Intra-Species Transmission Zoonosis

Intra-species Influenza A virus is transmission remarkable for the frequency and breadth of zoonoses: many species of birds, humans, pigs, horses, dogs Morens and Taubenberger

1299 with an equine epizootic43 coincident with a wide- spread European influenza epidemic. In this epizootic: ‘‘the horse carried his head drooping, would eat nothing, and ran from the eyes’’.22 Twenty-nine years later, in 1328, an equine epizootic in Yemen, later attributed to influenza,1,56 coincided with a major European epidemic of apparent influenza. In 1404, yet another equine epizootic was associated with a major European influenza epidemic.34,38,45 Between 1581 and 1728, a 147- year period in which pandemic influenza seemingly disappeared,5 countless local and regional influenza outbreaks were documented in Europe, the New World, and elsewhere. During this time, a close temporal–geographic association between equine and human influenza-like disease activity began to be documented in hundreds of medical, epidemiologic, and other publications. The first well-documented occurrence in the Renaissance followed a modest 1647–1648 human outbreak at the end of the Thirty Years’ War in Spain, in French soldiers in Germany, and in their cavalry horses.1,23,43 A decade later, in 1657–1658, one of the era’s most significant European influenza epidemics caused high human mortality [Oliver Cromwell (1599–1658) appar- ently died of it] accompanied by cases of influenza-like 35 Figure 3. The first page of Chapter 17, ‘‘On the catarrh and coughing illnesses in horses. epidemic’’, in Daniel Sennert’s account of the 1580 influenza From 1658 until the early 20th century, outbreaks con- pandemic.48 Sennert refers obliquely to a ‘‘coryza of chickens’’, but it is sistent with equine influenza were associated with major not known whether avian influenza occurred during the pandemic. human outbreaks, epidemics, and pandemics more often than not. Considering several hundred published reports of major influenza occurrences between 1688 and 1888 that causing ‘‘chicken coryza’’ noted in 1580 is not known, we have examined (obviously subject to errors related to, although the same term had been used at the time of the among other things, the possibility of not recognizing out- 1557 pandemic, possibly in referring to croup-like respira- breaks that occurred), and considering only those with tory signs in children.49 Neither HPAI nor Newcastle dis- reasonably complete historical information, by our count ease was recognized until more than 300 years later. We there were 112 (of 200) years in which either significant are unaware of other reports of similar avian illnesses in epidemics ⁄ pandemics or equine epizootics of apparent the voluminous literature on the 1580 pandemic which, influenza were documented in Europe. Of these 112 years, however, has rarely been examined systematically, and combined equine epizootics ⁄ epidemics were documented never to our knowledge for evidence of animal disease. in 67; equine epizootics only in 25; and epidemics only in 20. The pattern in the western hemisphere during the same An association between human and interval was somewhat different: of 56 years in which equine influenzas, 1648–1916 human or equine influenza was documented, both horses and humans were involved in 21 years; humans only in An H7N7 equine IAV was first isolated during a 1956 epi- 25 years, and horses only in 10 years. zootic50 but is now likely extinct;51 an H3N8 equine IAV, Regardless of geographic locale, equine influenza typi- detected in the early 1960s52 still circulates enzootically to cally appeared about 3 weeks before human influenza,42,57 cause significant disease and economic burden.53 Equine a pattern first noted in 1688 in England and Ireland.43 But H3N8 viruses can infect humans experimentally,54, and it in a few instances, this pattern was reversed, e.g., in 1776 has been noted by several groups that human birth cohorts and 1780 human outbreaks preceded equine out- from the late 19th century, particularly those born before breaks.31,42,43,47,57 Commenting on a 1688 influenza epi- about 1893, demonstrated serologic reactivity with equine demic, one chronicler wrote: ‘‘…an epidemic catarrh H3N8 viruses many decades later.55 followed all over Europe, beginning among horses and end- Before the virologic era, equine epizootics highly sugges- ing with men as is frequently the case’’,57 then observed tive of influenza were documented repeatedly, beginning in the same pattern five and again 10 years later [1693 and

330 ª 2010 Blackwell Publishing Ltd, Inﬂuenza and Other Respiratory Viruses, 4, 327–337 Morens and Taubenberger

Figure 1. Adonirum B. Judson’s map of the equine panzootic as it occurred in Canada and the United States, 1872–1873,1 showing progressive dates of outward expansion from the point of origin near Markham, Canada, in late September 1872, to its detection in Washington State in May 1873.

combinations (e.g., ‘‘chicken epizootic’’, ‘‘chicken disease’’, ‘‘fowl disease’’, and others), we searched two web-based newspaper archival sources (http://www.Newspaperarchives. com; http://www.Proquest.com) with optical character recognition (OCR) capability, visually scanned the OCR summaries for each of over 3200 ‘‘hits’’, and then examined, printed, and read newspaper pages containing relevant information.

Results Lethal outbreaks in domestic chicken flocks associated with the equine panzootic were first reported in Poughkeepsie, New York, on November 15, 1872.6 Newspapers generally referred to the epizootic as ‘‘the chicken disease’’, but many different names, some of them fanciful or humorous, were used in different parts of the country, including chicken epidemic [sic], chicken epihippic [sic; epihippic being a less common term for epizootic equine influenza], chickizooty, epiornithosis, henfluenza, ornicephalzymosis, and many others (Figure 27). In some Pennsylvania locales with large Figure 2. New York Herald headline (Monday, November 18, 1872, German-speaking populations, the avian epizootic disease page 3, column 6), reporting outbreaks of fatal avian disease in birds became known as Pferdhu¨hnkrankheit (‘‘horse-chicken-dis- brought from surrounding areas into New York City and elsewhere.30 ease’’8). It was universally believed that the avian disease, often referred to as Whole flocks were usually struck simultaneously, with ‘‘henfluenza’’, as it is here, was being transmitted to poultry from horses ill with equine influenza. The term ‘‘fatal epizootic’’ refers to fatality at or near 100%. Prominent features included upper equine influenza, which during the 1872–1873 panzootic was more respiratory signs of ‘‘a cold or influenza’’, slimy discharge often called ‘‘the epizootic’’ or ‘‘the epizooty’’ when seen in horses, emitted from the beak, dark streaks in the neck, ‘‘dizziness’’ mules, or humans. The reference to ‘‘mansard [Mansart] roofs’’ appears or ‘‘staggering fits’’, crawling away from the flock into to be a pun related to the ‘‘great’’ Boston fire 10 days earlier, in which 7 holes or corners, and death within 12–18 hours.9–12 In a large part of the city burned to the ground. The press blamed not only horse-less fire department wagons, pulled by teams of human many cases, the head swelled markedly before death, volunteers, for responding too slowly, but also alleged poor fire prompting poultry vendors to decapitate ill birds and rush repellant properties of new mansard roofs on storehouses that first 10,11 them into city markets for sale. Suspicious buyers caught fire.

374 ª 2010 Blackwell Publishing Ltd, Inﬂuenza and Other Respiratory Viruses, 4, 373–377 Pandemics since 1700

The 1918 pandemic was totally out of scale and has been called `the greatest medical holocaust in history.’ 40-50 million deaths but that was only a few percent of the infected (20% of the world’s population).

The 1918 “Spanish flu” pandemic

Policemen in Seattle during the 1918 pandemic

. , whp ,digs - tr. -- pit .for others shall of the men: who sltilecl wiEti htill;-*and 14Faith is the substance fail into•it bijavpsie: is theinZidern direeted!, themL:in measures that even-, hoped Ter."—Heb, • 7 1n. - their sate . tuated : -Mark Twain 'has .humorot - aloe of Pails* 7: xs, is, taildinS . 7tIvon shore, is what he tared . faith as • "heiloVing .0erautny..)344 h Oft, and is now told them explanatiOn. Of "MS. own knew ain't . so." Such is about to fall into- leaderir are confidence and courage , and hope. and notion of-religious . faithl.-. th her exponents. "The World shall' have az.:a.:reason why they should banish stttious credulity. _Faith, si hes% coat it actorfilair: to German fear. and take heart :attain: "There the giving of subistanCe miniuree* . 'Proclalined • the _German - stood ine this -night the angel Of -sired.. Thus defined faith .-- -poe‘--PhiliPt errnany-shall- west- We God whpse I am and whorit I serve, nyrn for idea,Ilinn. ,It is a a -Civilian snit- cut according. -to .Wil- saying, Fear not; there shalk,not 'be moral and ethical, achiever hatel .,:prineipleC, "Might is the su- the loss . of any man's-life, 4 intri be- faith . is dynamic with life, preme •ight," claimed General Bern lieve* God that it shall be even .so as it purpose and conforms to harititiliter.-the law of 'force is crushing was told- Me.". ..• • known reality. GeirMan l'''Ne(ferisity knows ACcording 'to thts-testimony; -given - Let •us note three things no law," explained. Chancellor - Beth- . thus fn 'circumstances that . tried the type of faith:, . main-Hollweg;' sa necessity; demands souls . of men to the utmost, the I. Faith as a dynamic the •Cessation of 7 rip*: Arnage and apostle's courage and -confidence and world war -fOr democracy' murder.. "Woe and - :,death to those f---ePtettnd-eanity-ef- -ftt-miliar'svfth -•.bi fderatii-. ; my",WITT7- 7-crCed the7arts.- grew elk of his faith in God. His faith Unit our army has 'gone -it tocratie kaisier:: In 1914; so democracy 1 , hi God was a great-asset in that hour of b tie to fight- for 1101 is coMpelling .tistis Narcr*to. drink - his of supreine trial. It .made the differ- human v. Th e ideals an own cup. of pcdOon. World dominion is • etee between him and the three score of faith; ey are an ex receiving its death blow. . and fifteen that sailed with him. It' conditions as we think th The present German.- chancellor re- banished feel., and gave hope and cour,,, be. In fact, faith hai rear cently said:..that "just terms" wOuld age and wisdom. It became' conta-" rind- con,ocious religious pt be acceptable, but: :he prevaricated. giOus, and filled the whole company gaspel of unselfish ministr Germany does trot want justice: - Jus- with courage and cheer and hope of the hesitant message of tice "*would demand the return of All deliverance.. has -become the • evangel her stolen prOPerty,, restoration •Of all world alliance. . devasta ii1thee, and_.fear. .exclude• each other. --nrear.--h-rtfh 'torment:- but faith casts z. ..eattb....hs, creatR W - hot**, cities and Cathedrals :and the. out fear and all its torments, and Sub- enough to have ideals; tit _ x (feu o constructive proAT-erns. . A ha,ve It stitutes hope and courage and. forti- "id aliem^--ofiiiir re would mean the doom of the nation. tude and calm treason and confidence' of oiir' in the ifitireme Good Will. But-it must theturietg display. 'I The kaiser. Would have to -be. crucified, creative; such faith gets the Pottiklam. gang, hung, the ariestoc- be faith that is faith indeed—faith yLassumes_ but assures by. ,suits. , The, American Re( *glven suhitance of its' fall people impriSoned. I sj iritual revelation ' that ‘"God is, and dOrd Reading re- ministry by.'the creation.o DAY' MOEt - eent17- idEld ' that: '"Jiistice is :merciful that He is the rewarder of them that 'diligently seek Him." agency ..of mercy. The when-there are 'exterruating circum- Work council haS given s'; stances, but wpen there'ire none, it The times through which we are now.. : , 7741!".••!17...! passing' call for that kind of faith—a. our common faith in the f • behooves Justice to be Stern."- you' ,of .Christian idealism uy find the "extenuating circumstances?" faith that makes us strong and of good -Practical..wOrkable, therm tierrnany must make an uncondi- . courage and hoPe_isnd cheer. As 'the brave old- .psalmist :_sages:%--I'lle that., -Of unselfish tervide . to the tional surrender and the allied powers army and:siayY.. must -,Notate the terme of'peace. Truth dwelleth 'in 'the - . secret plablc of the • 'Most High Shall abide tinder.. the 3. Faiths - AS,- a religious (lecher.* it, Justice demands it' . and reality.. Faith is not bell( conscience confirms it. "Be not de- shadow •of:the 'Almighty. I will say of Jehovah, He is my refuge and my 'bellevable;:, but bflitif 'fn ceived; God is not mocked, for What- the deal soever fortress, my God, In Whom 1 trust." able;,the.possable. a man noweth, that shall he Li not dreaming bat on aluo reap" is aisb true of any nation. May that faith be ours. 'and the strong, glad,. hopeful. — spirit -Which is born. Of znie .NOr.--lis .fait .. .The philc reefs ahead was heard..it It. was then to, solve the riddle of exli . Germany Soon Will Fall . Vint all hope dtidelIverance was taken Faith Is a Systoi vm • • I We Need a Faith That _ denial of reality. He (.1( awa y - - ItitO.. Pit Which She Dug . . And it was then, that one of , • for Idealism,alit); of He physical Says pain an Casts Outthe All whetters Fears atood up. cairn amid the • .• • _ ' • Churchless Sundays May dr. ys On the . sharp poi , ...... C__ _ - storm, ,fearless In that mad war of the oreineair,.confident that Revivethere should the Family Altar spikes driven . through ar. t !2‘f. REV: J;. J. • HUTCHISON. BY REV.- W. T:LOcKwOQD..his flesh with sacred fire BY REV. J. N. MACLEAN,'D,nuL s..lutlr_fall..fram_ths..liend D. sif _aux_ —. , mf0E-77::: •or.e•of••them.o. and-restored-the morale • -7 -. Of . th-e . 1 M.6.-!!*!140..g.tp!!it .cseeks1 1.01sch' to ..•., . r of the Prelebyterian Ghorch. "He whg,gi r:g'ItIt :ter', • ethers. .• shall „.,of tho.• men who ighilidSY. withRSV. him, CHARLES andht ;D.1''I0 ltCROUCHtlyt t.la,_ ITL7, 1t.. elintAir.ther,7_, or. 011144 14rees,4 which for fall into:Itisiimaifit-10-the-ma"There stoodern• by vet•- me direeted•-tItent-in--measursa this night the that even. d organising 'them man of First ,Methodist Episcopal. Church. in prepr 'don of Pealialangel 7:114.18. 6f God whose tuated .1. am andIn theirwhom Isafe . landing 111)011 II Murk Twain 'has humorously-.ends. - Thus, carIca- serve,- ergyiggf, ,Year not." Acts 27: SZ, "For I know Him That. linspitabia will shore. And corn- lids is What hointelligeft tured faith as "believing faith what demandri you ... clermarig..hag tiht •Wel- Thee*• lartaitsipit. and were' Is now sPoken told •thentin clrc umin axplastarttnt - 'mand His of children..and.Ver his own know ait. beuseholdn' -se:" Ruch merit is theof every popular heiman_nti alietit to•fatil intosts.n it.' elseHer thatgaveleaderm oreno opportunitysten(Idence_ forand courageafter HIM." and hope. - (Geneile end notion . 10:10). of - religiotur faith;by scienoe this is ;toitiper- 'safeguard Iia exporieata "Thecam ou world neve. shall They have represent us. a reason gelled whyne theyAt, should noon, asbanish' Abram* mations eat credulity.the door Faith.-than saystrust l'atil,'zienze Is 4211073 ;;resat ;tart' ariOordlnit.human experience. to German They (ear were and. Spoken. tike heart of hit'again: tent • "'Chore three mein the sIvIng 'dressed of_etihStance in asita414b. to things de- -procialinedon beard thea storm-driven German stood ship, by *hen me all this shining night the garments angel of hood sired. 0foni Titus defined .faith Is a eyno-, orrailleope:o. ---Oreps:feef-DO • Germany being shall saved, wear the God Whose I amThey- and whom 1 Nerve, nyrn--bonier for 'Idealism. w ,It Is a method of a' °Fenian 'tedt 01:"cut oneaccording man, towas WO, -taken saying, ..awar. roar Ter. not; nod.there fovea abate to notdwell be ismoral the truly and ethicalhappy achievement.-Astottenattra True ism's principles.fourteen "Might days Is' andthe nu-nights the the lose storm of pre-any man'shome. Ilfikland His anicobi 1 be- violt fulfil „homes is dynamic where with lire,• (-to restive_ Hope in • in F • pram: right," claimedvailed, General"neither Berri- sun nor neve stars Clod appear- that It shallhusbknds be even love so theiras It purposewives and''where anti conforms to the lawn of - hari11; 16;•4he lawing" of forcethrough is 'crushing the clouds was, and told darkness. me."'.. wivesi , • loveknown their hatbands, reality. *rid Where German militarism. 441dessity_knows children honor their parents. Ituf..h ax Viers were seventy-six souls.. According onboard, to this testininny; given Let .us- note three thingsWY about this no law," egplahrit• Cheapener Beth- borne is in harmony with God's will RSV. T. V. AP correlating of ship's crewthus and In paseen- circumstances that tried the type of faith: • of the divappol!oatt with . man•liellerig; soSara becwqlty Sad some demands soldiers in charge of a and God's law. AngelsI. can Faith go wher-us gouts of men to the utmost, the a dynamic ideal. The the •beesatkin of rape„ .4e.rnage • and • for priliOnnyeen the wayapostle'* to Rome courage for everand confidenceGod, the rather, and world the great war forhead democracy of rer has madeiv*** us that I •:latitriler7: "151419 istepss4h to those beftster-_-_iibeAlleposerraseresalty._nt jpilt was "plrlethe theante. family sanity in of mindHeaven. familiar' le reOpootod.. with .hi g, ideals:of thip:.priiiiiatit - We -are told Moo it irittr Ail over our volt land citsiost.ioCil ts atarht. grewthe out roar of his of faith in God. lily faith that our army he "Orbigone Into. ties111PItted the field with lh tocratic kaiser In 914; democracy hl (rod was a greatof assetSpanish. in that influenSa hour of 1s battle rigging, to fight fol- forshall honor, bur _Mello, revetaled in • • Is comp•lling-OM Nero'te- Mink his of supreme trial. lowedIt made by the a deadly differ- form humanity. of pnenleonla. These Ideals t:11. are • postulator' own..ouRieg notop, Witrid dominion Is el,c• between1111allulP him Obedientand the threeto the score orders of faith;of the they public nro an"Cbriirtianity expression ofis a re receiving ittl'death hlOW. ' v 1 and fifteen 'that sailed with hint. It conditions as we think they ought to -The present German' chancellor re- hanlithed tear, and gore hope and cour, he. In foci. faith has Touched far be- .that "Just terms' would 11,11. and . Wisdom. it became uonta- y•md canneloni, religious purposes. The_ bi acceptable, imt••he prevaricated. mous, and filled the whole einnottnY gospel of unselfish ministry, yesterday Germany-•ries clot went Juritice, - Jus- 'with Courage anti_ cheer and - hope of the hesitant mensage of the church, tice Would demand the return of all deliverance. has become the .evangel of _41. great her_ratchut--piopertYr-relitAnalen of Oil •alth and fear egelude each other. world alliance. . . davatititted rtattfiding..,of all "I'Ver• hath'termentr but faith casts 2. Path' ig,• creative.' • 'It' Is hh8. . homes, cities end 'Cathedrals end' the be out fear and all its-tormentc'and sub- enough to have Ideals; there• must . . .0r....all. *V criminals, who . Px.out10.11 . fort!: strUctive programs. Alt too much larolteuter-hationalLister....lt. stitutea hope and courageand bavg Coalition:0 Or.Otit-Idgeltiliiiirinir-illiritidit-il Tidily would tu'ileIiiiil- Zrtlin7iiiiiiiiiiii " :rode- mean -the (tooth of the nation. supreme Good Will. But It must —Is• rhetoricaldisplay. -True faith Is Tl^a kaiser haVt.to be. crucified, In the - - mug)" faith-tete _concrete re l-- the Pottilarw•gang litittg, the &Hattie- bn faith that' 14 faith Indeed-LIMO. creative; _f_Viliii. . The,' American Bed Cross hid racy electroCiuted and---the common tull-aubt,-allauli1011...buLarsure...bx, .that Online! revelation: that 'God is, and given substance it Its faith 'in•huftutn PetoOle ImPrisolted.. Lard Beading re- -a p-Ciiiiii-thil-,- cently /tart lie Is the rewarder of them that Ministry by 'the creation of raid' qua: !Vogler, Is merciful agency_ of mercy. The United War when there are - - 'diligently seek 'Vol.' eictruraating council-hart - to ....the timer! through which we are now Work given substance sauces, there%are .noneo it -faith--aonr.eoramon faith In the fundamentals passinscall .. for that-kind of-faith behooves 'justice to be -stOrn.-". Clen you -Chrlathan., Idealism by orgitelsing Lit faith that find the .".4tatenualing- eirotimstanoesr' makes us strong and vt rood pt. thorough program .courage and holgikatt . cheer. As the, Prketicil..workable. GartnanY roust nitrite an uncondi- Of tinselflah s•evIde. te . the Men of Cur- tion !l surrender and : the allied poWers bravo old. lasalmlet 4:104,;).-t'll• that, . - . mits4 ti__In" the', seorat.•..pliegai of'. the- :army and navy -' - '' • • .. . 1.11elate the terms oepilace. -Truth religious Most Hitt' , 'Man . -abide under the . -; :/• .. Veith"l'e' a °°1"r"ch to declares it. justice,. demands' It and shadow of 'the 'Almighty.. I will stay reality. Faith Is not belief in the 1111,7 conschinca-coaftrass . It. "Be; not de- of JeheVali, lie is my refuge and my :hellevable;%; but belief in the' reason.' e/OY.gdi- Cihd Is hot mocked, for What-' id..In lflumt _I. trust." 4106;:the W. . 11 b 10. the delire410. reith sower ; 1^• man ..ioWeth 'that: shall- ftrerlsolt.111)!-.4.1 Li .pot. dreaming1'14 creative, dypit- May that faith. 0•' 714.*nd the string; . map" is alsb.leue Oenny. nation. ',00,1 Ania_Idtrallsm..:.NorLis faith, a:disulal or glad;- itelatiftltitigrit:WhietrIarliorit -Alf •-•.• reality:., The IlindtriAlloeophor seeks .: ...-._ It - _ • to solve the riddle of exietence by the We Need a Faithl'hat . - ...__ -.. .„., dental_ of reality. He denies the re . Casts 'Out-Ail:Years Chard lest .:-. 4.11ty',joys -Ofon• phytrical the tbart," pain andpoint.. Oita- of lie iron for Reytie the Ifitalti. Altar 74. iittge-0 driven lblOttglla. plank, or bake his ;flesh 'with loitered' flies. •-: BY REV. J. N. PAAGLEAN, D. D. ',Faith, maths, ,to knew' reality 0,nd to , "•. 44-Pr! . .Hrr,hidOW.6114.04:e• -- - ' SY,- oigv. CHARLES' 0:-CROUCH, ceritAir.t.19,../41MMt which .eavirOa:,41(e. 'There stood br'aisi -=-144.-rilitht--the organising Ifilihi" for zeorgi:inett - livtItitat at oil% b.f . God, Whom "se, and_ wilia, , .0i First Methodist felsoepal ..Chureh. ends. Thus In the pressint *Olden* "tor I-1filow Trim that. Ile will own- Intelligent faith domande the employ - serve('Illefrtni',•"eir,itat, '" nits - It:it, ...,..... Tho ssirnillirwartirinaken in circhrin- mend . Htivehildien. and Ili. household moot of , eviry human means pro.!;10.40 stan— that'Na' y. no:opportunity for aftel,Iiiiii.777(Cleneelii-•111:15), -7---7 by ',clone', to 'eaf4guarti life rather 0 , ..1_'They represent genuine At .noon, as .Abrenr but rii the door_ than trudill'crionti-•bllad ' providence to human erportenca • They were 'spoken of his, tent •• three men. 'dreamed 'eatebileli___a___Mental_AtliArantine.. .. •.ort-Voittrat storm driven /thin. when all shtning *animas stoodiiKeir• •I / fit ,v/z- -; //

Genec Changes Among Inﬂuenza Viruses: DRIFT Associated with human EPIDEMIC

Mutations X X x X

Human Human Genec Changes Among Inﬂuenza Viruses: REASSORTMENT=SHIFT Associated with human PANDEMIC

H5 H3 H5 X =

N1 N2 N1

Avian Human Human Mutant The 1918 “Spanish flu” pandemic Table from Simonsen et al, The Journal of Infectious Diseases, 178:53 (1998) 1957 “Asian ﬂu” H2N2 pandemic

Thought to have originated in Southeast Asia. Less lethal than the 1918 pandemic. H2N2 replaced the circulating H1N1.

Table from Simonsen et al, The Journal of Infectious Diseases, 178:53 (1998) The 1968 “Hong Kong ﬂu” H3N2 pandemic H3N2 pandemic was even less lethal than the H2N2 pandemic

Table from Simonsen et al, The Journal of Infectious Diseases, 178:53 (1998) 1977 “Russian Flu” H1N1 pandemic

Emerged on the Soviet / Chinese border in 1977, was identical to H1N1 from the 1950’s.

This H1N1 did not replace the H3N2 virus. Instead, they co- circulated in the human population.

dos Reis et al. Journal of Molecular Evolution, 69:333 (2009)

The 2009 swine-origin H1N1 strain

Garten et al, Science, 2009 highly pathogenic H5N1 avian inﬂuenza: deadly but not (at least yet) transmissible in humans Cumulative number of confirmed human cases for avian influenza A(H5N1) reported to WHO, 2003-2012

Country 2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 Total cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths cases deaths Azerbaijan 000000 85000000000000 85 Bangladesh 00000000001 000002 0 3 0 6 0 Cambodia 00004 4221110 1 0 118822 20 18 China 11008 5 13 8 534474211111 42 28 Djibouti 000000 1 0 000000000000 1 0 Egypt 000000 18 10 25 9 8 4 39 4 29 13 39 15 9 5 167 60 Indonesia 000020 13 55 45 42 37 24 20 21 19 9 7 12 10 6 6 189 157 Iraq 000000 32000000000000 32 Lao People's Democratic Republic 00000000220 0 0 0000000 22 Myanmar 000000001 00000000000 1 0 Nigeria 00000000110 0 0 0000000 11 Pakistan 00000000310 0 0 0000000 31 Thailand 0017 12 5 2 3 3 000000000000 25 17 Turkey 000000 12 4 000000000000 12 4 Viet Nam 3 3 29 20 61 19 00856555720042123 61 Total 4 4 46 32 98 43 115 79 88 59 44 33 73 32 48 24 62 34 25 16 603 356

Total number of cases includes number of deaths WHO reports only laboratory cases All dates refer to onset of illness 356 / 603 = 59% case fatality rate

May be an overestimate due to under- reporting of non-lethal infections.

Source: WHO/GIP, data in HQ as of 02 May 2012 Could H5N1 become transmissible in humans? Ferrets as a model for airborne transmissibility. Example: the 2009 swine-origin pandemic transmits by respiratory droplets in ferrets, but its immediate swine precursor viruses do not.

Yen et al, PNAS, 108:14264-14269 (2011)

LETTER doi:10.1038/nature10831

Experimental adaptation of an influenza H5 HA confers respiratory droplet transmission to a reassortant H5 HA/H1N1 virus in ferrets

Masaki Imai1, Tokiko Watanabe1,2, Masato Hatta1, Subash C. Das1, Makoto Ozawa1,3, Kyoko Shinya4, Gongxun Zhong1, Anthony Hanson1, Hiroaki Katsura5, Shinji Watanabe1,2, Chengjun Li1, Eiryo Kawakami2, Shinya Yamada5, Maki Kiso5, Yasuo Suzuki6, Eileen A. Maher1, Gabriele Neumann1 & Yoshihiro Kawaoka1,2,3,5 H5N1 Highly pathogenic avian H5N1 influenza A viruses occasionally before a pandemic. Therefore, we studied the molecular features that infect humans, but currently do not transmit efficiently among would render H5-HA-possessing viruses transmissible in mammals. humans. The viral haemagglutinin (HA) protein is a known Previous studies suggested that HA has a major role in host-range second small protein, PB1-F2, which has been REPORThost-range determinant as it mediates virus binding to host- restriction of influenza A viruses1–3.TheHAofhumanisolatespreferen- implicated in the induction of cell death (4, 5). specific cellular receptors1–3. Here we assess the molecular changes tially recognizes sialic acid linked to galactose by a2,6-linkages in HA that would allow a virus possessing subtype H5 HA to be (Siaa2,6Gal), whereas the HA of avian isolates preferentially recognizes Segments 4 and 6 encode the viral surface glyco- transmissible among mammals. We identified a reassortant H5 sialic acid linked to galactose by a2,3-linkages (Siaa2,3Gal)3. A small proteins hemagglutinin (HA) and neuraminidase HA/H1N1 virus—comprising H5 HA (from an H5N1 virus) with number of avian H5N1 viruses isolated from humans show limited Airbornefour mutations and the remaining Transmission seven gene segments from a binding to human-type of receptors, Influenza a property conferred by several amino (NA), respectively. HA is responsible for binding 2009 pandemic H1N1 virus—that was capable of droplet transmis- acid changes in HA4–9. None of the H5N1 viruses tested transmitted to sialic acids (SAs), the viral receptors on host sion in a ferret model. The transmissible H5 reassortant virus efficiently in a ferret model10–13, although, while our paper was under A/H5N1preferentially recognized human-type Virus receptors, Between replicated effi- review, one study Ferrets14 reported that a virus with a mutant H5 HA and a cells, and for fusion of the viral and host cell ciently in ferrets, caused lung lesions and weight loss, but was neuraminidase (NA) of a human virus in the H5N1 virus background membranes upon endocytosis. NA is a sialidase, Sandernot highly Herfst, pathogenic1 Eefje and did J. not A. cause Schrauwen, mortality. These1 Martin results Linster,caused respiratory1 Salin droplet Chutinimitkul, transmission in one1 ofEmmie two contact de ferrets. Wit,1* indicate that H5 HA can1 convert to an HA that supports1 efficient To identify1 novel mutations in avian2 H5 HAs that confer human-1,2,3 responsible for cleaving SAs from host cells and Vincentviral transmission J. Munster, in mammals;* Erin however, M. Sorrell, we do not knowTheo whether M. Bestebroer,type receptor-bindingDavid preference, F. Burke, we introducedDerek random J. Smith, mutations virus particles. Segment 5 codes for the nucleo- Guusthe four F. mutationsRimmelzwaan, in the H51 HAAlbert identified D. here M. would E. Osterhaus, render a into1 Ron the globular A. M. head Fouchier (amino acids1† 120–259 (H3 numbering), which wholly avian H5N1 virus transmissible. The genetic origin of the includes the receptor-binding pocket) of A/Vietnam/1203/2004 capsid protein (NP) that binds to viral RNA and, remaining seven viral gene segments may also critically contribute (H5N1; VN1203) HA (Supplementary Fig. 1). Although this virus Highlyto transmissibility pathogenic in avian mammals. influenza Nevertheless, A/H5N1 as H5N1 virus viruses can causewas isolated morbidity from a human, and mortalityits HA retains in avian-type humans receptor-binding but thus far together with the polymerase proteins, forms the continue to evolve and infect humans, receptor-binding variants properties6,15. We also replaced the multibasic HA cleavage sequence ribonucleoprotein complexes (RNPs). Segment 7 hasof H5N1not acquired viruses with the pandemic ability potential, to be including transmit avian–humanted by aerosolwith a or non-virulent-type respiratory droplet cleavage sequence,(“airborne allowing transmission us to per- ”) betweenreassortant humans. viruses as To tested address here, the may concern emerge. Our that findings the virusform could studies acquire in biosafety this level ability 2 containment under natural (http://www.who.int/ conditions, codes for the viral matrix structural protein M1 emphasize the need to prepare for potential pandemics caused by csr/resources/publications/influenza/influenzaRMD2003_5.pdf). The and the ion-channel protein M2 that is incorpo- weinfluenza genetically viruses modifiedpossessing H5 A/H5N1 HA, and will virus help by individuals site-direct con- edmutated mutagenesis polymerase and chain subsequentreaction (PCR) products serial werepassage cloned in into ferrets.ducting The surveillance genetically in regions modified with circulating A/H5N1 H5N1 virus viruses acquired to RNA mutations polymerase I during plasmids16 passagecontaining in the ferrets, VN1203 HA ultimately comple- rated in the viral membrane. Segment 8 encodes recognize key residues that predict the pandemic potential of iso- mentary DNA, which resulted in Escherichia coli libraries representing the nonstructural protein NS1 and the nucleic- becominglates, which airborne will inform transmissible the development, in production ferrets. Noneand distri- of thetherecipient randomly generated ferrets died HA variants. after airborne Sequence infection analysis of with48 thebution mutant of effective A/H5N1 countermeasures. viruses. Four amino acid substitutionsrandomly in selected the host clones receptor-binding indicated an average protein of 1.0 amino acid export protein (NEP) previously known as NS2. Although H5N1 viruses continue to cause outbreaks in poultry and changes per HA globular head (data not shown). To generate an hemagglutinin,there are cases of human and one infection in inthe Indonesia, polymerase Vietnam, complex Egypt and protH5N1ein virus basic library, polymerase plasmids for the 2, synthesis were consistently of the mutated HA present gene NS1 is an antagonist of host innate immune re- inelsewhere airborne-transmitted (http://www.who.int/influ viruses.enza/human_animal_interface/H5N1_ The transmissible virusesand the were unmodified sensitive NA gene to of the VN1203 antiviral were transfected drug oseltamivir into human sponses and interferes with host gene expression, cumulative_table_archives/en/index.html), they have not acquired the embryonic kidney (293T) cells together with plasmids for the synthesis andability reacted to cause well human-to-human with antisera transmission. raised Investment against in H5 H5N1 influenzaof the six vaccine remaining strains. viral genes Thus, of A/Puerto avian Rico/8/34 A/H5N1 (H1N1; influenza PR8), a whereas NEP is involved in the nuclear export vaccines has therefore been questioned. However, because humans laboratory-adapted human influenza A virus. of RNPs into the cytoplasm before virus assem- viruseslack immunity can acquire to influenza the viruses capacity possessing for anairborne H5 HA, the emergence transmissionTurkey between red blood mammals cells (TRBCs; without which possess recombination both Siaa2,6Gal inand an intermediateof a transmissible host H5-HA-possessing and therefore virus constitute would probably a risk cause for a humanSiaa2,3Gal pandemic on their surface influenza. (data not shown)) were treated with bly (2, 3). pandemic. To prepare better for such a scenario, it is critical that we Salmonella enterica serovar Typhimurium LT2 sialidase, which pref- Influenza A viruses show pronounced genetic understand the molecular changes that may render H5-HA-possessing erentially removes a2,3-linked sialic acid (that is, avian-type receptors), virusesnfluenza transmissible A viruses in mammals. have Such beenknowledge isolated would allow from us to creatingriiformes TRBCs (gulls, that predominantly terns, and possess waders) Siaa2,6Gal are thought on the cell to variation of the surface glycoproteins HA and on February 7, 2013 monitor circulating or newly emerging variants for their pandemic surface (Siaa2,6-TRBCs; Supplementary Fig. 2). The virus library NA (1). Consequently, the viruses are classified potential,many focus host eradication species, efforts including on viruses humans, that already pigs, have wasform then the adsorbed virus to Sia reservoira2,6-TRBCs in at nature 4 uC and ( extensively1). Influenza washed A acquiredhorses, subsets dogs, of molecular marine changes mammals, critical for and transmission a wide in toviruses remove nonspecifically belong to or theweakly family bound viruses. Orthomyxoviridae; Bound viruses were based on the antigenic variation of the HA and Imammals, stockpile antiviral compounds in regions where such viruses eluted by incubation at 37 uC for 30 min, and then diluted to approxi- NA proteins. To date, 16 major antigenic variants rangecirculate, of domestic and initiate vaccinebirds, generation yet wild and birds large-scale in the production orders matelythese, viruses0.5 viruses have per well an (on RNA the basis genome of a pilot experiment consisting that of of HA and 9 of NA have been recognized in wild Anseriformes1Department of Pathobiological (ducks, Sciences, geese, University and of Wisconsin-Madison, swans) and Madison, Charad- Wisconsin 53711, USA.eight2ERATO gene Infection-Induced segments Host Responses (2, Project,3). Saitama Segments 332-0012, Japan. 13Department to 3 en- of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. 4Department of Microbiology and Infectious Diseases, birds and are found in numerous combinations 5 code the polymerase proteins: basic polymerase6 1 Kobe University, Hyogo 650-0017, Japan. Division of Virology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan. Health Science DepartmentHills, College of Life of and Virology, Health Sciences, Erasmus Chubu University, Medical Kasugai, Center, Aichi 487-8501, Rotterdam, Japan. 2(PB2),basicpolymerase1(PB1),andacidic designated as virus subtypes (for instance, H1N1, The Netherlands. 2Department of Zoology, University of Cam- 3 polymerase (PA),00 MONTH respectively. 2012 | VOL 000 These | NATURE proteins | 1 H5N1, H7N7, and H16N3), which are used in bridge, Cambridge, UK. Fogarty International©2012 Macmillan Center, NationalPublishers Limited. All rights reserved Institutes of Health (NIH), Bethesda, MD 20892, USA. form the RNA-dependent RNA polymerase com- influenza A virus classification and nomenclature

(1, 6). This classification system is biologically www.sciencemag.org †To whom correspondence should be addressed. E-mail: plex responsible for transcription and replication [email protected] of the viral genome. Segment 2 also encodes a relevant, as natural host antibodies that recognize

AB C D 66! A/H5N1 6! A/H5N1 6! 6! A/H5N1 N182K A/H5N1 Q222L G224S N182K Q222L G224S 5! 5! 5! 5! 44! 4! 4! 4! 3! 3! 3! 3! 22! 2! 2! 2! 1! 1! 1! 1! 0! 0! 0! 0! log 10 TCID 50 / ml 0 Downloaded from

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8 8 8 8 8 8 8 8 7 7 7 7 7 7 7 7 6 6 6 6 6 6 6 6 5 5 5 5 5 5 5 5 4 4 4 4 4 4 4 4 3 3 3 3 3 3 3 3 2 2 2 2 2 2 2 2 tissue 1 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 NT T L NT T L NT T L NT T L NT T L NT T L NT T L NT T L log 10 TCID 50 / g tissue 3 dpi 7 dpi 3 dpi 7 dpi 3 dpi 7 dpi 3 dpi 7 dpi Fig. 1. In experiment 1, we inoculated groups of six ferrets intranasally with shedding from the URT as determined by virus titers in nasal and throat swabs 6 1 × 10 TCID50 of (A)influenzaA/H5N1wildtype virus and the three mutants (B) was highest in A/H5N1wildtype-inoculated animals. The mutant that yielded the A/H5N1HA N182K,(C)A/H5N1HA Q222L,G224S,and(D)A/H5N1HA N182K,Q222L,G224S. highest virus titers during the 7-day period was A/H5N1HA Q222L,G224S,buttiters Three animals were euthanized at day 3 for tissue sampling and at day 7, when were ~1 log lower than for the A/H5N1wildtype-inoculated animals. In the first this experiment was stopped. Virus titers were measured daily in nose swabs 3days,whensixanimalspergroupwerepresent, no significant differences (top) and throat swabs (middle) and also on 3 and 7 dpi in respiratory tract were observed between A/H5N1HA N182K-andA/H5N1HA Q222L,G224S-inoculated tissues (bottom) of individual ferrets. Virus titers in swabs and nasal turbinates animals, as calculated by comparing the viral titer (Mann-Whitney test, P = (NT), trachea (T), and lungs (L) were determined by end-point titration in MDCK 0.589 and 0.818 for nose and throat titers, respectively). (Bottom row) No cells. [One animal inoculated with A/H5N1HA N182K,Q222L,G224S died at 1 dpi due marked differences in virus titers in respiratory tissues were observed between to circumstances not related to the experiment (D).] (Top two rows) Virus the four groups. Each bar color denotes a single animal.

1534 22 JUNE 2012 VOL 336 SCIENCE www.sciencemag.org Benefits

1. Now we know that H5N1 has the potential to become airborne transmissible in mammals.

2. Now we know some of the specific mutations that might enable this to happen.

3. This information might aid in surveillance of the viruses ongoing evolution.

4. This information might aid in vaccine development against specific H5N1 strain lineages.

5. This information increases our mechanistic understanding of the basis for influenza transmissibility. Risks

1. Someone could recreate the virus using reverse genetics, and then release it intentionally or accidentally.

2. There is the potential for accidental release of the virus (currently all authorized work is restricted to BSL3).

3. Controversy about this research could impede other important types of inquiry into influenza and other diseases. Zoonosis

Intra-species Influenza A virus is transmission remarkable for the frequency and breadth of zoonoses: many species of birds, humans, pigs, horses, dogs