pandemic before it entered Taiwan. The other mutant, E374K located at the stalk region of HA2 was firstly found at week 34 in Taipei and rose as a major circulated strain at post-peak of the epidemic (64.65%, 64/99). In addition, 14.94% and 3.44% of 174 isolates had one and two amino acids changes in the four antigenic sites, respectively but they did not persist through all the epidemic periods. Only 6 strains had variations at receptor binding sites (4 at 220-loop and 2 at 190-helix) and another 2 strains showed variations in the loss of one N-linked glycosylation site of HA (2.56%, 2/78). The NA of 40 strains retained all N-linked glycosylation sites without H275Y mutation responsible for Tamiflu resistance. Taken together, most of the pH1N1 had conserved antigenicity, N-linked glycosylation sites of HA and NA and variations in HA (S203T, Q293H, D222G, N125D and R205K) were not associated with clinical severity.
The unique adaptive E374K mutant was first detected at 3 weeks before the epidemic peak in Taipei and 6 weeks later (40th week) in Kaohsiung and then increased significantly higher in the post-peak than those in the pre-peak period of the epidemic [64.65% (64/99) vs 9.28% (9/97), p＜0.0001] in both cities. The frequency of E374K reached to 85.7% (96/112) in 2010-2011 winter [wild type E374: 1.8% (2/112), E374G:12.5%, (14/112)].The E374K was not associated with clinical severity [mild vs severe cases: 34.8% (37/116) vs 37.3% (19/52), p=0.6]. The tempo-spatial spreading of E374K mutants was more concentrated during the post–peak (41st-52nd week) in seven districts of Taipei City. Multivariate logistic regression analysis confirmed that higher odds ratios (OR) occurred in later time periods (OR=1.53, p <0.001) and in areas with spatial clustering (OR=4.57, p=0.047), after controlling age and population density.
After the three major interventions, the E374K variant did not disappear but was even associated with increasing percentages after the usage of Tamiflu since August 1, 2009 [0% (0/17) vs 40.78% (73/179), p<0.001]. Such a phenomenon was not found in other mutations in the four antigenic sites [29.4% (5/17) vs 17.8% (28/157), p=0.32]. During the 2nd peak of class suspension (week 41-45), the E374K reached 90% (9/10) with tempo-spatial clusters within weeks of 41-52. Finally, these E374K mutants increased after vaccination (22.9%, 32/140 vs 72.3%, 41/56, p<0.001) with persistently high frequency through 10 months post-vaccination on November 1 16, 2009. Vaccination also significantly elevated Sa mutants (2.4%, 3/127 vs 23.4%, 11/47, p<0.001).
To investigate the mechanism of the survival of E374K in human, 7 E374K strains were firstly tested for HI or MNt antibodies, using the three anti-pH1N1-HI(+) serum samples from human, sheep and pig. No significant difference in sero-titers between E374K and E374 (≦2 fold), indicated that E374K did not survive through immune escape. The growth curve of E374K in MDCK cells showed a similar pattern to that of E374 without significant difference. The replication advantage of E374K needs to be further tested in human respiratory tract cells. Lastly, co-mutation analyses revealed that 8 E374K viruses isolated from June, 2009 to October, 2010 had 100% (8/8) unique co-mutations at T257A of PB1 but such a co-mutation was totally replaced by other sites [PA: N321K (81.82%, 9/11), A343T (54.55%, 9/11); M: V89I (81.82%, 9/11); PB1: I397M (54.55%. 6/11), I435T (63.64%. 7/11)] in E374K viruses obtained from November 1, 2010 to February 2011. All co-mutations were absent in E374 viruses.
Taken together, the Taiwan’s finding on temporal increase in E374K percentage from this study was consistent with observations in several high population countries (Singapore, UK, China and India). It is very likely that E374K evolved through natural evolution under low selection pressure and obtained evolutionary advantages at specific sites with temp-spatial clusters of cases in areas with high population density, possibly through co-mutations in other genes and thus facilitating better viral replication capability in human respiratory cells and fast human-to-human transmission to become a dominant mutant. Future efforts need to increase sample size and examine the E374 replication in different human respiratory cells for further confirmation.
This is the first study examining viral changes during the naïve phase of a pandemic of influenza through integrated virological/serological/clinical surveillance, tempo-spatial analysis, and intervention policies. Our results enlighten to carefully monitor amino acids of HA and NA and co-mutations in other segments of pandemic influenza viruses isolated at exponential/peak phases in areas with high cluster cases.

並列關鍵字

human 2009 pdm influenza virus A (H1N1) ； pandemic ； public healt ； hemagglutinin ； neuraminidase ； epidemiological attributes for antigenic drif ； amino acid changes ； mutation ； epidemiology ； Taiwan

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