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1 Journal of Bacteriology and Virology Vol. 42, No. 4 p Review Article Antiviral Agents Against Influenza Viruses Sehee Park 1,2, Jin Il Kim 1,2 and Man-Seong Park 1,2 * 1 Department of Microbiology, 2 Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon, Gangwon-do, Korea In annual epidemics and occasional pandemics, influenza viruses cause acute respiratory illnesses in infected humans. Vaccines and antiviral drugs are two main arsenals available for a fight against influenza viruses. However, vaccines often exhibit a limited efficacy in high risk populations, and antiviral drugs are always concerned for mutations, which confer viral resistance. Here we review current advances and knowledge in relation to the usage of antiviral drugs as a prophylactic or therapeutic and the mechanism of resistant variants mainly against the neuraminidase inhibitors. Comprehensive understanding of the resistant mechanism will pave a road for developing new antivirals and/or finding medical or natural alternatives inducing less frequent resistance, and application of combination therapy using two or three different kinds of antivirals can suggest a useful medical intervention against both of seasonal and highly pathogenic influenza viruses including resistant variants. In this review, we provide insights of antiviral drugs for the control and prevention of influenza viruses. Key Words: Antiviral drugs, Combination therapy, Influenza, Neuraminidase, Resistance 서론인플루엔자바이러스 (influenza virus) 는호흡기질환을일으키는바이러스중하나로서, 세계보건기구 (World Health Organization, WHO) 에따르면매년계절성인플루엔자바이러스 (seasonal influenza virus) 에의해약 25만 ~50만명이사망하는것으로알려져있다 (1). 인플루엔자바이러스는 Orthomyxoviridae 과 (family) 에속하며 A, B, C형으로분류된다 (2). A형은사람뿐아니라돼지, 말, 개등의포유류, 그리고야생조류및가금류등을포함하는다수의동물이감염숙주로알려져있으나, 이와달리 B형과 C형바이러스는사람만을감염시키는것으로알려져있다 (3). 한편, A형바이러스는입자표면에존재하는당단백질 인 hemagglutinin (HA) 과 neuraminidase (NA) 의항원성에따라각각 17개와 10개의아형 (subtype) 으로분류된다 (2). 이러한인플루엔자바이러스에의한감염은인후염 (laryngopharyngitis), 고열 (high fever), 두통 (headache) 등의심각한호흡기감염질환을유발하며, 폐렴균등의 2차감염에의한합병증으로사망하는경우도있다 (4). 해마다반복되는계절성유행성독감과달리간헐적으로발생하는인플루엔자대유행 (influenza pandemic) 은단기간에많은생명을위협할수있는것으로, 지난 20세기에는 H1, H2 및 H3 아형바이러스에의해 3차례발생하였다 (3). 조류유래의인플루엔자바이러스유전자유입으로발발된세차례의대유행중 (5), 가장피해가심했던대유행은 1918년스페인독감 (Spanish influenza, H1N1) 이다. 1차세계대전의혼란시기에유행한스페인독감은 Received: October 15, 2012/ Revised: November 17, 2012/ Accepted: November 21, 2012 * Corresponding author: Man-Seong Park, Ph.D. Department of Microbiology, Center for Medical Science Research, College of Medicine, Hallym University, Chuncheon, Gangwon-do, , Korea. Phone: , Fax: , manseong.park@gmail.com ** This study was supported by a grant from the Korea healthcare technology R&D project of the Ministry of Health & Welfare, Republic of Korea (Grants No. A103001). CC This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( 284

2 Antiviral Agents Against Influenza Viruses 285 발생몇개월이내에전세계로빠르게전파되어약 5천만명의사망자가발생한것으로확인되고있다 (6). 이후 1957년아시아인플루엔자 (Asian influenza, H2N2) 대유행은주로영유아와노인에게집중적으로발생되어약 100만명이사망하였으며, 1968년홍콩인플루엔자 (Hong Kong influenza, H3N2) 대유행시에는약 100만명의사망자가발생하였다 (5). 이대유행사례처럼, 서로다른아형바이러스간의유전자재편성 (genetic reassortment) 으로유발되는항원대변이 (antigenic shift) 는새로운인플루엔자바이러스를발생시키는주요원인이다. 21세기최초의인플루엔자대유행은돼지유래의 H1N1 바이러스에의해 2009년북미에서처음발생하였다 (7). 하지만, 발생초기의급속한전파속도와세계각지에서의환자속출로인한우려와는달리 2009년대유행인플루엔자바이러스 (2009 ph1n1) 는과거발생한대유행에비해다소경미하였다. 동물모델에서 2009 ph1n1의병원성은다소높았지만치사율 (mortality) 은계절성인플루엔자바이러스와유사하였다 (8, 9). 그리고 1997년이후 H5N1 고병원성조류인플루엔자 (Highly Pathogenic Avian Influenza, HPAI) 바이러스에의해지속적인인체감염사례가보고되고있다. 이에최근 HPAI H5N1 바이러스와 2009 ph1n1 간의유전자재편성에의한항원대변이발생에관한우려가많아유전자재편성바이러스를직접제조하여사람집단내전파력 (human-to-human transmission) 을분석하기도하였다 (10, 11). 또한, 최근보고된돼지유래 (swine origin) 의 H3N2 (swh3n2) 및조류유래 (avian origin) 의 H7N3 (avh7n3) 바이러스에의한인체감염사례는지속적인동물인플루엔자바이러스에대한감시체계의필요성을역설하고있다 (12, 13). 그리고동물유래바이러스에의한인체감염의경우, 항원대변이에의해새로이생겨난바이러스처럼인체는보통면역부재현상을나타내어, 결국사용할수있는의학적수단은항바이러스제뿐이다. 실제로 HPAI H5N1, swh3n2 및 avh7n3 감염환자에대한치료법은항바이러스제를우선적으로적용하는것이다. 물론, 인플루엔자바이러스감염의최우선예방책은백신접종이다. 하지만, 인플루엔자백신접종은끊임없는항원소변이 (antigenic drift) 또는항원대변이에의한면역회피 (immune evasion) 와대유행같은상황에서는백신공급의지연및부족등의문제가나타날수있다. 또한, 1997년과 2005년에나타난사람집단에유행하는균주와백신균주의불일치 (vaccine mismatch) 현상은현재사용되고 있는삼가백신 (trivalent influenza vaccine, TIV) 의한계점이다 (14, 15). 그러므로백신에문제점이있을경우항바이러스제는감염의확산을막는유용한방편이될수있다. 실제로항바이러스제가적용된경우인플루엔자바이러스감염치료및전파력억제에효과적인것으로보고되었다 (16, 17). 특히노인, 임신부, 만성폐질환자, 심장질환자등의면역저하자에서발생하는인플루엔자감염은세균성폐렴과같은합병증의유발이나앓고있던만성질환의악화로이어지는경우가빈번하기때문에, 감염초기항바이러스제의사용은합병증및병증의악화예방에필수적인것으로보고되고있다 (18). 하지만지속적인항바이러스제의사용은내성바이러스의출현을야기할수있다. 이에본논고에서는내성바이러스의출현기전및이를제어할수있는한방법인항바이러스제병합요법 (combination therapy) 에관해논의하고자한다. 1. 항바이러스제및내성바이러스의유전자변이인플루엔자감염환자에게사용되는인플루엔자치료제는크게 M2 이온통로억제제 (M2 ion channel inhibitor, M2I) 와 NA 억제제 (NA inhibitor, NAI) 가있다 (Fig. 1). M2I는 A 형인플루엔자바이러스의 M2 단백질기능을억제하는것으로알려져있다. Amantadine (Symmetrel ) 과 rimantadine (Flumadine ) 등이포함된 M2I는일반적으로 endosome 에서바이러스내로유입되는수소이온 (H+) 통로를차단하여바이러스의외피 (envelope) 가탈피 (uncoating) 되는것을방해한다. 이것은결국바이러스의복제효소복합체 (ribonucleoprotein complex) 가세포질내로방출되는것을막아바이러스의증식을억제할수있다 (3). 하지만, 최근유행하는대부분의 A형바이러스들은 M2I에대한내성유전자변이 (S31N) 을보유하고있고, B형과 C형바이러스에는효과가없어일차적약제로선택되지않고있다 (19). Oseltamivir (Tamiflu ) 와 zanamivir (Relenza ) 로대표되는 NAI는인플루엔자바이러스의표면당단백질인 NA의활성부위 (active site) 에부착하여 NA의효소기능을억제하는약물로, 동물모델과사람에서치료효과가입증되어현재임상에서가장많이사용되고있다 (20). 그리고 NAI는백신처럼바이러스의감염을직접예방할수없지만, 감염초기에사용하면치료효과가좋고약물의안전성또한뛰어나예방적처방까지이루어질수있다 (21, 22). 최근또다른 NAI인 peramivir (Perami Flu ) 가개발

3 286 S Park, et al. Figure 1. Antiviral agents against influenza virus. Sialidase fusion protein blocks the binding of influenza virus to its cellular receptors in the cell surface. M2 ion channel inhibitors (M2I) block the ion channel produced by M2 protein. Neuraminidase inhibitors (NAI) block the process of release of influenza viruses from infected cells. Polymerase inhibitor (PI) inhibits influenza virus polymerase activity. 되어일본과우리나라에서만승인, 사용되고있다. 이처럼 NAI는백신과더불어인플루엔자감염을효과적으로억제하는수단으로간주되고있다. 그러나현재 NAI 사용으로인한내성바이러스 (resistant variant) 가출현하고있어 (Table 1) 이에대한적극적인감시가이루어질필요성이있다 (23). 1999년 NAI의처방이시작된이후초기약 3년간의통계에따르면, NA의 N274Y (mutation of asparagine into tyrosine at NA residue 274, N2 numbering) 변이로대표되는 NAI 내성주의분리율은 0.33% (8/2,287 검체 ) 에불과했다 (24). 하지만, 2008년에이르러노르웨이를비롯한유럽각국에서확인된 NAI 내성주의분리율은약 20% 에이르렀다 (25). 그리고 2009년 H1N1 대유행발생직전에는 NAI 내성주가전체 H1N1 바이러스의약 95% 를차지한것으로확인되었으나 (26), 이런내성바이러스의출현은 NAI의사용건수에정비례하여나타난것은아니었다 (27, 28). 그러므로 NAI 내성주들의발생기전및사람집단내의전파효율을이해하는것은내성주 의출현에대한대응전략을마련하는데매우중요하다. 일반적으로 NA 단백질활성부위에서일어나는 H274Y 유전자변이는 oseltamivir 에대한내성을유도하는변이로, NA의효소활성을감소시키는것으로알려졌다. 그러므로 NA 단백질내 H274Y 변이는감염세포로부터자손바이러스의방출이감소되는현상이발생할수있어이는진화적으로바이러스생존에유리한선택은아니다 (29). 또한, zanamivir에대한내성변이로확인된 E119G/D/A (mutation of glutamic acid into glycine, aspartic acid, or alanine at NA residue 119) 변이는 NA 단백질의효소활성과는무관한구조틀부위 (framework site) 에서일어난다. 이유전자변이로 zanamivir의 guanidinium 잔기와 NA 단백질 119 번아미노산인글루탐산 (E119) 간의상호작용이상실되어내성이유발된다 (3). 이렇듯바이러스는내성을획득할수있는유전자변이를통해 NAI에의한압력으로부터탈출에성공할수있다. 그러나이런내성변이는바이러스의생존에유리한선택이아닐수도있어, 이를보

4 Antiviral Agents Against Influenza Viruses 287 Table 1. Summary of NA mutations conferring resistance to neuraminidase inhibitors. Viral phenotypes against NAI (based on the IC 50 value) Virus NA mutations a Oseltamivir Zanamivir Peramivir A/H1N1 A/H3N2 A/H5N1 B E119A/D/E/V S/R b R R Q136K S S H274Y R S R H274Y + I222V/R R S/I/R R H274Y + R221Q + V233M R N294S R S I/R E119I/V R S/I/R S/R E119V + I222V R S S Q136K S R D151A/D S R Deletion R I S R292K R R N294S R E119G I R D198G I R H274Y R S N294S R S R152K R R R D198N R R S H273Y R S R R371K R R a N2 numbering b S, susceptible; I, intermediate (5-fold < wild-type IC 50 < 10-fold); R, resistant (>10-fold increase in wild-type IC 50 ); based on the NAI susceptibility definition of Neuraminidase Inhibitor Surveillance Network ( 완하기위해바이러스는적절한보상변이 (complementary mutation) 를나타내는것으로알려졌다. 즉 NA 단백질내의 R221Q (mutation of arginine into glutamine at NA residue 221) 와 V233M (mutation of valine into methionine at NA residue 233) 변이는 H274Y 변이로인한 NA 단백질의효소활성저하를회복시켜주는것으로확인되었고 (30), I222V (mutation of isoleucine into valine at NA residue 222) 변이는 H274Y 또는 E119V 내성변이에대한보상변이로각각 H1N1, H3N2 아형바이러스의생존에도움을주는것으로나타났다 (31, 32). 그리고 HPAI H5N1 아형바이러스에서확인된 NA 단백질내 N294S (mutation of asparagine into serine at NA residue 294) 변이는 H274Y 변 이처럼 NAI에대한내성을유발하지만보상변이가일어나지않아도감염된족제비 (ferret) 에서병원성이증가하는것으로확인되었다 (33). 인플루엔자바이러스표면당단백질인 HA와 NA 단백질들의균형 (balance) 은바이러스의증식에매우중요한요소이다 (34). NAI 억압을회피하는데성공한 escape mutant 바이러스의경우 NAI의표적인 NA 단백질뿐만아니라 HA 단백질에서도유전자변이가일어나는것이관찰되는데, 이것은 HA 단백질의변화로 HA-NA 단백질간의균형이조절되어바이러스의생존에효과적이기때문이다. 즉 H1N1 아형바이러스의 HA 단백질내 82, 141, 또는 189 (H3 numbering) 번의아미노산에변이가있을경

5 288 S Park, et al. 우 NA 단백질 H274Y 변이를갖는내성바이러스가더효과적으로증식할수있는것으로확인되었다 (35). 그리고 NAI에내성인 NA 유전자를가진바이러스는 NAI 에감수성인 NA 유전자를가진바이러스보다더강한전파력을지닌것으로기니픽을이용한실험에서판명되었다 (36). 즉 NA 유전자이외의나머지 7개유전자보다 NA 유전자에내성바이러스의전파력을결정짓는중요한요소가들어있음을알수있다. 2007년-2009년에만연한 H1N1 아형내성바이러스는이런특성을토대로전세계적으로바이러스가확산된걸로여겨지고있다. 그러므로현재사용되고있는 NAI에대한내성바이러스를효과적으로감시하기위해서는내성을결정짓는유전자, 내성변이를보완하는보상유전자, 그리고전파력에영향을미치는유전자등이들에대한종합적인검토가이루어져야한다고사료된다. 2. 신규항바이러스제 (1) 숙주세포내시알산비활성화제 (sialidase fusion protein) DAS181은 Fludase (Nexbio, Inc., San Diego, CA) 상품명으로개발되어, 호흡기상피세포의표면에존재하는시알산 (sialic acid) 을비활성화하여바이러스가숙주세포에감염되는첫단계인부착 (attachment) 과정을억제하는것으로알려져있다 (Fig. 1) (37). DAS181은 2009 H1N1 대유행바이러스, H1N1, H3N2 아형의계절성인플루엔자바이러스, 그리고 HPAI H5N1 바이러스에대해모두광범위한효과를나타내었다 (38~40). DAS181에대한제1상임상시험에서는중증이상반응없이흡입이가능하였으며, 현재미국에서임상2상시험중에있다. (2) 바이러스중합효소억제제 (polymerase inhibitor, PI) Respiratory syncytial virus (RSV) 에의한감염질환치료제로승인된 ribavirin은다른 DNA 및 RNA 바이러스에도효과가있는광범위뉴클레오시드유사체 (nucleoside analogue) 이다 (41, 42). 하지만 ribavirin은바이러스뿐만아니라숙주세포의 DNA 혹은 RNA 합성을방해하며, 골수전구세포 (bone marrow progenitors) 에독성을나타내는것으로알려져임상에서의적용은제한적이다. 같은뉴클레오시드유사체계열의항바이러스제인 Favipiravir (T-705) 는 ribavirin과는달리바이러스의유전자복제과정에작용하는항바이러스제로일본의 Toyama Chemical에 서개발되었다. T-705는 H1N1, H2N2, H3N2, H5N1 아형, B형, C형인플루엔자바이러스, 그리고 H1N1 NAI 내성바이러스에서효과가입증되었다 (43). 동물실험에서도효과가입증되어, 바이러스감염후최대 96시간이지난후 favipiravir를투여하였을때에도 88.9% 의생존율을보였다. 또한인플루엔자바이러스뿐만아니라 arenavirus, West Nile virus, 그리고 yellow fever virus에대해서도효능을보였다. 현재인플루엔자바이러스에대하여미국에서임상2상, 그리고일본에서임상3상시험중에있다. (3) 새로운 NA 억제제 (neuraminidase inhibitor, NAI) 일본과우리나라에서허가된 peramivir는새로운정맥내주사용 (intravenous, I.V.) NAI 제제이다. 2009년부터국내에서인플루엔자감염으로위독한환자들에게사용되고있고, 당뇨병, 만성폐질환, 그리고면역억제제로치료받는인플루엔자감염환자들에서약물의효과및안전성이확인되었다 (44). 또한, 이들환자들에게서특별한부작용없이인플루엔자로인한유병기간을단축시킨것으로판명되었다 (45). 하지만, H274Y 변이를갖고있는 oseltamivir 내성바이러스에교차내성을보이기때문에내성바이러스로인한감염치료에는 peramivir의사용이제한적이다 (46). CS-8958로알려진 laninamivir는 zanamivir와유사한분자구조를가지는 NAI이며, 건강한성인남성을대상으로한약동학 (pharmacokinetics) 평가결과, 분말형태로흡입을하였을때약물의반감기는약 3일이며체내잔존시간이약 144시간인것으로확인되었다 (47). H1N1, H3N2 계절성인플루엔자바이러스, 그리고 HPAI H5N1 및 B형인플루엔자바이러스에광범위한효과를보인 laninamivir는임상시험에서 20~40 mg의단독투여만으로도 5일간 2회씩투여한 oseltamivir와비슷한항바이러스효능을나타냈다. 현재일본에서승인되어 Inavir (Daiichi Sankyo, Tokyo, Japan) 의상품명으로판매중이다. 또다른경구용 NAI인 A 는인플루엔자바이러스에감염된마우스에서효과가입증되었으나아직임상시험에진입하지못하여개발여부가불확실한것으로알려져있다. 3. 인플루엔자감염치료보조제로서면역조절제 (immunomodulator) 사용최근보고된족제비모델에서 HPAI H5N1 바이러스의공기전파 (aerosol transmission) 가능성은 HPAI H5N1 바이러스로인한대유행발생의우려를다시금부각시켰다

6 Antiviral Agents Against Influenza Viruses 289 Table 2. Summary of reported combination therapies against influenza viruses. M2I NAI PI Virus Reference Amantadine Rimantadine Oseltamivir Peramivir Rivabirin Favipiravir A/NWS/33 (54) A/NWS/33 (55) A/H1N1 A/NWS/33 (56) A/Puerto Rico/8/34 (57) A/California/04/09 (58) A/Aichi/2/68 (59) A/H3N2 A/Victoria/3/75 (55) A/Victoria/3/75 (60) A/Duck/MN/1525/81 (61) A/Duck/MN/1525/81 (61) A/Duck/MN/1525/81 (61) A/H5N1 A/Duck/MN/1525/81 (55) A/Vietnam/1203/04 (62) A/Vietnam/1203/04 (63) A/Turkey/15/06 (63) B B/Sichuan/379/99 (64) (10, 11). 1997년이후로계속되고있는 HPAI H5N1 바이러스에의한인체감염사례는숙주의면역체계에서유발된과도한 'cytokine storm' 에의한병원성증가에기인한것으로알려져있다 (48, 49). 하지만, 이러한 hypercytokinemia (cytokine storm) 를조절한다고해서 HPAI H5N1 바이러스의병원성이낮아지는것은아니다 (50). 또한, glucocorticoids를처방받은 HPAI H5N1 감염환자의예후가상반된결과를도출한점에서논란이있긴하지만 (51, 52), HPAI H5N1에의한감염에효과적인대응책은바이러스를표적으로하는치료제와과도하게유발된면역반응을조절하는치료제를병합하여사용하는것일수있다. 미국식품의약품안전청 (Food and Drug Administration, FDA) 에서승인된 COX-2 inhibitor (celebcoxib, PF ), macrolide (azithromycin, erythromycin, etc.), glucocorticoids, 그리고 statins 등의면역조절약물을이용한인플루엔자감염질환치료의효용성이입증된경우가있긴하지만, 앞으로더많은연구를통해임상에서의적용여부가평가되어야할것으로판단된다 (53). 4. 병합요법 (combination therapy) 인플루엔자바이러스에의한감염질환을치료하는데사용되는항바이러스제의단일요법은효과가다소제한적이고, 항바이러스제에대한내성주를유발할수있다는단점이있다. 이에대한대안으로복수의항바이러스제를이용하거나인플루엔자바이러스억제에효능을보인천연물등의보조제를이용한병합요법이대두되고있다 (Table 2) (54~64). C형간염바이러스 (hepatitis C virus, HCV) 환자및사람면역결핍바이러스 (human immunodeficiency virus, HIV) 에감염된환자들을대상으로그효용성이확인된병합요법처럼 (65, 66), 인플루엔자의치료에병합요법이적용될경우, 투여되는항바이러스제의용량을최소화할뿐만아니라, 서로다른두약제간의상가 (additive effect) 또는상승 (synergy effect) 작용을기대할수있고, 내성바이러스의출현을낮출수있다는장점을기대할수있다 (67). 인플루엔자바이러스에의한감염치료에이용되는병합요법은주로 M2I, NAI, 그리고 PI의세가지제제를

7 290 S Park, et al. 사용한다 (Table 2). 두가지 NAI를이용한병합요법에선 oseltamivir와 peramivir 사이에나타날수있는길항작용 (antagonism) 에대한우려가있었으나, 오히려인플루엔자바이러스를억압하는상승작용을나타내는것으로확인되었고 (56), NAI와 M2I를이용한마우스병합요법실험에서 peramivir와 rimantadine의병합요법은바이러스억제에있어서단일요법보다우수한상승작용을나타내었다 (60). 또한 PI인 favipiravir와 oseltamivir의 NAI를이용한병합요법역시바이러스에감염된마우스의생존율을높여, 각약물의단독요법보다는더효과적으로바이러스를억제하는것을확인하였다 (55). 두가지약물의조합뿐만아니라서로다른기전을표적으로하는 NAI, M2I 그리고 PI의세가지제제모두를이용한병합요법은각약물의단일요법보다더높은상승작용을나타내었고, 내성바이러스에대해서도효능이입증되어, 현재임상시험중에있다 (68). 하지만, 2009년발발한 H1N1 대유행바이러스에감염된중증환자를대상으로시행된 oseltamivir 와 zanamivir를이용한병합요법은단독요법보다효용성이낮은것으로확인되어 (69), 향후임상환자에효과적으로적용될수있는최적화된병합요법안을마련하는연구가진행되어야할필요성이있다. 결론인플루엔자바이러스는사람에서급성호흡기질환을유발하는병원체이다. 해마다반복되는계절성인플루엔자바이러스와간헐적으로발생하는대유행은인류에게큰폐해를끼치고있다. 현재인플루엔자감염질환에는백신과항바이러스제가모두개발되어있어감염질환을억제하는데매우유용하게사용되고있다. 그러나변신의귀재인인플루엔자바이러스에좀더광범위하게작용하는효능 (broad-spectrum efficacy) 은그해유행하는바이러스에만작용하는백신보다항바이러스제에의해나타날수있다. 특히대유행같은위기시에는항바이러스제가신속하고효과적인임상대응책이될수있다. 그리고항바이러스제사용으로인한내성바이러스의출현기전을규명하고항바이러스효능을상승시킬수있는병합요법의개발은항바이러스제의임상적가치를극대화시킬수있는또다른중요수단이될수있다. 그러므로이를위한지속적이고심도있는연구가절실히필요한실정이다. 참고문헌 1) WHO. Fact sheet: influenza (seasonal). mediacentre/factsheets/fs211/en/ ) Palese P, Shaw ML. Fileds Virology, Ch.47 Orthomyxoviridae: The viruses and their replication. 5th ed: Lippincott Williams & Wilkins, ) Wright PF, Neumann G, Kawaoka Y. Fileds Virology, Ch.48 Orthomyxoviruses. 5th ed: Lippincott Williams & Wilkins, ) Clark NM, Lynch JP 3rd. Influenza: epidemiology, clinical features, therapy, and prevention. Semin Respir Crit Care Med 2011;32: ) Palese P. Influenza: old and new threats. Nat Med 2004;10: S ) Taubenberger JK, Reid AH, Janczewski TA, Fanning TG. Integrating historical, clinical and molecular genetic data in order to explain the origin and virulence of the 1918 Spanish influenza virus. Philos Trans R Soc Lond B Biol Sci 2001; 356: ) Garten RJ, Davis CT, Russell CA, Shu B, Lindstrom S, Balish A, et al. Antigenic and genetic characteristics of swine-origin 2009 A (H1N1) influenza viruses circulating in humans. Science 2009;325: ) Maines TR, Jayaraman A, Belser JA, Wadford DA, Pappas C, Zeng H, et al. Transmission and pathogenesis of swine-origin 2009 A (H1N1) influenza viruses in ferrets and mice. Science 2009;325: ) Dawood FS, Iuliano AD, Reed C, Meltzer MI, Shay DK, Cheng PY, et al. Estimated global mortality associated with the first 12 months of 2009 pandemic influenza A H1N1 virus circulation: a modelling study. Lancet Infect Dis 2012;12: ) Russell CA, Fonville JM, Brown AE, Burke DF, Smith DL, James SL, et al. The potential for respiratory droplettransmissible A/H5N1 influenza virus to evolve in a mammalian host. Science 2012;336: ) Herfst S, Schrauwen EJ, Linster M, Chutinimitkul S, de Wit E, Munster VJ, et al. Airborne transmission of influenza A/H5N1 virus between ferrets. Science 2012;336: ) CDC. Swine-origin influenza A (H3N2) virus infection in two cildren--indiana and Pennsylvania, July-August MMWR

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