臺灣博碩士論文加值系統

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。

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.

Contents

Contents Page No

Signature page (口試委員會審定書)………………………………………….…… I
Acknowledgement (致謝)…………………………………………………………....II
Chinese Abstract (中文摘要)……………………………………................………..III
English Abstract (英文摘要)…………………………………………….…….……VI
Contents………………………………………………..............................................1
Contents of Figures……………………………………………………..…………..9
Contents of Tables………………………………………………………………....10
Chapter 1: Introduction.……………………………………………………...…..12
Chapter 2: Literature Review……………………………….…………………....16
2.1. Epidemiological Studies of the 2009 Pandemic
influenza A virus H1N1 (pH1N1)……………………………………..........16
2.1.1. General Aspects of epidemiology…………….……………………...…16
2.1.1.1. Global Issues about the Pandemic Spreading………….……….….16
A. Cross-Country Spreading at the Initial Stage of
the 2009 Pandemic…………………………………….….16
B. Mortality and Case Fatal rates ………………………………………17
1. Mortality Rates, Case Fatality Rates………………………………17
2. Risk Factors for Mortality…………………………………………18.
C. Attack Rates………………………………………………………….19
1. Hospitalization and/or ICU Admission……………………………19
2. Schools………………………………………………...…………..20
3. Households…………………………………………………………21
2.1.1.2. Unique Epidemiological Characteristics…………………..………..22
A. Age……………………………………………………………………22
B. Obesity……………………………………………………………… .22
C. Pregnancy………………………………………………………….….23
D. Underlying Diseases……………………………………………….….24
2.1.2. Risk Factors Facilitate Viral Transmission
of pH1N1…………………………………………………….………...24
2.1.2.1. Risk factors related to facilitate Human-to-Human
Transmission………………………………………………..………..24
A. Population density…………………………………………………….24
B. Close contacts…………………………………………………………25
C. Public transportation……………………………………………..……25
2.1.2.2. Environmental Factors Facilitate Viral Transmission of pH1N1
A. Temperature…………………………………………………….……...26
B. Humidity…………………………………………………………..…...26
2.1.3. Tempo-spatial Transmission of pH1N1…………………………………....27
2.1.3.1. Unique Characteristics…………………………………………..........27
A. Geographical variation of pH1N1 Viruses………………………….….27
B. Tempo-spatial Clustering and Transmission……………………………28
2.2. Molecular and Phenotypic Variations of pH1N1………..…………………......29
2.2.1 Global Status…………………………………………….…………..……..29
A. America……………………………………………….………..….…...30
1. The First Wave in the USA and Mexico………………………….....30
2. Early Epidemic Period in Mexico City……………………...……....31.
3. The 1st and the 2nd Waves in New York State, Milwaukee
of Wisconsin and Houston of Texas…………………………………31
4. The First and Second Waves in Canada………………………………32
5. The South American…………………………………………………..33
B. Europe……………………………………………….……………………33
1. Finland…………………………………………………………….......34
2. The United Kingdom (UK)……………………………………….......34
3. Spain………………………………………………………………….35
4. Greece………………………………………………………………..36
C. Asia………………………………………………….…………………..36
1. SARS Non-affected Countries/Areas……………………………….36
a) Japan……………………………………………………………36
b) India…………………………………………………………….37
c) Malaysia…………………………………………………….….38
2. SARS-affected Countries………………………….………………...38
a) Singapore………………………………………………………38
b) Hong Kong………………………………………………….....39
c) China………………………………………………………......39.
d) Taiwan………………………………………………………....41

2.2.2. Clinical Severity and Viral Virulence………………….……………..…42
A. Clinical Severity……………………………………..……………....42
B. Viral Virulence in Humans and Animal Experiments….……….…...43
2.2.3. Antigenic Variants………………………………………..…………..…44
2.2.4. Receptor Binding Site Variants…………………………………………44
2.2.5. Replication and Polymerase Variations……………………...……….....44
2.2.6. Co-evolutions of Mutations……………………..………....………..….45
2.2.7. Viral Fitness and Adaptations…………………...….………...……..…...45
2.2.8. Other Biological Features with Public
Health Significance…………………………………..………………….46
A. Drug Resistance……….……………………………………………...46
B. Glycosylation Sites…………………………………………………...46
C. Herd Immunity……………………………………………………….47

2.3. Public Health Interventions to Mitigate the 2009 pH1N1 Epidemic………..47

2.3.1. School Closure…………………………………………………............47
2.3.2. Antiviral Drug Intervention…………………………………………….48
2.3.3. Vaccination Campaign………………………………..………………..49
2.3.4. Summary of Public Health Interventions……………………………...50
A. The Effectiveness of Intervention on the Control of
pH1N1 Epidemics…………………………………………………50
B. The Role of Intervention in the Variations of pH1N1……………....51
Chapter 3: Objective, Specific Aims and Hypothesis…………………………..52
Chapter 4: Material and Methods………………………………………………54
4.1. Study Design and Study Populations………………………………………54
4.2. Comparison in viral sequence variations of HA and NA of pH1N1
through different periods of the 2009-2010 epidemic.…………………..55
4.2.1. Isolation of pH1N1 Viruses……………………………………….……….55
4.2.2. Nucleotide Sequencing of HA and NA Genes of
pH1N1 Viruses………………………..………………………...……….....56
4.2.3. Antigenicity Analysis……………………………………………………..57
4,2.3.1. Hemagglutination Inhibition (HI) Test.………………………………57
4.3. The spreading of Taipei’s HA-E374K mutants associated
with epidemiological characteristics public health interventions…………...58
4.3.1. Temporal comparison the frequency of E374K…………………….…….58
4.3.2. Tempo-Spatial Analyses and Statistic tests……………………………....58
4.3.3. Public Health Interventions………………………………………...........60
4.3.4. Epidemiological Data Analyses on Risk Factors for E374K…………….60
4.3.5. Global Comparison on the Temporal
Distributions of pH1N1-HA-E374K Mutants……………….…………..61
4.4. Possible Mechanisms of the Fitness of E374K Mutants
4.4.1. Antigenicity Analysis…………………………………………………....62
4.4.1.1. Hemagglutination Inhibition (HI) Test……………………………...62
4.4.1.2. Micro-Neutralization (MNt) Assay……………………..…………..62
4.4.2. The Replication of pH1N1 in MDCK Cell……………………………...63
4.4.2.1. Quantitative Real-Time RT-PCR…………………………………...64
4.4.3. Analysis the Co-Mutations of PB1, PB2, PA, NP, M and NS
of Two Fixed Mutants (E374K and S203T)………………….................64
4.4.3.1. E374K Mutants……………………………………………………..64
4.4.3.2. S203T……………………………………………………………....65

Chapter 5: Results…………………………………………………………….........66
5.1. Comparison in viral sequence variations of HA and NA of
pH1N1 through different periods of the 2009-2010 epidemic ……………..66
5.1.1. Comparison of the positive detection rates of pH1N1
in northern and southern Taiwan……………………………………….66
5.1.2.The diversity of amino acid residues and dynamic changes
in antigenic sites and receptor-binding sites in HA1 of pH1N1 ……….…....67
5.1.3.The diversity of amino acid residues of NA of pH1N1 isolates.………..…..71
5.1.4. Amino acid changes at N-glycosylation sites of HA and NA…………..….73
5.1.5. pH1N1 HA1 virus variants and clinical severity…………………….…….73
5.1.6. Summary of pH1N1 Variants in HA and NA……………………….……...74
5.2. The spreading of Taipei’s HA-E374K mutants associated with
epidemiological characteristics public health interventions…………………..74
5.2.1. Emergence of pH1N1-HA E374K mutants related to different
intervention strategies in various phases over the epidemic period……........74

5.2.2.Tempo-spatial analysis of dynamic changes of E374K mutants……………77
5.2.3. Global comparison on the temporal distributions of pH1N1-
HA-E374K Mutants in different influenza transmission zones in
2009……………………………………………………………..…………78
5.3. The selection mechanisms of E374K for better fitness…………………….……79
5.3.1. Effect of the E374K mutation on the antigenicity and neutralization
activity of HA…………………………………………………………….79
5.3.2. Compare the growth curve of E374K and E374 in MDCK cells………...81
5.3.3. Analysis of co-mutation of the PB2, PB1, PA, NS, NP and M gene
with HA E374K mutation…………………………………………………81
5.3.4. The S203T co-mutations in the PB2, PB1, PA, NS, NP, M genes –
Searching for possible common mechanisms for better fitness
of E374K………………………………………………………………….82

Chapter 6: Discussion…………….………………………………………………... 83

6.1. Dynamic Temporal Changes of pH1N1 and Virus Evolution in Taiwan……..83
6.1.1. Evolution of Influenza Viruses in General and pH1N1 Viruses……….….84
6.1.2. Phenotypic Variations in Influenza Viruses………………………………85
A. Antigenic Variations of HA and Immune Pressure………………………85
B. Clinical Correlations and HA Variants of pH1N1………………..………..87
C. NA Variants of pH1N1………………………………………………..…...87
6.1.3. Three pH1N1 Fixed Variants of HA and NA……………………………..88
6.2. The Epidemiology of HA2-E374K Mutants and Public
Health Interventions……………………………………………………………89
6.2.1. E374K Mutants and the Three Taiwan’s Public Health
Interventions…………………………………………….………………..89
6.2.2. Epidemiology of E374K Mutants………………………………………..90
6.2.3. E374K Evolution through both Natural Transmission and
Public Health Interventions in Taiwan…………………………………..91
6.2.4. Global Epidemiology of E374K Mutants………………………………..92
6.3. Possible Mechanisms Involved in Selection and
Fitness of E374K Mutants………………………………………………………92
6.3.1. Antibody-Escape Mechanism…………………………………………….93
6.3.2. Replication Advantage Mechanism………………………………............93
6.3.3. E374K and Co-mutations in Other Segments……………………………94
6.4. Limitations of the Study………………………………………………….……95
6.5. Conclusion………………………………………………………………….….96
6.6. Future Directions……………………………………………………….……..97

Chapter 7. Recommendation………………………………………………………98
References……………………………..…………………………………………... 100
Autobiography……………………………………………………………………...140

References

1.Ginsberg M, Hopkins J, Maroufi A, Dunne G, Sunega DR, et al. (2009) Swine influenza A (H1N1) infection in two children-Southern California, March-April 2009. MMWR 58: 400-402.
2.Novel Swine-Origin Influenza A (H1N1) Virus Investigation Team (2009) Emergence of a novel swine-origin influenza A (H1N1) virus in humans. N Engl J Med 360: 2605-2615.
3.Smith GJ, Vijaykrishna D, Bahl J, Lycett SJ, Worobey M, et al. (2009) Origins and evolutionary genomics of the 2009 swine-origin H1N1 influenza A epidemic. Nature 459:1122-1125.
4.Drake JW (1993) Rates of spontaneous mutation among RNA viruses. Proc Natl Acad Sci U S A 90: 4171-4175.
5.Ortín J, Nájera R, López C, Dávila M, Domingo E (1980) Genetic variability of Hong Kong (H3N2) influenza viruses: spontaneous mutations and their location in the viral genome. Gene 11: 319-331.
6.Huang JW, King CC, Yang JM (2009) Co-evolution positions and rules for antigenic variants of human influenza A/H3N2 viruses. BMC Bioinformatics. Jan 30;10 Suppl 1:S41
7.Steinhauer DA, Skehel JJ (2002) Genetics of influenza viruses. Annu Rev Genet 36: 305–332.
8.Webster RG, Bean WJ, Gorman OT, Chambers TM, Kawaoka Y (1992) Evolution and ecology of influenza A viruses. Microbiol Rev 56(1):152-179.
9.Zambon MC (2001) The pathogenesis of influenza in humans. Rev Med Virol 11: 227-241.
10.de Wit E , Munster VJ, van Riel D, Beyer WE, Rimmelzwaan GF, et al. (2010) Molecular determinants of adaptation of highly pathogenic avian influenza H7N7 viruses to efficient replication in the human host. J Virol 84: 1597-1606.
11.Kobasa D, Wells K, Kawaoka Y (2001) Amino acids responsible for the absolute sialidase activity of the influenza A virus neuraminidase: relationship to growth in the duck intestine. J Virol 75: 11773–11780.
12.Hughes MT, McGregor M, Suzuki T, Suzuki Y, Kawaoka Y (2001) Adaptation of influenza A viruses to cells expressing low levels of sialic acid leads to loss of neuraminidase activity. J Virol 75: 3766-3770.
13.Boni MF (2008) Vaccination and antigenic drift in influenza. Vaccine 26 (Suppl 3): C8-14.
14.Boni MF, Gog JR, Anderson V, Feldman MW (2006) Epidemic dynamics and antigenic evolution in a single season of influenza A. Proc Biol Sci 273: 1307-1316.
15.Lee CW, Senne DA, Suarez DL (2004) Effect of vaccine use in the evolution of Mexican lineage H5N2 avian influenza virus. J Virol 78: 8372–8381.
16.Zepeda HM, Perea-Araujo L, Zarate-Segura PB, Vázquez-Pérez JA, Miliar-García A, et al. (2010) Identification of influenza A pandemic (H1N1) 2009 variants during the first 2009 influenza outbreak in Mexico City. J Clin Virol 48: 36-39.
17.Glinsky GV (2010) Genomic analysis of pandemic (H1N1) 2009 reveals association of increasing disease severity with emergence of novel hemagglutinin mutations. Cell Cycle 9: 958-970.
18.van der Vries E, Stelma FF, Boucher CA (2010) Emergence of a multidrug-resistant pandemic influenza A (H1N1) virus. N Engl J Med 363: 1381-1382.
19.Melidou A, Gioula G, Exindari M, Chatzidimitriou D, Diza E, et al. (2010) Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections. Virus Res 151: 192-199.
20.Puzelli S, Facchini M, Spagnolo D, De Marco MA, Calzoletti L, et al. (2010) Transmission of hemagglutinin D222G mutant strain of pandemic (H1N1) 2009 virus. Emerg Infect Dis 16: 863-865
21.Maurer-Stroh S, Chuen Lee RTC, Eisenhaber F, Lin Cui L, et al. (2010) A new common mutation in the hemagglutinin of the 2009 (H1N1) influenza A virus. PLoS Curr 2: RRN1162.
22.Tscherne DM, García-Sastre A (2011) Virulence determinants of pandemic influenza viruses. J Clin Invest 121(1):6-13
23.Dept of Internal Affairs, Taiwan, R.O.C. [http://www.ris.gov.tw/ch4/static/y0s609800.xls]
24.Ho TS, Wang SM, Liu CC (2010) Historical review of pandemic influenza A in Taiwan, 2009. Pediatr Neonatol 51: 83−88.
25.Huseh PR, Lee PI, Chiu AWH, Yen MY (2010) Pandemic (H1N1) 2009 vaccination and class suspensions after outbreaks, Taipei City, Taiwan. Emerg Infect Dis 16:1309-1311.
26.Huang WT, Hsu CC, Lee PI, Chuang JH (2010) Mass psychogenic illness in nationwide in-school vaccination for pandemic influenza A(H1N1) 2009, Taiwan, November 2009-January 2010. Euro Surveill 15(21): 19575.
27.Jhung MA, Swerdlow D, Olsen SJ, Jernigan D, Biggerstaff M, Kamimoto L, Kniss K, Reed C, Fry A, Brammer L, Gindler J, Gregg WJ, Bresee J, Finelli L (2011) Epidemiology of 2009 pandemic influenza A (H1N1) in the United States. Clin Infect Dis 52 Suppl 1:S13-26.
28.Chowell G, Echevarría-Zuno S, Viboud C, Simonsen L, Tamerius J, Miller MA, Borja-Aburto VH (2011) Characterizing the epidemiology of the 2009 influenza A/H1N1 pandemic in Mexico PLoS Med 8(5):e1000436.
29.Graham M, Liang B, Van Domselaar G, Bastien N, Beaudoin C, et al. (2011) Nationwide molecular surveillance of pandemic H1N1 influenza A virus genomes: Canada, 2009. PLoS One 6: e16087.
30.Galiano M, Agapow PM, Thompson C, Platt S, Underwood A, Ellis J, Myers R, Green J, Zambon M (2011) Evolutionary pathways of the pandemic influenza A (H1N1) 2009 in the UK. PLoS One 6(8):e23779.
31.Shiino T, Okabe N, Yasui Y, Sunagawa T, Ujike M, Obuchi M, Kishida N, Xu H, Takashita E, Anraku A, Ito R, Doi T, Ejima M, Sugawara H, Horikawa H, Yamazaki S, Kato Y, Oguchi A, Fujita N, Odagiri T, Tashiro M, Watanabe H (2010) Molecular evolutionary analysis of the influenza A(H1N1) pdm, May-September, 2009: temporal and spatial spreading profile of the viruses in Japan. PLoS One 5(6):e11057.
32.Wu JT, Cowling BJ, Lau EH, Ip DK, Ho LM, Tsang T, Chuang SK, Leung PY, Lo SV, Liu SH, Riley S (2010) School closure and mitigation of pandemic (H1N1) 2009, Hong Kong. Emerg Infect Dis 16(3):538-41.
33.Bishop JF, Murnane MP, Owen R (2009) Australia''s winter with the 2009 pandemic influenza A (H1N1) virus. N Engl J Med 361(27):2591-4
34.Opatowski L, Fraser C, Griffin J, de Silva E, Van Kerkhove MD, Lyons EJ, Cauchemez S, Ferguson NM(2011) Transmission characteristics of the 2009 H1N1 influenza pandemic: comparison of 8 Southern hemisphere countries. PLoS Pathog7(9):e1002225
35.Nishiura H (2011) The virulence of pandemic influenza A (H1N1) 2009: an epidemiological perspective on the case-fatality ratio. Expert Rev Respir Med.4(3):329-38.
36.Presanis AM, De Angelis D; New York City Swine Flu Investigation Team, Hagy A, Reed C, Riley S, Cooper BS, Finelli L, Biedrzycki P, Lipsitch M (2009) The severity of pandemic H1N1 influenza in the United States, from April to July 2009: a Bayesian analysis. PLoS Med 6(12):e1000207.
37.Donaldson LJ, Rutter PD, Ellis BM (2009) Mortality from pandemic A/H1N1 2009 influenza in England. BMJ 2009, 339:b5213-b5213.
38.Kasowski EJ, Garten RJ, Bridges CB(2011) Influenza pandemic epidemiologic and virologic diversity: reminding ourselves of the possibilities. Clin Infect Dis. 2011 Jan 1;52 Suppl 1:S44-9.
39.Fraser C, Donnelly CA, Cauchemez S, Hanage WP, Van Kerkhove MD et al. (2009) Pandemic potential of a strain of influenza A (H1N1): early findings. Science 324(5934):1557-61.
40.Steens A, Waaijenborg S, Teunis PF, Reimerink JH, Meijer A, van der Lubben M, Koopmans M, van der Sande MA, Wallinga J, van Boven M (2011) Age-dependent patterns of infection and severity explaining the low impact of 2009 influenza A (H1N1): evidence from serial serologic surveys in the Netherlands. Am J Epidemiol 174(11):1307-15.
41.Centers for Disease Control and Prevention (CDC) (2011) Severe illness from 2009 pandemic influenza A (H1N1)--Utah, 2009-10 influenza season. Morb Mortal Wkly Rep 60(38):1310-4.
42.Helferty M, Vachon J, Tarasuk J, Rodin R, Spika J, Pelletier L(2010) Incidence of hospital admissions and severe outcomes during the first and second waves of pandemic (H1N1) 2009. CMAJ 182(18):1981-7.
43.Li T, Liu Y, Di B, Wang M, Shen J, Zhang Y, Chen X, Yuan J, Wu J, Li K, Lu E, Wu Y, Hao A, Chen X, Wang Y, Liu J, Pickerill S, Zheng B (2011) Epidemiological investigation of an outbreak of pandemic influenza A (H1N1) 2009 in a boarding school: serological analysis of 1570 cases. J Clin Virol 50(3):235-9.
44.Huai Y, Lin J, Varma JK, Peng Z, He J, Cheng C, Zhong H, Chen Y, Zheng Y, Luo Y, Liang W, Wu X, Huang Z, McFarland J, Feng Z, Uyeki TM, Yu H (2010) A primary school outbreak of pandemic 2009 influenza A (H1N1) in China. Influenza Other Respi Viruses 4(5):259-66.
45.Jackson ML, France AM, Hancock K, Lu X, Veguilla V, Sun H, Liu F, Hadler J, Harcourt BH, Esposito DH, Zimmerman CM, Katz JM, Fry AM, Schrag SJ (2011) Serologically confirmed household transmission of 2009 pandemic influenza A (H1N1) virus during the first pandemic wave--New York City, April-May 2009. Clin Infect Dis 53(5):455-62.
46.Leung YH, Li MP, Chuang SK (2011) A school outbreak of pandemic (H1N1) 2009 infection: assessment of secondary household transmission and the protective role of oseltamivir. Epidemiol Infect 139(1):41-4.
47.Dellagi K, Rollot O, Temmam S, Salez N, Guernier V et al. (2011) Pandemic influenza due to pH1N1/2009 virus: estimation of infection burden in Reunion Island through a prospective serosurvey, austral winter 2009. PLoS One 2 6(9):e25738.
48.Kerkhove MDV, Vandemaele KA, Shinde V, Jaramillo-Gutierrez G, Koukounari A, et al. Risk factors for severe outcomes following 2009 influenza A (H1N1) infection: a global pooled analysis. PLoS Med 8(7):e1001053
49.Ward KA, Spokes PJ, McAnulty JM (2011) Case-control study of risk factors for hospitalization caused by pandemic (H1N1) 2009. Emerg Infect Dis. 201117(8):1409-16.
50.Liu Y, Wang W, Li X, Wang H, Luo Y, Wu L, Guo X (2011) Geographic distribution and risk factors of the initial adult hospitalized cases of 2009 pandemic influenza A (H1N1) virus infection in mainland China. PLoS One.;6(10):e25934.
51.Iuliano AD, Dawood FS, Silk BJ, Bhattarai A, Copeland D, Doshi S, France AM, Jackson ML, Kennedy E, Loustalot F, Marchbanks T, Mitchell T, Averhoff F, Olsen SJ, Swerdlow DL, Finelli L (2011) Investigating 2009 pandemic influenza A (H1N1) in US schools: what have we learned? Clin Infect Dis 52 Suppl 1:S161-7.
52.Sikora C, Fan S, Golonka R, Sturtevant D, Gratrix J, Lee BE, Jaipaul J, Johnson M (2010) Transmission of pandemic influenza A (H1N1) 2009 within households: Edmonton, Canada. J Clin Virol 49(2):90-3.
53..Foxwell AR, Roberts L, Lokuge K, Kelly PM (2011) Transmission of influenza on international flights, may 2009. Emerg Infect Dis 17(7):1188-94.
54.Cui F, Luo H, Zhou L, Yin D, Zheng C, Wang D, Gong J, Fang G, He J, McFarland J, Yu H. (2011) Transmission of pandemic influenza A (H1N1) virus in a train in China. J Epidemiol. 2011 21(4):271-7.
55.Tarabbo M, Lapa D, Castilletti C, Tommaselli P, Guarducci R et al.(2009) Retrospective investigation of an influenza A/H1N1pdm outbreak in an Italian military ship cruising in the Mediterranean Sea, May-September 2009. PLoS One 6(1):e15933.
56.Dublineau A, Batéjat C, Pinon A, Burguière AM, Leclercq I, Manuguerra JC (2011) Persistence of the 2009 pandemic influenza A (H1N1) virus in water and on non-porous surface. PLoS One 6(11):e28043.
57.Mubareka S, Lowen AC, Steel J, Coates AL, García-Sastre A, Palese P (2009) Transmission of influenza virus via aerosols and fomites in the guinea pig model. J Infect Dis. 2009 Mar 15;199(6):858-65.
58.Shaman J, Kohn M (2009) Absolute humidity modulates influenza survival, transmission, and seasonality. Proc Natl Acad Sci U S A.106(9):3243–3248.
59.Shaman J, Pitzer VE, Viboud C, et al. Absolute humidity and the seasonal onset of influenza in the continental United States [electronic article]. PLoS Biol. 2010;8(2):e1000316.
60.Shaman J, Goldstein E, Lipsitch M (2011) Absolute humidity and pandemic versus epidemic influenza. Am J Epidemiol 173(2):127-35.
61.Steel J, Palese P, Lowen AC (2011) Transmission of a 2009 pandemic influenza virus shows a sensitivity to temperature and humidity similar to that of an H3N2 seasonal strain. J Virol 85(3):1400-1402.
62.Nelson M, Spiro D, Wentworth D, Beck E, Fan J, et al. (2009) The early diversification of influenza A/H1N1pdm. PLoS Curr 1: RRN1126.
63.Nelson MI, Tan Y, Ghedin E, Wentworth DE, St George K, Edelman L, Beck ET, Fan J, Lam TT, Kumar S, Spiro DJ, Simonsen L, Viboud C, Holmes EC, Henrickson KJ, Musser JM(2011) Phylogeography of the spring and fall waves of the H1N1/09 pandemic influenza virus in the United States. J Virol 85(2):828-34.
64.Zhang X, He J, Li L, Zhu X, Ke C, Ni H, Hou N, Zhong H, Wu J (2011) Serologic survey of the pandemic H1N1 2009 virus in Guangdong Province, China: a cross sectional study. PLoS One 6(8):e23034.
65.Chowell G, Viboud C, Munayco CV, Gómez J, Simonsen L, Miller MA, Tamerius J, Fiestas V, Halsey ES, Laguna-Torres VA(2011) Spatial and temporal characteristics of the 2009 A/H1N1 influenza pandemic in Peru. PLoS One 6(6):e21287.
66.Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, et al. (2009) Antigenic and genetic characteristics of swine-origin 2009 A(H1N1) influenza viruses circulating in humans. Science 325(5937):197-201
67.Goñi N, Moratorio G, Ramas V, Coppola L, Chiparelli H, Cristina J.Phylogenetic analysis of pandemic 2009 influenza A virus circulating in the South American region: genetic relationships and vaccine strain match. Arch Virol 156(1):87-94.
68.Ikonen N, Haanpää M, Rönkkö E, Lyytikäinen O, Kuusi M, et al. (2010) Genetic diversity of the 2009 pandemic influenza A(H1N1) viruses in Finland. PLoS One 5: e13329.
69.Ledesma J, Pozo F, Reina G, Blasco M, Rodríguez G, Montes M, López-Miragaya I, Salvador C, Reina J, Ortíz de Lejarazu R, Egido P, López Barba J, Delgado C, Cuevas MT, Casas I; Spanish Influenza Surveillance System (SISS) (2012) Genetic diversity of influenza A(H1N1)2009 virus circulating during the season 2010-2011 in Spain. J Clin Virol 53(1):16-21
70.Melidou A, Gioula G, Exindari M, Chatzidimitriou D, Diza E, Malisiovas N (2010) Molecular and phylogenetic analysis of the haemagglutinin gene of pandemic influenza H1N1 2009 viruses associated with severe and fatal infections. Virus Res 151(2):192-9.
71.Morlighem JÉ, Aoki S, Kishima M, Hanami M, Ogawa C, et al. (2011) Mutation analysis of 2009 pandemic influenza A(H1N1) viruses collected in Japan during the peak phase of the pandemic. PLoS One 6: e18956.
72.Mullick J, Cherian SS, Potdar VA, Chadha MS, Mishra AC (2011) Evolutionary dynamics of the influenza A pandemic (H1N1) 2009 virus with emphasis on Indian isolates: evidence for adaptive evolution in the HA gene. Infect Genet Evol 11(5):997-1005.
73.Balraj P, Sidek H, Suppiah J, Khoo AS, Saat Z(2011) Molecular analysis of 2009 pandemic influenza A(H1N1) in Malaysia associated with mild and severe infections. Malays J Pathol 33(1):7-12.
74.Barr IG, Cui L, Komadina N, Lee RT, Lin RT (2010) A new pandemic influenza A(H1N1) genetic variant predominated in the winter 2010 influenza season in Australia, New Zealand and Singapore. Euro Surveill 15(42) pii: 19692.
75.Poon LL, Chan KH, Chu DK, Fung CC, Cheng CK, et al. (2011) Viral genetic sequence variations in pandemic H1N1/2009 and seasonal H3N2 influenza viruses within an individual, a household and a community. J Clin Virol 52(2): 146-150.
76.Zhao JR, Li YD, Pan LM, Zhu N, Ni HX, Xu GZ, Jiang YZ, Huo XX, Xu JQ, Xia H, Han N, Tang S, Zhang Z, Kou Z, Rayner S, Li TX (2011) Genetic characteristics of 2009 pandemic H1N1 influenza a viruses isolated from Mainland China. Virol Sin 26(6):418-27.
77.Farooqui A, Lei Y, Wang P, Huang J, Lin J, Li G, Leon AJ, Zhao Z, Kelvin DJ (2011) Genetic and clinical assessment of 2009 pandemic influenza in southern China. J Infect Dev Ctries 5(10):700-10.
78.Xu L, Bao L, Zhou J, Wang D, Deng W, Lv Q, Ma Y, Li F, Sun H, Zhan L, Zhu H, Ma C, Shu Y, Qin C (2011) Genomic polymorphism of the pandemic A (H1N1) influenza viruses correlates with viral replication, virulence, and pathogenicity in vitro and in vivo. PLoS One 6(6):e20698.
79.Yang JR, Huang YP, Lin YC, Su CH, Kuo CY, Hsu LC, Wu HS, Liu MT (2011) Early findings of oseltamivir-resistant pandemic (H1N1) 2009 influenza A viruses in Taiwan. Antiviral Res 88(3):256-62.
80.Ertek M, Durmaz R, Guldemir D, Altas AB, Albayrak N, et al. (2010) Epidemiological, demographic, and molecular characteristics of laboratory-confirmed pandemic influenza A (H1N1) virus infection in Turkey, May 15-November 30, 2009. Jpn J Infect Dis 63(4): 239-245.
81.Nougairède A, Ninove L, Zandotti C, Salez N, Mantey K, Resseguier N, Gazin C, Raoult D, Charrel RN, de Lamballerie X (2010) Novel virus influenza A (H1N1sw) in South-Eastern France, April-August 2009. PLoS One 5(2):e9214.
82.Chutinimitkul S, Herfst S, Steel J, Lowen AC, Ye J et al. (2010) Virulence-associated substitution D222G in the hemagglutinin of 2009 pandemic influenza A(H1N1) virus affects receptor binding. J Virol 84(22):11802-13.
83.Ilyushina NA, Khalenkov AM, Seiler JP, Forrest HL, Bovin NV, et al. (2010) Adaptation of pandemic H1N1 influenza viruses in mice. J Virol 84:8607-16.
84.Duan S, Boltz DA, Seiler P, Li J, Bragstad K, Nielsen LP, Webby RJ, Webster RG, Govorkova EA (2010) Oseltamivir-resistant pandemic H1N1/2009 influenza virus possesses lower transmissibility and fitness in ferrets. PLoS Pathog 6(7):e1001022
85.Eshaghi A, Patel SN, Sarabia A, Higgins RR, Savchenko A, Stojios PJ, Li Y, Bastien N, Alexander DC, Low DE, Gubbay JB (2011) Multidrug-resistant pandemic (H1N1) 2009 infection in immunocompetent child. Emerg Infect Dis. 2011 Aug;17(8):1472-4
86.Zhou JJ , Tian J , Fang DY , Liang Y , Yan HJ , Zhou JM , Gao HL , Fu CY , Liu Y , Ni HZ , Ke CW , Jiang LF (2011) Analysis of antigen epitopes and molecular pathogenic characteristics of the 2009 H1N1 pandemic influenza A virus in China. Acta Virol 55 (3): 195-202.
87.Walter D, Böhmer MM, Heiden M, Reiter S, Krause G, Wichmann O (2011) Monitoring pandemic influenza A(H1N1) vaccination coverage in Germany 2009/10 - results from thirteen consecutive cross-sectional surveys. Vaccine 29(23):4008-12.
88.Mak DB, Daly AM, Armstrong PK, Effler PV (2010) Pandemic (H1N1) 2009 influenza vaccination coverage in Western Australia. Med J Aust 193(7):401-4.
89.Ortqvist A, Berggren I, Insulander M, de Jong B, Svenungsson B (2011) Effectiveness of an adjuvanted monovalent vaccine against the 2009 pandemic strain of influenza A(H1N1)v, in Stockholm County, Sweden. Clin Infect Dis 52(10):1203-11.
90.Halder N, Kelso JK, Milne GJ (2011) Cost-effective strategies for mitigating a future influenza pandemic with H1N1 2009 characteristics. PLoS One 6(7):e22087.
91.Wu JT, Cowling BJ, Lau EH, Ip DK, Ho LM, Tsang T, Chuang SK, Leung PY, Lo SV, Liu SH, Riley S (2010) School closure and mitigation of pandemic (H1N1) 2009, Hong Kong. Emerg Infect Dis 16(3):538-41.
92.Morimoto T, Ishikawa H (2010) Assessment of intervention strategies against a novel influenza epidemic using an individual-based model. Environ Health Prev Med 15(3):151-61.
93.Hens N, Ayele GM, Goeyvaerts N, Aerts M, Mossong J, Edmunds JW, Beutels P (2009) Estimating the impact of school closure on social mixing behaviour and the transmission of close contact infections in eight European countries. BMC Infect Dis 9:187.
94.Higuera Iglesias AL, Kudo K, Manabe T, Corcho Berdugo AE, Corrales Baeza A, Alfaro Ramos L, Guevara Gutiérrez R, Manjarrez Zavala ME, Takasaki J, Izumi S, Bautista E, Perez Padilla JR (2011) Reducing occurrence and severity of pneumonia due to pandemic H1N1 2009 by early oseltamivir administration: a retrospective study in Mexico. PLoS One 6(7):e21838.
95.Valenciano M, Kissling E, Cohen JM, Oroszi B, Barret AS et al. (2011) Estimates of pandemic influenza vaccine effectiveness in Europe, 2009-2010: results of Influenza Monitoring Vaccine Effectiveness in Europe (I-MOVE) multicentre case-control study. PLoS Med :e1000388.
96.Kissling E, Valenciano M, Cohen JM, Oroszi B, Barret AS et al.(2011) I-MOVE multi-centre case control study 2010-11: overall and stratified estimates of influenza vaccine effectiveness in Europe. PLoS One 6(11):e27622.
97.Kenah E, Chao DL, Matrajt L, Halloran ME, Longini IM Jr (2011) The global transmission and control of influenza. PLoS One 6(5):e19515.
98.Suh M, Lee J, Chi HJ, Kim YK, Kang DY, Hur NW, Ha KH, Lee DH, Kim CS (2010) Mathematical modeling of the novel influenza A (H1N1) virus and evaluation of the epidemic response strategies in the Republic of Korea. J Prev Med Public Health 43(2):109-16.
99.Hoffmann E, Stech J, Guan Y, Webster RG, Perez DR (2001) Universal primer set for the full-length amplification of all influenza A viruses. Arch Virol 146: 2275-2289.
100.Lee SS, Wong NS (2011) The clustering and transmission dynamics of pandemic influenza A (H1N1) 2009 cases in Hong Kong. J Infect 2011 May 7 [Epub ahead of print]
101.Igarashi M, Ito K, Yoshida R, Tomabechi D, Kida H, et al. (2010) Predicting the antigenic structure of the pandemic (H1N1) 2009 influenza virus hemagglutinin. PLoS One 5(1): e8553.
102.Yang H, Carney P, Stevens J (2010) Structure and Receptor binding properties of a pandemic H1N1 virus hemagglutinin. PLoS Curr 2: RRN1152.
103.Bradley KC, Jones CA, Tompkins SM, Tripp RA, Russell RJ, et al. (2011) Comparison of the receptor binding properties of contemporary swine isolates and early human pandemic H1N1 isolates (Novel 2009 H1N1). Virology. 413:169-82.
104.Colman PM, Varghese JN, Laver WG. (1983) Structure of catalytic and antigenic sites in influenza A neuraminidase. Nature 303: 41-44.
105.Das SR, Puigbò P, Hensley SE, Hurt DE, Bennink JR, et al. (2010) Glycosylation focuses sequence variation in the influenza A virus H1 hemagglutinin globular domain. PLoS Pathog 6(11): e1001211.
106.Cherry JL, Lipman DJ, Nikolskaya A, Wolf YI (2009) Evolutionary dynamics of N-glycosylation sites of influenza virus hemagglutinin. PLoS Curr 1:RRN1001.
107.Rabadan R, Robins H (2007) Evolution of the influenza a virus: some new advances. Evol Bioinform 3: 299–307.
108.WHO (2009) Pandemic (H1N1) 2009 briefing note 17. Public health significance of virus mutation detected in Norway. 20 November. http://www.who.int/csr/disease/swineflu/notes/briefing_20091120
109.Chan Paul KS, Lee N. Joynt GM, Choi KW, Cheung Jo LK, et al. (2011) Clinical and virological course of infection with haemagglutinin D222G mutant strain of 2009 pandemic influenza A (H1N1) virus. J Clin Virol 50: 320-324.
110.Xu L, Bao L, Lv Q, Deng W, Ma Y, et al. (2010) A single-amino-acid substitution in the HA protein changes the replication and pathogenicity of the 2009 pandemic A (H1N1) influenza viruses in vitro and in vivo. Virol J 7: 325-331.
111.Ye J, Sorrell EM, Cai Y, Shao H, Xu K, et al. (2010) Variations in the hemagglutinin of the 2009 H1N1 pandemic virus: potential for strains with altered virulence phenotype? PLoS Pathog 6(10): e1001145
112.WHO (2011) http://www.who.int/csr/disease/swineflu/Influenza_Transmission_ Zones. pdf
113.Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, et al. (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324(5924): 246-51.
114.Sui J, Hwang WC, Perez S, Wei G, Aird D, et al. (2009) Structural and functional bases for broad-spectrum neutralization of avian and human influenza A viruses. Nat Struct Mol Biol 16: 265-273.
115.Archetti I, Horsfall FL (1950) Persistent antigenic variation of influenza A virus after incomplete neutralizationin ovo with heterologus immune serum. J Exp Med 92: 441-462.
116.Nelson MI, Holmes EC (2007) The evolution of epidemic influenza. Nat Rev Genet 8: 196-205.
117.Kuroda M, Katano H, Nakajima N, Tobiume M, Ainai A, et al. (2010) Characterization of quasispecies of pandemic 2009 influenza A virus (A/H1N1/2009) by de novo sequencing using a next-generation DNA sequencer. PLoS One 23; 5(4): e10256
118.Chen H, Wen X, To KK, Wang P, Tse H, et al. (2010) Quasispecies of the D225G substitution in the hemagglutinin of pandemic influenza A(H1N1) 2009 virus from patients with severe disease in Hong Kong, China. J Infect Dis 15; 201: 1517-21.
119.Brookes SM, Núñez A, Choudhury B, Matrosovich M, Essen SC (2010) Replication, pathogenesis and transmission of pandemic (H1N1) 2009 virus in non-immune pigs. PLoS One 5(2): e9068.
120.Rambaut A, Pybus OG, Nelson MI, Viboud C, Taubenberger JK, et al. (2008) The genomic and epidemiological dynamics of human influenza A virus. Nature 453: 615-619.
121.Ferguson NM, Galvani AP, Bush RM (2003) Ecological and immunological determinants of influenza evolution. Nature 422; 428-433.
122.Hoelzer K, Murcia PR, Baillie GJ, Wood JLN, Metzger SM, et al. (2010) Intrahost evolutionary dynamics of canine influenza virus in naïve and partially immune dogs. J Virol 84: 5329–5335
123.Murcia PR, Baillie GJ, Daly J, Elton D, Jervis C, et al. (2010) Intra- and interhost evolutionary dynamics of equine Iinfluenza virus. J Virol 84: 6943-6954.
124.Kilander A, Rykkvin R, Dudman S, Hungnes O (2010) Observed association between the HA1 mutation D222G in the 2009 pandemic influenza A(H1N1) virus and severe clinical outcome, Norway 2009-2010. Euro Surveill 15(9) pii: 19498.
125.Malato L, Llavador V, Marmier E, Youssef J, Balick Weber C, et al. (2011) Pandemic influenza A(H1N1) 2009: molecular characterisation and duration of viral shedding in intensive care patients in Bordeaux, south-west France, May 2009 to January 2010. Euro Surveill 16(4) pii: 19776.
126.WHO (2010) Preliminary review of D222G amino acid substitution in the haemagglutinin of pandemic influenza A(H1N1) 2009 viruses. Wkly Epidemiol Rec 85(4) :21-22.
127.Jayaraman A, Pappas C, Raman R, Belser JA, Viswanathan K, et al. (2011) A single base-pair change in 2009 H1N1 hemagglutinin increases human receptor affinity and leads to efficient airborne viral transmission in ferrets. PLoS One 6(3): e17616.
128.Abe Y, Takashita E, Sugawara K, Matsuzaki Y, Muraki Y, et al. (2004) Hongo S Effect of the addition of oligosaccharides on the biological activities and antigenicity of influenza A/H3N2 virus hemagglutinin. J Virol 78(18): 9605-9611.
129.Wang CC, Chen JR, Tseng YC, Hsu CH, Hung YF, et al. (2009) Glycans on influenza hemagglutinin affect receptor binding and immune response. Proc Natl Acad Sci U S A 106(43):18137-18142.
130.Blackburne BP, Hay AJ, Goldstein RA (2008) Changing selective pressure during antigenic changes in human influenza H3. PLoS Pathog 4(5):e1000058.
131.Nunes B, Pechirra P, Coelho A, Ribeiro C, Arraiolos A, Rebelo-de-Andrade H (2008) Heterogeneous selective pressure acting on influenza B Victoria- and Yamagata-like hemagglutinins. J Mol Evol 67:427-435.
132.Wilson IA, Cox NJ (1990) Structural basis of immune recognition of influenza virus hemagglutinin. Annu Rev Immunol 8:737-771.
133.Shih AC, Hsiao TC, Ho MS, Li WH (2007) Simultaneous amino acid substitutions at antigenic sites drive influenza A hemagglutinin evolution. Proc Natl Acad Sci U S A. 104:6283-6288.
134.Pan C, Cheung B, Tan S, Li C, Li L et al. (2010) Genomic signature and mutation trend analysis of pandemic (H1N1) 2009 influenza A virus. Plos One 5: e9549.
135.To KK, Chan KH, Li IW, Tsang TY, Tse H, et al. (2010) Viral load in patients infected with pandemic H1N1 2009 influenza A virus. J Med Virol 82:1-7.
136.Li IW, Hung IF, To KK, Chan KH, Wong SS, et al. (2010) The natural viral load profile of patients with pandemic 2009 influenza A(H1N1) and the effect of oseltamivir treatment. Chest 137:759-768.
137.Balcan D, Hu H, Goncalves B, Bajardi P, Poletto C, et al. (2009) Seasonal transmission potential and activity peaks of the new influenza A(H1N1): a Monte Carlo likelihood analysis based on human mobility. BMC Med 7:45-56.
138.Chi CY, Liu CC, Lin CC, Wang HC, Cheng YT, et al. (2010) Preexisting antibody response against 2009 pandemic influenza H1N1 viruses in the Taiwanese population. Clin Vaccine Immunol 17(12):1958-1962.
139.Skountzou I, Koutsonanos DG, Kim JH, Powers R, Satyabhama L, et al. (2010) Immunity to pre-1950 H1N1 influenza viruses confers cross-protection against the pandemic swine-origin 2009 A (H1N1) influenza virus. J Immunol. 185(3):1642-1649.
140.Potter CW. (2001) A history of influenza. J Appl Microbiol 91:572-579.
141.Ansart S, Pelat C, Boelle PY, Carrat F, Flahault A, et al. (2009) Mortality burden of the 1918–1919 influenza pandemic in Europe. Influenza Other Respi Viruses 3: 99–106.
142.Miller MA, Viboud C, Balinska M, Simonsen L (2009) The signature features of influenza pandemics-implications for policy. N Engl J Med 360: 2595–2598.
143.Wen TH, Lin NH, Chao DY, Hwang KP, Kan CC, Lin KC, Wu JT, Huang SY, Fan IC, King CC (2010) Spatial-temporal patterns of dengue in areas at risk of dengue hemorrhagic fever in Kaohsiung, Taiwan, 2002. Int J Infect Dis 14(4):e334-43.
144.Müller V, Maggiolo F, Suter F, Ladisa N, De Luca A, et al. (2009) Increasing clinical virulence in two decades of the Italian HIV epidemic. PLoS Pathog 5(5):e1000454.
145.Mitchell T, Dee DL, Phares CR, Lipman HB, Gould LH, et al. (2011) Non-pharmaceutical interventions during an outbreak of 2009 pandemic influenza A (H1N1) virus infection at a large public university, April-May 2009. Clin Infect Dis. 52 Suppl 1:S138-45.
146.Lee VJ, Yap J, Cook AR, Chen MI, Tay JK, et al. (2010) Effectiveness of public health measures in mitigating pandemic influenza spread: a prospective sero-epidemiological cohort study. J Infect Dis 202:1319-1326.
147.Pelletier I, Rousset D, Enouf V; GROG, Colbere-Garapin F, van der Werf S, Naffakh N (2011) Highly heterogeneous temperature sensitivity of 2009 pandemic influenza A(H1N1) viral isolates, northern France. Euro Surveill 16(43). pii: 19999
148.Huang JW, King CC, Yang JM (2009) Co-evolution positions and rules for antigenic variants of human influenza A/H3N2 viruses. BMC Bioinformatics 10 Suppl 1:S41.
149.Langley WA, Thoennes S, Bradley KC, Galloway SE, Talekar GR, et al. (2009) Single residue deletions along the length of the influenza HA fusion peptide lead to inhibition of membrane fusion function. Virology 394(2):321-330.
150.Ekiert DC, Bhabha G, Elsliger MA, Friesen RH, Jongeneelen M, et al. (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324(5924):246-251.
151.To KK, Hung IF, Li IW, Lee KL, Koo CK, et al. (2010) Delayed clearance of viral load and marked cytokine activation in severe cases of pandemic H1N1 2009 influenza virus infection. Clin Infect Dis 50(6): 850-9
152.Seyer R, Hrincius ER, Ritzel D, Abt M, Mellmann A, Marjuki H, Kühn J, Wolff T, Ludwig S, Ehrhardt C (2012) Synergistic adaptive mutations in the hemagglutinin and polymerase acidic protein lead to increased virulence of pandemic 2009 H1N1 influenza A virus in mice. J Infect Dis 205(2):262-71.
153.Ghedin E, Laplante J, DePasse J, Wentworth DE, Santos RP, et al. (2011) Deep sequencing reveals mixed infection with 2009 pandemic influenza A (H1N1) virus strains and the emergence of oseltamivir resistance. J Infect Dis 203(2):168-174.
154.Tsai KN, Chen GW (2011) Influenza genome diversity and evolution. Microbes Infect 13(5): 479-488.
155.Höper D, Hoffmann B, Beer M (2011) A comprehensive deep sequencing strategy for full-length genomes of influenza a. PLoS One 6(4):e19075.
156.Chan MC, Chan RW, Yu WC, Ho CC, Yuen KM, et al. (2010) Tropism and innate host responses of the 2009 pandemic H1N1 influenza virus in ex vivo and in vitro cultures of human conjunctiva and respiratory tract. Am J Pathol 176:1828-1840.
157.Ozawa M, Basnet S, Burley LM, Neumann G, Hatta M, et al. (2011) Impact of amino acid mutations in PB2, PB1-F2, and NS1 on the replication and pathogenicity of pandemic (H1N1) 2009 influenza viruses. J Virol. 85(9):4596-601.
158.Mehle A, Doudna JA (2009) Adaptive strategies of the influenza virus polymerase for replication in humans. Proc Natl Acad Sci U S A 106(50):21312-6.
159.Chan KH, Lai ST, Poon LL, Guan Y, Yuen KY, Peiris JS (2009) Analytical sensitivity of rapid influenza antigen detection tests for swine-origin influenza virus (H1N1).J Clin Virol 45(3):205-7.
160.Xu Z, Zhou R, Jin M, Chen H (2010) Selection pressure on the hemagglutinin gene of influenza A (H1N1) virus: adaptation to human and swine hosts in Asia.Acta Virol 54(2):113-8