Zoonotic and pandemic influenza A viruses: lessons from H5N1 and H2N2

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(1)Zoonotic and Pandemic Influenza A Viruses: Lessons from H5N1 and H2N2. Martin Linster.

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(4) The research described in this thesis was conducted at the Department of Viroscience of the Erasmus Medical Center, Rotterdam, The Netherlands and within the Postgraduate School of Molecular Medicine. Cover design: ‘aerosol spray’ by the author Printed by PrintPartners Ipskamp B.V. © Martin Linster, 2020.

(5) =RRQRWLFDQG3DQGHPLF,QÀXHQ]D$9LUXVHV /HVVRQVIURP+1DQG+1 =R|QRWLVFKHHQSDQGHPLVFKHLQÀXHQ]D$YLUXVVHQ leren van H5N1 en H2N2. 3URHIVFKULIW. ter verkrijging van de graad van doctor aan de Erasmus Universiteit Rotterdam RSJH]DJYDQGHUHFWRUPDJQL¿FXV Prof. dr. R.C.M.E. Engels en volgens het besluit van het College voor Promoties. De openbare verdediging zal plaatsvinden op woensdag 12 februari 2020 om 09:30 uur door 0DUWLQ/LQVWHU geboren te Salzkotten, Duitsland.

(6) Promotiecommissie: promotor 3URIGU5$0)RXFKLHU overige leden Prof. dr. M.D. de Jong Prof. dr. M.P.G. Koopmans 3URIGU$'0(2VWHUKDXV copromotor Dr. S. Herfst.

(7) &KDSWHU 1 2 3 4 5 6 7 8 9. Title Introduction $LUERUQHWUDQVPLVVLRQRILQIOXHQ]D$+1 virus between ferrets Characterization, and Natural Selection of 0XWDWLRQV'ULYLQJ$LUERUQH7UDQVPLVVLRQRI $+1YLUXV $GDSWDWLRQRI3DQGHPLF+1YLUXVHVLQ Humans 7KH0ROHFXODU%DVLVIRU$QWLJHQLF'ULIWRI +XPDQ$+1,QÀXHQ]D9LUXVHV Transmissibility of human and avian H2N2 LQIOXHQ]D$YLUXVHVEHWZHHQIHUUHWV Summarizing Discussion 1HGHUODQGVH6DPHQYDWWLQJ Deutsche Zusammenfassung 3RUWIROLR'DQNZRRUG5HIHUHQFHV. 3DJHV 8-19 22-62 66-104 108-133 136-151 154-163 166-179 182-193 196-226.

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(11). Erin M. Sorrell Eefje J. Schrauwen Martin Linster Miranda de Graaf Sander Herfst 5RQ$0)RXFKLHU.

(12) chapter p 1: Introduction. ,QÀXHQ]D$YLUXVHV&ODVVL¿FDWLRQ ,QÀXHQ]D $ YLUXV LV D VHJPHQWHG QHJDWLYH VWUDQGHG 51$ YLUXV ZLWKLQ WKH $OSKDLQÀXHQ]DYLUXV JHQXV RI WKH IDPLO\ 2UWKRP\[RYLULGDH 7KLV IDPLO\ IXUWKHU FRQWDLQV WKH JHQHUD %HWDLQÀXHQ]DYLUXV *DPPDLQÀXHQ]DYLUXV DQG 'HOWDLQÀXHQ]DYLUXV DQG WKH WKUHH QRQLQÀXHQ]D JHQHUD 7KRJRWRYLUXV ,VDYLUXVDQG4XDUDQMDYLUXV

(13) ,QÀXHQ]D$YLUXVHVDUHGLYLGHGLQWRVXEW\SHV based on antigenic differences between the major surface glycoproteins KHPDJJOXWLQLQ +$

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(17) 7KHFRPELQDWLRQRI+$DQG1$RIDYLUXVOHDGVWRWKH common abbreviations such as H1N1, H2N2, H5N1, H7N9, H16N9 to specify YLUXV VXEW\SHV ,Q DGGLWLRQ WZR LQÀXHQ]D$OLNH YLUDO JHQRPHV KDYH EHHQ LGHQWL¿HG LQ IUXLW EDWV ZKLFK FRQWDLQ DQWLJHQLFDOO\ DQG IXQFWLRQDOO\ GLVWLQFW surface glycoproteins and have been provisionally designated “HL17NL10” DQG ³+/1/´ ZLWK +/ DQG 1/ LQGLFDWLQJ +$OLNH DQG 1$OLNH VXUIDFH JO\FRSURWHLQV

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(19) ,QWKHVLQÀXHQ]D$YLUXVHVZHUHLGHQWL¿HGWRFDXVHGLVHDVH in human and swine (7). The fact that these viruses from birds, pigs, and KXPDQVZHUHFORVHO\UHODWHGZDVQRWUHFRJQL]HGXQWLOPXFKODWHU,QÀXHQ]D virus isolates from different species have been collected since 1902 to cover the pandemic outbreaks of H2N2 in 1957, H3N2 in 1968, H1N1 in 2009 as well as zoonotic events in humans of H5N1 from 1997 and H7N9 from 2013 onwards respectively.. ,QÀXHQ]D$YLUXVHV*HQRPHFRQVWHOODWLRQDQGYLULRQVWUXFWXUH ,QÀXHQ]D$YLUXVSDUWLFOHVDUHSOHRSPRUSKLFVSKHULFDOWR¿ODPHQWRXVZLWKD diameter of about 80-120 nm, and a lipid bilayer envelope that is derived from WKHKRVWFHOOPHPEUDQH )LJXUH

(20) 7KHWZRPDMRUJO\FRSURWHLQV+$DQG1$ cover the viral surface and have important functions during virus attachment and release. M1 proteins line the inner part of the viral membrane mainly conferring stability to the virion. M2 proteins are transmembrane proteins DFWLQJDVLRQFKDQQHOV7KHJHQRPHRILQÀXHQ]D$YLUXVHVFRQVLVWVRIJHQH VHJPHQWVRIQHJDWLYHVWUDQGHG51$HQFRGLQJDWOHDVWYLUDOSURWHLQV(DFK 51$VHJPHQWLQWKHLQIHFWLRXVYLUXVSDUWLFOHLVDVVRFLDWHGZLWKQXFOHRSURWHLQ (NP) and the polymerase proteins basic polymerase 1 and 2, and acidic SRO\PHUDVH 3% 3% DQG 3$

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(24) chapter p 1: Introduction. M2 PA. PB1. PB2. M1 HA NP. PB2 PB1 PA. HA NP NA M NS. NA. )LJXUH6WUXFWXUHRIWKHLQÀXHQ]D$YLULRQ The viral genome consisting of eight viral RNPs is surrounded by a lipid bilayer lined by M1 SURWHLQV+$1$DQG0DUHVXUIDFHPROHFXOHVDQFKRUHGLQWKHPHPEUDQH)LJXUHWDNHQIURP Subbarao et al. (9).. ,QÀXHQ]D$YLUXVLQIHFWLRQDQGUHSOLFDWLRQ ,QÀXHQ]D$YLUXVHVDWWDFKWRFHOOVWKURXJKLQWHUDFWLRQRIWKH+$SURWHLQZLWK terminal sialic acid containing receptors located on the cell surface. The YLUXVHVDUHVXEVHTXHQWO\LQWHUQDOL]HGLQWRHQGRVRPHVE\DSURFHVVFDOOHG receptor-mediated endocytosis (10). The lowering of the pH in the endosome WULJJHUVDFRQIRUPDWLRQDOFKDQJHRIWKH+$DOORZLQJWKHYLUDODQGHQGRVRPDO membranes to fuse and the viral RNPs to be released into the cytosol. The RNPs are then transported to the nucleus, leading to the production of three GLIIHUHQWW\SHVRI51$YLUDO51$ Y51$

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(28) 6XUIDFHSURWHLQV+$1$ and M2 are transported via the Golgi apparatus to the cellular membrane. This is where new infectious particles are assembled and released through FOHDYDJHRIUHPDLQLQJOLQNDJHVEHWZHHQ+$DQGWKHVLDOLFDFLGUHFHSWRUE\ 1$

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(30) chapter p 1: Introduction. )LJXUH5HSOLFDWLRQF\FOHRILQÀXHQ]D$YLUXVHV 8SRQELQGLQJWRWKHVLDOLFDFLGUHFHSWRULQÀXHQ]D$YLUXVLVLQWHUQDOL]HGE\UHFHSWRUPHGLDWHG endocytosis, their genomes are replicated in the nucleus, and new virus particles are DVVHPEOHGDWWKHPHPEUDQHEHIRUHWKH\FDQEXG)LJXUHDGDSWHGIURP1HXPDQQHWDO

(31) . The viral polymerases and nucleoprotein 7KH WKUHH ORQJHVW JHQH VHJPHQWV RI LQÀXHQ]D $ YLUXVHV HQFRGH WKH SRO\PHUDVHSURWHLQV3%3%DQG3$ZKLFKWRJHWKHUIRUPWKHSRO\PHUDVH FRPSOH[ 6LQFH WKHVH YLUDO 51$ GHSHQGHQW 51$ SRO\PHUDVHV ODFN proofreading activity, the resulting high mutation rate can ultimately lead to rapid adaptation of the virus to a new host. PB2 binds to a cap, the altered QXFOHRWLGHDWWKH¶HQGRIVRPH51$VDQGWKLVFDSELQGLQJVWHSLVLPSRUWDQW for transcription initiation (12). PB2 also harbors the well-studied glutamic acid (E) to lysine (K) substitution at position 627, which is associated with mammalian host-adaptation (13). The PB1 protein is the catalytic subunit RI WKH SRO\PHUDVH FRPSOH[ DQG DOORZV V\QWKHVLV RI 51$ 3$ LV LPSRUWDQW IRU WKH LQWHUDFWLRQ RI DOO VXEXQLWV LQ WKH FRPSOH[ DQG H[HUWV HQGRQXFOHDVH function. In addition to these 3 polymerase subunits, NP is essential for viral JHQRPHUHSOLFDWLRQLWLVDVVRFLDWHGZLWKDQGSK\VLFDOO\SURWHFWVYLUDO51$V IURPGHJUDGDWLRQE\KRVWSURWHLQV

(32) )RUDIXOOOLVWRILQÀXHQ]D$YLUXVJHQH products, please refer to Table 1.. 10.

(33) chapter p 1: Introduction. The hemagglutinin molecule 7KH +$ VXUIDFH JO\FRSURWHLQ LV ERWK D PDMRU WDUJHW IRU DQWLERGLHV DQG D FUXFLDOGHWHUPLQDQWRISDWKRJHQLFLW\RILQÀXHQ]D$YLUXVHV7KH+$PROHFXOH is a homotrimer (i.e. consisting of three identical subunits or monomers) and harbors the receptor binding site (RBS) (15), a discontinuous ‘groove’ RIDPLQRDFLGVLQWHUDFWLQJZLWK6$UHFHSWRUVRQKRVWFHOOV+$LVHVVHQWLDO for infection, because it induces fusion between the membranes of the virus and the endosome allowing the release of the viral genetic material into the F\WRVRO7KLVIXQFWLRQLVGHSHQGHQWRQSUHYLRXVFOHDYDJHRIWKH+$E\KRVW SURWHDVHV$OO LQÀXHQ]D$ YLUXVHV FRQWDLQ D FOHDYDJH VLWH WKH DPLQR DFLG SRVLWLRQDWZKLFKWKHELRORJLFDOO\LQDFWLYH+$PROHFXOHLVVSOLWLQWR+$DQG +$/RZSDWKRJHQLFDYLDQLQÀXHQ]D /3$,

(34) YLUXV+$FDQRQO\EHFOHDYHGE\ WU\SVLQOLNHSURWHDVHVSULPDULO\H[SUHVVHGLQWKHUHVSLUDWRU\WUDFWRIPDPPDOV DQGLQWHVWLQDOWUDFWRIELUGVUHVWULFWLQJ+$SURFHVVLQJWRWKHVHRUJDQV\VWHPV

(35) +LJKO\SDWKRJHQLFDYLDQLQÀXHQ]DYLUXVHV +3$,YLUXVHV

(36) FDQHPHUJH after circulation in poultry. They contain a multi basic cleavage site (MBCS) - a stretch of several basic amino acids – that can be cleaved by a range of SURWHDVHVWKDWDUHXELTXLWRXVO\H[SUHVVHGWKURXJKRXWWKHERG\RIPDPPDOV and birds, allowing systemic virus replication and spread. Glycosylation, the DGGLWLRQRIVXJDUPRLHWLHVWRWKH+$DWVSHFL¿FSRVLWLRQV HVSHFLDOO\WKRVH close to the RBS) can modify binding of antibodies, innate immune sensing DQG UHFHSWRU ELQGLQJ XOWLPDWHO\ LQÀXHQFLQJ LQIHFWLYLW\ SDWKRJHQLFLW\ DQG SRWHQWLDOO\WUDQVPLVVLELOLW\

(37) 6LQFHJO\FRV\ODWLRQRIWKH+$GHSHQGVRQWKH FHOO PDFKLQHU\ GLIIHUHQFHV LQ JO\FRV\ODWLRQ SDWWHUQ H[LVW LQ GLIIHUHQW KRVWV including cell lines and embryonated chicken eggs (18).. 5HFHSWRUVSHFL¿FLW\RI+$ Sialic acids at the terminal position of carbohydrate chains on the host cell VXUIDFHDUHWKHWDUJHWRIWKH+$PROHFXOHIRUDWWDFKPHQW7KHUHFHSWRUELQGLQJ VLWH LV ORFDWHG LQ WKH KHDGGRPDLQ RI WKH +$ DQG LQGLYLGXDO DPLQR DFLGV within the binding pocket can engage in bonds with the sialic acid molecule. &RQVHTXHQWO\VLQJOHVXEVWLWXWLRQVLQWKH5%6FDQLQÀXHQFHUHFHSWRUELQGLQJ VSHFL¿FLW\DQGDI¿QLW\

(38) $YLDQDQGKXPDQDGDSWHGLQÀXHQ]D$YLUXVHV vary in their preference for the type of linkage of the sialic acid molecule. The ‘human-type’ receptor consists of an oligosaccharide with the terminal sialic DFLGERXQGWRJDODFWRVHLQDQĮFRQIRUPDWLRQZKHUHDVWKHµDYLDQW\SH¶ UHFHSWRU WHUPLQDO VXJDUV DUH DUUDQJHG LQ DQ Į PDQQHU

(39) $OWKRXJK human tissue contains both groups of glycans, severity of disease can be DWOHDVWSDUWLDOO\DWWULEXWHGWRGLIIHUHQWLDOH[SUHVVLRQRIUHFHSWRUVWKURXJKRXW the respiratory tract. In both the ferret and human upper respiratory tract 857

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(44) chapter p 1: Introduction. 7DEOH,QÀXHQ]D$YLUXVSURWHLQVDQGWKHLUPDMRUIXQFWLRQ V

(45) 7DEOHDGDSWHGIURP)LHOGV9LURORJ\9,WKHGLWLRQ

(46) . gene. gene. QXPEHU. VHJPHQW. 1. PB2. 2. PB1. JHQHSURGXFW Polymerase Basic 2. internal protein,. (PB2). SDUWRISRO\PHUDVHFRPSOH[. Polymerase Basic 1. internal protein,. (PB1). SDUWRISRO\PHUDVHFRPSOH[. 3RO\PHUDVH%DVLF) 3%)

(47). induction of apoptosis. Polymerase Basic 1-N40. 3. +$. 5. NP. 6. 1$. (PB1-N40). N-terminally truncated PB1, no polymerase function. 3RO\PHUDVH$FLGLF 3$

(48). internal protein, SDUWRISRO\PHUDVHFRPSOH[. 3RO\PHUDVH$FLGLF;. internal protein,. 3$;

(49). host response modulation. 3RO\PHUDVH$FLGLF1. 1WHUPLQDOO\WUXQFDWHG3$. 3$1

(50). no polymerase function. 3RO\PHUDVH$FLGLF1. 1WHUPLQDOO\WUXQFDWHG3$. 3$1

(51). no polymerase function. Hemagglutinin +$

(52). surface glycoprotein, membrane fusion viral attachment, antigenic determinant. Nucleoprotein. nucleocapsid protein,. (NP). 51$FRDWLQJQXFOHDUWDUJHWLQJ. Neuraminidase. surface glycoprotein, virion release, antigenic determinant. 1$

(53) 0DWUL[. 8. 12. targets mitochondria,. 3$. 4. 7. PDMRU IXQFWLRQV. M. (M1). membrane protein, virion stability. 0DWUL[. membrane protein. (M2). ion channel. 0DWUL[. M2-like membrane protein,. (M42). ion channel. Nonstructural 1. internal protein. (NS1). multifunctional, immune modulatory. NS Nonstructural 2. internal protein. (NS2 or NEP). QXFOHDUH[SRUWRI513V.

(54) <HDU)LUVW Detected. 1958. 1959. 1979. 1992. 1997. 1998. 2003. 2004. 2004. 2006. 2007. 2013. 2013. 2013. 2014. 2017. 2018. 6XEW\SH*URXS. H1N1v. +3$,+1. /3$,+1. H3N2v. +3$,+1. /3$,+1. /3$,+1. +3$,+1. /3$,+1. /3$,+1. H1N2v. /3$,+1. /3$,+1. /3$,+1. +3$,+1. +3$,+1. /3$,+1. 2018. 2017. 2018. 2013. 2014. 2018. 2017. 2006. 2012. 2012. 2017. 2015. 2019. 2019. 2013. 2003. 2018. <HDU/DVW Detected. China. China, Taiwan. China. Taiwan. China. Canada, China, Malaysia, Taiwan. Brazil, Philippines, US. United Kingdom. $XVWUDOLD(J\SW. &DQDGD0H[LFR. United Kingdom, US. 1. 8. 22. 1. 3. 1568. 26. 1. 4. 4. 7. 39. 861. $]HUEDLMDQ%DQJODGHVK&DPERGLD&DQDGD&KLQD'MLERXWL(J\SW+RQJ.RQJ ,QGRQHVLD,UDT/DRV0\DQPDU1HSDO1LJHULD3DNLVWDQ7KDLODQG7XUNH\9LHWQDP %DQJODGHVK&KLQD(J\SW+RQJ.RQJ6$5. 435. 5. $XVWUDOLD&DQDGD+RQJ.RQJ6$51HWKHUODQGV869LHWQDP. US, Italy, United Kingdom. 91. 43. Canada, China, Czechoslovakia, Italy, Netherlands, Russia, Spain, Switzerland, Thailand, US $XVWUDOLD861HWKHUODQGV. 1XPEHURI &RQILUPHG &DVHV. &RXQWULHVRI2FFXUUHQFH. 0. 4. 6. 0. 2. 616. 0. 1. 0. 0. 0. 1. 455. 2. 0. 1. 7. 1XPEHURI &RQILUPHG )DWDOLWLHV. 7DEOH=RRQRWLFLQÀXHQ]DYLUXVLQIHFWLRQVLQKXPDQVUHSRUWHGZRUOGZLGH 7DEOHGDWDDGDSWHGIURP:LGGRZVRQHWDO

(55) DQGXSGDWHGEDVHGRQ:+2PRQWKO\ULVNDVVHVVPHQWVXPPDULHVXQWLO0D\QG 9LUXVHVDUHVRUWHGE\¿UVW\HDURIGHWHFWLRQ. chapter p 1: Introduction. 13.

(56) chapter p 1: Introduction. Genetic drift and shift 7ZR GLVWLQJXLVKDEOH PHFKDQLVPV OHDG WR JHQHWLF YDULDWLRQ RI LQÀXHQ]D $ viruses. The term ‘drift’ denotes the successive accumulation of mutations WKDWEHFRPH¿[HGLQWKHYLUXVSRSXODWLRQHLWKHUVWRFKDVWLFDOO\RUGXHWRWKHLU ¿WQHVVDGYDQWDJHµ6KLIW¶UHIHUVWRWKHUHDUUDQJHPHQWRIJHQHWLFPDWHULDODOVR termed reassortment and was the main underlying mechanisms that led to the pandemics of 1957, 1968 and 2009 (24, 25). Coinfection of a single cell ZLWK WZR RU PRUH GLIIHUHQW LQÀXHQ]D$ YLUXVHV LV UHTXLUHG IRU UHDVVRUWPHQW WR KDSSHQ 'XULQJ YLULRQ IRUPDWLRQ JHQH VHJPHQWV RI LQÀXHQ]D$ YLUXVHV can be redistributed in arrangements different from the two or more parental YLUXVHV3LJVDUHZLGHO\FRQVLGHUHGWRIRUPµPL[LQJYHVVHOV¶EHFDXVHWKH\ FDQEHLQIHFWHGZLWKDUDQJHRIKXPDQDQGDYLDQDGDSWHGLQÀXHQ]D$YLUXVHV (26). Reassortment is obviously the more drastic change in virus evolution and led to the formation of several - if not all - pandemics of the last century )LJXUH

(57) ,QGLYLGXDOVXEVWLWXWLRQVVHHPWRµ¿QHWXQH¶WKHYLUDOSKHQRW\SHWR yield optimal host adaptation.. =RRQRWLFLQÀXHQ]D$YLUXVHV ,QFLGHQWDOLQIHFWLRQRIPDPPDOVZLWKLQÀXHQ]D$YLUXVHVRIYDULRXVVXEW\SHV QRUPDOO\IRXQGLQDYLDQVSHFLHVFDQRFFXU,QWKHSDVWFHQWXU\LQÀXHQ]D$ YLUXVVXEW\SHV++DQG+ZHUHFDSDEOHRIFDXVLQJSDQGHPLFV H[WHQGHG outbreaks on multiple continents), while H5, H6, H7, H9, and H10 viruses FDXVHG]RRQRVHV FRQ¿QHGLQIHFWLRQVDWWKHKXPDQDQLPDOLQWHUIDFH

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(59) =RRQRWLFHYHQWVKDYHEHHQUHSRUWHGIUHTXHQWO\ recently, partially due to increased awareness, increased surveillance and improved detection methods. +3$,DYLDQ+1YLUXVFLUFXODWLQJLQ6RXWK(DVW$VLDVLQFHZDVUDSLGO\ UHFRJQL]HGDVWKHFDXVDWLYHDJHQWRIWKH¿UVWRXWEUHDNLQKXPDQVLQ

(60) ,Q$XJXVWWKHLQGH[KXPDQFDVHRI+3$,+1YLUXVLQIHFWLRQ ZDVUHSRUWHGKRVSLWDOL]HGSDWLHQWVLQ+RQJ.RQJ6SHFLDO$GPLQLVWUDWLYH Region were infected with viruses closely resembling those found in FKLFNHQ

(61) VL[RIWKHPVXFFXPEHGGXHWRWKHLQIHFWLRQ5LJRURXVFRQWURO measurements including the temporary closing down of live bird markets and culling of poultry prevented the further spread of H5N1 virus in Hong Kong. H5N1 virus seemed to have disappeared after that but reemerged in LQ&KLQD6LQFHWKHQWKHYLUXVKDVEHHQGHWHFWHGIUHTXHQWO\LQYDULRXV hosts in different parts of the world and continues to be detected to date. In DGGLWLRQWRGRPHVWLFSRXOWU\+3$,+1YLUXVZDVDOVRIRXQGLQSLJVFDWV dogs, martens, tigers and leopards. Human infections are detected from time to time, so far adding up 861 cases, 455 of which were fatal (30). The case fatality rate in hospitalized cases is 50-60% with the peak age group of infected humans at 10-19 years (31). However, the true case-fatality rate is SUREDEO\ORZHUDVDFRQVHTXHQFHRIXQGHUUHSRUWLQJ(SLGHPLRORJ\VWXGLHV. 14.

(62) chapter p 1: Introduction. LGHQWL¿HG SRXOWU\ PDUNHWV DV D PDMRU VRXUFH IRU KXPDQ LQIHFWLRQV

(63) ,Q 2015, clusters of human infections have been reported from Egypt totaling to 119 infected individuals by the end of March (33). In recent years, the number of H5N1 infections has been decreasing with only four laboratoryFRQ¿UPHGUHSRUWVLQDQGDVLQJOHFDVHLQ1HSDOLQ (LJKW\VHYHQ KXPDQ LQIHFWLRQV RI +1 YLUXV ZHUH FRQ¿UPHG LQ WKH Netherlands in 2003, one of which was fatal (34, 35). Human infections with +1YLUXVHVZHUHUHSRUWHG¿UVWLQ&KLQDLQ

(64) FXPXODWLQJWR human infections including 616 fatal cases by May 2019 (37). Sporadic human infections have also been detected in Hong Kong, Taiwan, Canada, DQG0DOD\VLD7KHGLVHDVHFDQUDSLGO\SURJUHVVIURPJHQHUDOLQÀXHQ]DOLNH symptoms to severe pneumonia (38). Poultry workers in China are at great ULVNIRU+1LQIHFWLRQ

(65) ,QIHFWLRQVVHHPWRDSSHDULQZDYHVZLWKWKH¿IWK wave currently ongoing (40). H7N9 displays limited transmission capability in the ferret model by the airborne route, but sustained transmission upon direct FRQWDFW

(66) 7KLV+1YLUXVKDVDFTXLUHGDQXPEHURIPXWDWLRQVWKDW are associated with human adaptation and resistance against antiviral drugs and could possibly lead to a future pandemic after further human adaptation

(67) ,Q)HEUXDU\&KLQHVHDXWKRULWLHVUHSRUWHGWKHGHWHFWLRQRIKXPDQ infections with highly-pathogenic H7N9 viruses in the Guangdong Province (44). These viruses contained a multiple basic cleavage site, but are not associated with increased disease severity in humans so far (45). Three other H7 virus subtypes (H7N2, H7N3, H7N4) have been associated ZLWK VSRUDGLF KXPDQ LQIHFWLRQ

(68) $Q +1 YLUXV ZDV LVRODWHG IURP one patient in Taiwan in 2013 (48). H6 viruses are capable of infecting mice DQGIHUUHWDQGDUHWUDQVPLVVLEOHEHWZHHQJXLQHDSLJV

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(70) KDV EHHQ FLUFXODWLQJ H[WHQVLYHO\ LQ WKH (DVWHUQ hemisphere for more than a decade. In 2013, H10N8 viruses caused three human infections in China, two of which were fatal (54). Numbers mentioned above are correct as of 8 May 2019.. 3DQGHPLFLQÀXHQ]D$YLUXVHV ,QUDUHFDVHV]RRQRWLFLQÀXHQ]D$YLUXVHVFDQEHFRPHHVWDEOLVKHG LQWKH human population. Upon introduction in humans, the high error rate of the YLUDO SRO\PHUDVH DQG WKH FDSDFLW\ RI UHDVVRUWPHQW JHQH VHJPHQW PL[LQJ

(71) IDFLOLWDWH UDSLG DGDSWDWLRQ WR WKH QHZ KRVW 7R VSUHDG HI¿FLHQWO\ EHWZHHQ humans and cause a pandemic, dissemination via the airborne route (virus WUDQVPLVVLRQYLDDHURVRODQGRUUHVSLUDWRU\GURSOHWV

(72) DQGDVXI¿FLHQWO\ODUJH LPPXQRORJLFDOO\QDwYHSRSXODWLRQLVFUXFLDO,WZDVORQJDVVXPHGWKDWLQÀXHQ]D viruses capable of causing pandemics comprised subtypes H1, H2 and H3 only, since pandemics of the past were restricted to these three subtypes. )RUWKHSDQGHPLFVRI +1

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(75) chapter p 1: Introduction. mammalian. avian. H?N?. H?N?. seasonal human. H1N1. (PB2, PA, NP, MA, NS). avian. H2N2. (PB1, HA, NA). seasonal human. avian. seasonal human. H2N2. H3N?. H3N2. (PB2, PA, NP, NA, MA, NS) (PB1, HA). (PB1). avian. H1N1. (PB2, PA). classical swine. avian. H1N1. H1N1. (HA, NP, NS). eurasian swine. reassortant. H1N1. H1N?. (NA, MA). (PB2, PB1, PA, HA, NP, NS). reassortant. reassortant. reassortant. H1N1?. H2N2. H3N2 reassortant. H1N1. pandemic human. pandemic human. pandemic human. H1N1. H2N2. H3N2. before or in 1918 ‘Spanish influenza’ H1N1. before or in 1957 ‘Asian influenza’ H2N2. before or in 1968 ‘Hong Kong influenza’ H3N2. animal Influenza A viruses. H1N1. zoonotic human Influenza A viruses. before on in 2009 ‘Mexican influenza’ H1N1. continuous ‘cross species transmission’ of influenza A viruses. pandemic human. )LJXUH 6FKHPDWLF RYHUYLHZ RI JHQHWLF HYHQWV OHDGLQJ WR SDVW SDQGHPLF YLUXVHVDQGLQWHUVSHFLHVWUDQVPLVVLRQRI]RRQRWLFLQÀXHQ]DYLUXVHV Gene segments are indicated in decreasing size 3%3%3$+$131$0316

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(78) chapter p 1: Introduction. evidence is available (55). Viruses that caused the pandemic in 1918 (H1N1) were reconstructed from tissues of individuals that succumbed to the infection and were buried in the permafrost and from archived autopsy material (56). )RUWKH +1

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(81) SDQGHPLFVYLUXVZDV isolated from infected patients directly. $ FHQWXU\ DJR WKH GHDGOLHVW LQÀXHQ]D SDQGHPLF UHFRUGHG WR GDWH ZDV sparked by an H1N1 virus. This virus was most likely directly introduced LQ WKH KXPDQ SRSXODWLRQ IURP ELUGV

(82) $Q HVWLPDWHG WR PLOOLRQ LQGLYLGXDOVVXFFXPEHGGXULQJWKHµ6SDQLVKÀX¶SDQGHPLFLQWKHZLQWHUPRQWKV RIDQGUHPDUNDEO\WKHPDLQGHDWKWROOZDVREVHUYHGLQRWKHUZLVH healthy adults (58). +1LQÀXHQ]D$YLUXVHVZHUH¿UVWUHSRUWHGLQ6RXWKHDVW$VLDLQDQG caused a pandemic with an estimated mortality of 1 to 4 million people ZRUOGZLGH

(83) 5HDVVRUWPHQWEHWZHHQDYLDQDQGKXPDQLQÀXHQ]D$YLUXVHV OHGWRWKHIRUPDWLRQRI+1YLUXVHVFRQVLVWLQJRIWKH+$1$DQG3%JHQH VHJPHQWVIURPDYLDQLQÀXHQ]DYLUXVHVDQGWKHRWKHU¿YHJHQHVHJPHQWVIURP the human H1N1 virus that had been circulating since 1918 (60, 61). In 1968, after 12 years of circulation in the human population, H2N2 was replaced by H3N2, after one or several reassortment events with avian viruses. Since WKHQ LQÀXHQ]D$ YLUXVHV RI WKH +1 VXEW\SH DQG RWKHU FRPELQDWLRQV RI H2 and N2 keep circulating in waterfowl and pigs globally (62-65), thereby providing a reservoir from which H2N2 may be reintroduced into the human population. Since H2N2 viruses have been absent in the human population from 1968 onwards, many individuals, and certainly those born after 1968, KDYHQRVLJQL¿FDQWLPPXQRORJLFDOSURWHFWLRQDJDLQVWWKHVHYLUXVHV 6XEVHTXHQWO\LQDQHZ+1YLUXVVWUDLQDSSHDUHGLQ$VLD7KHYLUXV was a reassortant from the previously circulating H2N2 virus and contained JHQH VHJPHQWV 3% DQG +$ IURP DQ DYLDQ + YLUXV

(84) 7KH ¿UVW YLUXV isolate was detected in Hong Kong in July 1968, hence the name ‘Hong .RQJÀX¶SDQGHPLF

(85) In the years preceding 2009, a number of reassortment events involving human H3N2, avian H1N1 and swine H1N1 viruses led to the generation of DQHZUHDVVRUWDQW+1YLUXVWKDWZDVDWWKHVWDUWRIWKH¿UVWSDQGHPLFRIWKH VWFHQWXU\

(86) 7KHµ0H[LFDQÀX¶YLUXVZDV¿UVWGHWHFWHGLQ6RXWK$PHULFD and had direct precursors in pigs (68). Both H3N2 and the recent pandemic +1YLUXVDUHFLUFXODWLQJDVVHDVRQDOLQÀXHQ]D$YLUXVHVWRGDWH. 17.

(87) chapter p 1: Introduction. $LUERUQHWUDQVPLVVLRQRILQÀXHQ]D$YLUXVHVEHWZHHQPDPPDOV 8OWLPDWHO\ DOO SDQGHPLF LQÀXHQ]D$ YLUXVHV ZHUH RQFH ]RRQRWLF LQ QDWXUH EXWQRWDOO]RRQRWLFYLUXVHVKDYHDFTXLUHGRUZLOOHYHUDFTXLUHWKHSRWHQWLDO to become airborne transmissible and cause a pandemic. Human-adapted viruses usually cause infections that are restricted to the respiratory tract with severe cases leading to pneumonia, but they do generally not infect other organs (69). Mutagenesis of the RBS of pandemic 1918 H1N1, SDQGHPLF+1DQGVHDVRQDO+1+$UHVXOWHGLQDVZLWFKRIUHFHSWRU preference, from the human- to avian-type receptor, abrogating airborne transmission between ferrets (70-72). It might be speculated that the scarcity of avian receptors in human upper respiratory epithelium cannot prevent LQIHFWLRQZLWKLQÀXHQ]D$YLUXVHVGLVSOD\LQJDYLDQW\SHUHFHSWRUSUHIHUHQFH but does not allow airborne transmission. We previously hypothesized, after analyses of the processes leading to the H1N1 (1918), H2N2 (1957), H3N2 (1968), and H1N1 (2009) pandemic viruses, that three different virus properties may be crucial for aerosol transmission between mammals to occur; binding to cells in the upper respiratory tract 857

(88) UHSOLFDWLRQWRVXI¿FLHQWO\KLJKYLUDOWLWHUVLQWKHFHOOVRIWKH857DQG the generation of individual virus particles rather than virus aggregates (1). 9LUXVFRQWDLQLQJDHURVROVRUUHVSLUDWRU\GURSOHWVFDQEHH[KDOHGGLUHFWO\IURP the URT without settling of substantial portions of the particles on inanimate VXUIDFHVDIWHUWKHLUIRUPDWLRQ5HVSLUDWRU\GURSOHWVDUHFRPPRQO\GH¿QHGDV moisture particles >5 μm in diameter. They are attracted by gravity and settle UDWKHU TXLFNO\ $HURVROV DUH VSKHULFDO SDUWLFOHV VPDOOHU WKDQ P LQ VL]H that can stay in the air for prolonged periods. Both types of particles travel through the air and are formed by coughing, sneezing, breathing and talking. Since aerosols can travel larger distances in the air, this type of particle is suggested to be at the origin of pandemics when spiked with human-adapted LQÀXHQ]D$ YLUXVHV 7R HVWDEOLVK LQIHFWLRQ D VXI¿FLHQW QXPEHU RI DHURVROV needs to contain virus. High viral titers and the packaging of individual virions LQDLUERUQHGURSOHWVVHHPWREHUHTXLUHGIRUHIIHFWLYHDLUERUQHVSUHDG. Vaccines and antivirals ,QÀXHQ]DYLUXVLQIHFWLRQFDQFDXVHV\PSWRPVLQKXPDQVUDQJLQJIURPFRXJK sore throat, fatigue and nausea to pneumonia and acute respiratory distress V\QGURPH&XUUHQWO\OLFHQVHGYDFFLQHVDUHDYDLODEOHIRUVHDVRQDO$ +1

(89) SGPDQG+1LQÀXHQ]D$YLUXVHVDQGWKHWZROLQHDJHVRILQÀXHQ]D% virus. Vaccination is recommended for high-risk individuals including the elderly, people with chronic diseases, pregnant women and healthcare ZRUNHUV$OWKRXJKWKHHIIHFWLYHQHVVRIFXUUHQWYDFFLQHVLVIDUIURPSHUIHFW WKH\ KDYH DQ LPSRUWDQW UROH WR UHGXFH LQÀXHQ]D DVVRFLDWHG PRUELGLW\ DQG PRUWDOLW\

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(91) chapter p 1: Introduction. LPSURYHG SURWHFWLRQ &XUUHQWO\ +1 +1 DQG LQÀXHQ]D % YLUXVHV DUH circulating worldwide. Vaccines comprise strains of the above-mentioned YLUXVHV WKDW DUH VHOHFWHG WZLFH \HDUO\ E\ DQ H[SHUW JURXS XQGHU JXLGDQFH RI WKH :RUOG +HDOWK 2UJDQL]DWLRQ :+2

(92) 5HFHQWO\ DYDLODEOH WHWUDYDOHQW LQÀXHQ]DYDFFLQHVLQFOXGHERWK9LFWRULDDQG<DPDJDWDOLQHDJHLQÀXHQ]D% virus seed strains. $QWLYLUDOGUXJVDJDLQVWLQÀXHQ]D$YLUXVHVFRPSULVHWKHWZRQHXUDPLQLGDVH inhibitors oseltamivir and zanamivir as well as the two ion channel inhibitors amantadine and rimantadine. Immune compromised patients are likely to SUR¿WIURPWUHDWPHQWZLWKDQWLLQÀXHQ]DGUXJVWRUHGXFHYLUDOORDG5HVLVWDQFH mutations have been described for both classes of antivirals and are of growing concern nowadays (74).. Scope and outline of the thesis 7KLVWKHVLVSUHVHQWVUHVHDUFKRQLQÀXHQ]D$YLUXVVXEW\SHV+1DQG+1 to gain knowledge about zoonotic and pandemic viruses and potentially to DLGLQSDQGHPLFULVNPLWLJDWLRQLQWKHIXWXUH%RWKLQÀXHQ]D$YLUXVVXEW\SHV DUH FRQVLGHUHG WR EH SRVVLEOH FDQGLGDWHV IRU D IXWXUH LQÀXHQ]D SDQGHPLF Whereas H5N1 viruses circulate in avian species and cause incidental zoonotic infections, H2N2 virus was at the origin of a pandemic in 1957 and continued to circulate in waterfowl. Chapter 2 describes the natural selection of an altered H5N1 virus isolate in ferrets by repeated inoculation of naïve animals. The resulting passaged mutant virus was shown to be able to transmit via the air between ferrets. &KDSWHU GHVFULEHV WKH LGHQWL¿FDWLRQ RI WKH PXWDWLRQV HVVHQWLDO IRU WKLV DLUERUQH VSUHDG DQG WKH YLUDO SKHQRW\SHV DVVRFLDWHG ZLWK HDFK RI WKH ¿YH UHTXLUHG VXEVWLWXWLRQV 0RUHRYHU WKLV FKDSWHU GHPRQVWUDWHV WKDW DGDSWLYH changes in the H5N1 virus genome accumulated very rapidly during passage in ferrets. In chapter 4, the diversity of avian and human H2N2 virus isolates is highlighted in phylogenetic analyses, and sites within the viral genome that DUH XQGHU VHOHFWLRQ SUHVVXUHV DUH LGHQWL¿HG &KDSWHU IXUWKHU GHVFULEHV investigations to map the antigenic evolution of H2N2 viruses that circulated between 1957 and 1968, while chapter 6 depicts the potential to spread via the air for a range of avian and human H2N2 virus isolates. $VXPPDUL]LQJGLVFXVVLRQRIWKHVWXGLHVSUHVHQWHGLQWKLVWKHVLVLQWKHFRQWH[W of work of others is presented in chapter 7.. 19.

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(94) $LUERUQHWUDQVPLVVLRQRILQÀXHQ]D $+1YLUXVEHWZHHQIHUUHWV 6FLHQFH. Sander Herfst Eefje J.Schrauwen Martin Linster Salin Chutinimitkul Emmie de Wit Vincent J. Munster Erin M. Sorrell Theo M. Bestebroer 'DYLG)%XUNH Derek J. Smith *XXV)5LPPHO]ZDDQ $E'0(2VWHUKDXV 5RQ$0)RXFKLHU.

(95) chapter p 2: Generation of airborne A/H5N1. $%675$&7 +LJKO\ SDWKRJHQLF DYLDQ LQÀXHQ]D +1 YLUXV FDQ FDXVH PRUELGLW\ DQG PRUWDOLW\LQKXPDQVEXWWKXVIDUKDVQRWDFTXLUHGWKHDELOLW\WREHWUDQVPLWWHG by aerosol or respiratory droplet (“airborne transmission”) between humans. 7RDGGUHVVWKHFRQFHUQWKDWWKHYLUXVFRXOGDFTXLUHWKLVDELOLW\XQGHUQDWXUDO FRQGLWLRQVZHJHQHWLFDOO\PRGL¿HG+1YLUXVE\VLWHGLUHFWHGPXWDJHQHVLV DQGVXEVHTXHQWVHULDOSDVVDJHLQIHUUHWV7KHJHQHWLFDOO\PRGL¿HG+1YLUXV DFTXLUHGPXWDWLRQVGXULQJSDVVDJHLQIHUUHWVXOWLPDWHO\EHFRPLQJDLUERUQH transmissible in ferrets. None of the recipient ferrets died after airborne LQIHFWLRQ ZLWK WKH PXWDQW +1 YLUXVHV )RXU DPLQR DFLG VXEVWLWXWLRQV LQ the host receptor-binding protein hemagglutinin, and one in the polymerase FRPSOH[SURWHLQEDVLFSRO\PHUDVHZHUHFRQVLVWHQWO\SUHVHQWLQDLUERUQH transmitted viruses. The transmissible viruses were sensitive to the antiviral GUXJRVHOWDPLYLUDQGUHDFWHGZHOOZLWKDQWLVHUDUDLVHGDJDLQVW+LQÀXHQ]D YDFFLQHVWUDLQV7KXVDYLDQ+1LQÀXHQ]DYLUXVHVFDQDFTXLUHWKHFDSDFLW\ for airborne transmission between mammals without recombination in an intermediate host and therefore constitute a risk for human pandemic LQÀXHQ]D. 0$,17(;7 ,QÀXHQ]D$ YLUXVHV KDYH EHHQ LVRODWHG IURP PDQ\ KRVW VSHFLHV LQFOXGLQJ humans, pigs, horses, dogs, marine mammals, and a wide range of GRPHVWLFELUGV\HWZLOGELUGVLQWKHRUGHUV$QVHULIRUPHV GXFNVJHHVHDQG swans) and Charadriiformes (gulls, terns, and waders) are thought to form WKH YLUXV UHVHUYRLU LQ QDWXUH

(96) ,QÀXHQ]D$ YLUXVHV EHORQJ WR WKH IDPLO\ 2UWKRP\[RYLULGDHWKHVHYLUXVHVKDYHDQ51$JHQRPHFRQVLVWLQJRIHLJKW gene segments (76, 77). Segments 1 to 3 encode the polymerase proteins: basic polymerase 2 (PB2), basic polymerase 1 (PB1), and acidic polymerase 3$

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(101) 6HJPHQWFRGHVIRUWKHYLUDOPDWUL[ structural protein M1 and the ion-channel protein M2 that is incorporated in the viral membrane. Segment 8 encodes the nonstructural protein NS1 DQGWKHQXFOHLFH[SRUWSURWHLQ 1(3

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(104) &RQVHTXHQWO\ WKH YLUXVHV DUH FODVVL¿HG EDVHG RQ WKH DQWLJHQLF YDULDWLRQ RI WKH +$ DQG 1$ SURWHLQV 7R GDWH PDMRU DQWLJHQLF YDULDQWV RI +$ DQG RI 1$ KDYH EHHQ UHFRJQL]HG LQ ZLOG birds and are found in numerous combinations designated as virus subtypes IRULQVWDQFH+1+1+1DQG+1

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(111) 7KH 0%&6LQ+$FDQEHFOHDYHGE\XELTXLWRXVO\H[SUHVVHGKRVWSURWHDVHVWKLV cleavage facilitates systemic virus replication and results in mortality of up to 100% in poultry (16, 82). 6LQFH WKH ODWH V +3$, +1 YLUXVHV KDYH GHYDVWDWHG WKH SRXOWU\ industry of numerous countries in the Eastern Hemisphere. To date, H5N1 KDV VSUHDG IURP $VLD WR (XURSH $IULFD DQG WKH 0LGGOH (DVW UHVXOWLQJ in the death of hundreds of millions of domestic birds. In Hong Kong in WKH¿UVWKXPDQGHDWKVGLUHFWO\DWWULEXWDEOHWRDYLDQ+1YLUXVZHUH UHFRUGHG

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(114) :KHWKHUWKLVYLUXVPD\DFTXLUH the ability to be transmitted via aerosols or respiratory droplets among PDPPDOVLQFOXGLQJKXPDQVWRWULJJHUDIXWXUHSDQGHPLFLVDNH\TXHVWLRQ IRUSDQGHPLFSUHSDUHGQHVV$OWKRXJKRXUNQRZOHGJHRIYLUDOWUDLWVQHFHVVDU\ for host switching and virulence has increased substantially in recent years

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(117) LQÀXHQ]D+1wildtype virus and the three mutants (B) H5N1+$1., (C) H5N1+$4/*6, and (D) H5N1+$1.4/*6. Three animals were euthanized at day 3 for tissue sampling and at day 7, ZKHQWKLVH[SHULPHQWZDVVWRSSHG9LUXVWLWHUVZHUHPHDVXUHGGDLO\LQQRVHVZDEV WRS

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(121) 9LUXVVKHGGLQJIURPWKH857DVGHWHUPLQHGE\YLUXVWLWHUVLQQDVDODQGWKURDWVZDEVZDVKLJKHVWLQ+1wildtypeinoculated animals. The mutant that yielded the highest virus titers during the 7-day period was H5N1+$4/*6, but titers were ~1 log lower than for the H5N1wildtypeLQRFXODWHGDQLPDOV,QWKH¿UVWGD\VZKHQVL[DQLPDOVSHUJURXSZHUHSUHVHQWQRVLJQL¿FDQWGLIIHUHQFHVZHUHREVHUYHG between H5N1+$1. and H5N1+$4/*6-inoculated animals, as calculated by comparing the viral titer (Mann-Whitney test, P = 0.589 and 0.818 for nose and throat titers, respectively) (Bottom row). No marked differences in virus titers in respiratory tissues were observed between the four groups. Each bar color denotes a single animal.. chapter p 2: Generation of airborne A/H5N1. 24.

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(129) QRVHVZDEVFROOHFWHGGDLO\ until day 4, from ferrets inoculated with H5N1wildtype (blue) and H5N1+$4/*63%(. (red) WKURXJKRXW WKH VHULDO SDVVDJHV GHVFULEHG LQ )LJXUH 9LUXV WLWHUV ZHUH GHWHUPLQHG E\ HQGSRLQW WLWUDWLRQ LQ 0'&. FHOOV$IWHU LQRFXODWLRQ ZLWK +1wildtype, virus titers in the nasal WXUELQDWHV ZHUH YDULDEOH EXW KLJK UDQJLQJ IURP î 5 WR î 6 7&,'JUDP WLVVXH $

(130) ZLWKQRIXUWKHULQFUHDVHREVHUYHGZLWKUHSHDWHGSDVVDJH$IWHULQRFXODWLRQZLWK+1+$ YLUXVWLWHUVLQQDVDOWXUELQDWHVDYHUDJHGî4LQWKH¿UVWWKUHHSDVVDJHV Q222L,G224S PB2 E627K î 5 LQ SDVVDJHV IRXU WR VHYHQ DQG î 5 7&,'JUDP WLVVXH LQ WKH ODVW WKUHH SDVVDJHV VXJJHVWLYH RI LPSURYHG UHSOLFDWLRQ DQG YLUXV DGDSWDWLRQ $ VLPLODU SDWWHUQ RI adaptation was observed in the virus titers in the nose swabs of animals inoculated with H5N1+$ 4/*6 3% (. (B). These titers also increased during the successive passages, ZLWKSHDNYLUXVVKHGGLQJRIî57&,'DWGSLDIWHUSDVVDJHV$OWRJHWKHUWKHVHGDWD indicate that H5N1+$4/*63%(. DGDSWHGWRPRUHHI¿FLHQWUHSOLFDWLRQLQWKHIHUUHW857 upon repeated passage, with evidence for such adaptation by passage number 4. In contrast, analyses of the virus titers in the nose swabs of the ferrets collected at 1 to 4 dpi throughout the 10 serial passages with H5N1wildtype revealed no changes in patterns of virus shedding. $VWHULVNVLQGLFDWHWKDWDQRVHZDVKZDVFROOHFWHGEHIRUHWKHQRVHVZDEZDVWDNHQZKLFK PD\LQÀXHQFHWKHYLUXVWLWHUWKDWZDVGHWHFWHG 26.

(131) chapter p 2: Generation of airborne A/H5N1. )LJXUH Summary of the substitutions detected upon serial passage and airborne Transmission of H5N1+$ 4/*6 3% (. YLUXV LQ IHUUHWV 7KH HLJKW LQÀXHQ]D YLUXV JHQH VHJPHQWVDQGVXEVWLWXWLRQVDUHGUDZQDSSUR[LPDWHO\WRVFDOH WRSWRERWWRP3%3%3$ +$131$016

(132) 9LUXVHVVKRZQLQEOXHRUDQJHDQGUHGUHSUHVHQWWKHLQLWLDOUHFRPELQDQW H5N1+$4/*63%(. virus (P0), ferret passage-10 virus (P10), and P10 virus after airborne transmission to recipient ferrets, respectively. Viruses shown in gray indicate that virus was not WUDQVPLWWHGWRWKHUHFLSLHQWIHUUHW)LUVWZHWHVWHGZKHWKHUDLUERUQHWUDQVPLVVLEOHYLUXVHVZHUH present in the heterogeneous virus population of ferret P10. We inoculated four donor ferrets intranasally, which were then housed in transmission cages and paired with four recipient IHUUHWV7UDQVPLVVLEOHYLUXVHVZHUHLVRODWHGIURPWKUHHRXWRIIRXUUHFLSLHQWIHUUHWV )WR)

(133) 1H[W ZH WRRN D WKURDWVZDE VDPSOH IURP ) WKLV VDPSOH FRQWDLQHG WKH KLJKHVW YLUXV WLWHU among the positive recipient ferrets), and this sample was used to inoculate two more donor IHUUHWVLQWUDQDVDOO\,QDWUDQVPLVVLRQH[SHULPHQWWKHVHGRQRUVLQIHFWHGWZRUHFLSLHQWIHUUHWV YLDDLUERUQHWUDQVPLVVLRQ )DQG)

(134) 9LUXVLVRODWHGIURP)ZDVSDVVDJHGRQFHLQ0'&. FHOOVDQGZDVVXEVHTXHQWO\XVHGLQDWKLUGWUDQVPLVVLRQH[SHULPHQWLQZKLFKWZRLQWUDQDVDOO\ LQRFXODWHGGRQRUIHUUHWVWUDQVPLWWHGWKHYLUXVWRRQHRIWZRUHFLSLHQWIHUUHWV )

(135) 7KHJHQHWLF FRPSRVLWLRQRIWKHYLUDOTXDVLVSHFLHVSUHVHQWLQWKHQDVDOZDVKRIIHUUHW3ZDVGHWHUPLQHG E\ VHTXHQFH DQDO\VLV XVLQJ WKH 5RFKH *6)/; VHTXHQFLQJ SODWIRUP &RQYHQWLRQDO 6DQJHUVHTXHQFLQJZDVXVHGWRGHWHUPLQHWKHFRQVHQVXVVHTXHQFHLQRQHKLJKWLWHUQDVDO or throat-swab sample for each ferret. Thick and thin black vertical bars indicate amino acid and nucleotide substitutions, respectively; substitutions introduced by reverse genetics are VKRZQLQ\HOORZVXEVWLWXWLRQVGHWHFWHGLQSDVVDJHDQGDOOVXEVHTXHQWWUDQVPLVVLRQVDUH shown in green.. 27.

(136) chapter p 2: Generation of airborne A/H5N1. )LJXUH $LUERUQHWUDQVPLVVLRQRI+1YLUXVHVLQIHUUHWV 7UDQVPLVVLRQH[SHULPHQWVDUHVKRZQIRU+1wildtype $DQG%

(137) DQG+1+$4/*63%(. (C and D) after 10 passages (P10) in ferrets. Two or four ferrets were inoculated intranasally with nasal wash samples collected from P10 virus of H5N1wildtype and H5N1+$4/*63%(., UHVSHFWLYHO\DQGKRXVHGLQGLYLGXDOO\LQWUDQVPLVVLRQFDJHV $DQG&

(138) $QDwYHUHFLSLHQWIHUUHW was added to each transmission cage adjacent to a donor ferret at 1 dpi (B and D). Virus titers in throat (black bars) and nose swabs (white bars) were determined by end-point titration in MDCK cells. Geometric mean titers and SDs (error bars) of positive samples are shown. The number of animals infected via airborne transmission is indicated in (D) for each time point DIWHUH[SRVXUHWKHGURSIURPWKUHHDQLPDOVLQIHFWHGDWGD\WRRQHDQLPDODWGD\DQGQR DQLPDOVDWGD\LVH[SODLQHGE\WKHIDFWWKDWWKHDQLPDOVWKDWEHFDPHLQIHFWHGYLDDLUERUQH WUDQVPLVVLRQKDGFOHDUHGWKHYLUXVE\WKHHQGRIWKHH[SHULPHQWDQGWKHUHIRUHGHWHFWDEOH amounts of virus were no longer present. The dotted lines indicate the lower limit of virus detection.. 28.

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(146) )RUFRPSDULVRQSXEOLVKHGGDWDRQWUDQVPLVVLRQRI 2009 pandemic H1N1 virus between ferrets is shown in (E) to (H) (95). Data for individual WUDQVPLVVLRQ H[SHULPHQWV LV VKRZQ LQ HDFK SDQHO ZLWK YLUXV VKHGGLQJ LQ LQRFXODWHG DQG DLUERUQHYLUXVH[SRVHGDQLPDOVVKRZQDVOLQHVDQGEDUVUHVSHFWLYHO\)RUWKHWUDQVPLVVLRQ H[SHULPHQWV ZLWK DLUERUQHWUDQVPLVVLEOH +1 $ WR '

(147) QRVH RU WKURDW VZDEV ZHUH QRW collected at 2 dpi and 2 dpe. White circles and bars represent shedding from the nose; black circles and bars represent shedding from the throat. The asterisk indicates the inoculated animal that died 6 days after intranasal inoculation.. 29.

(148) chapter p 2: Generation of airborne A/H5N1. RILQÀXHQ]DYLUXVWUDQVPLVVLRQEHWZHHQDQLPDOVDQGRQWKHGHWHUPLQDQWVRI DLUERUQHWUDQVPLVVLRQDUHKLJKRQWKHLQÀXHQ]DUHVHDUFKDJHQGD The viruses that caused the major pandemics of the past century emerged XSRQUHDVVRUWPHQW WKDWLVJHQHWLFPL[LQJ

(149) RIDQLPDODQGKXPDQLQÀXHQ]D viruses (1). However, given that viruses from only four pandemics are available IRUDQDO\VHVZHFDQQRWH[FOXGHWKHSRVVLELOLW\WKDWDIXWXUHSDQGHPLFPD\ EHWULJJHUHGE\DZKROO\DYLDQYLUXVZLWKRXWWKHUHTXLUHPHQWRIUHDVVRUWPHQW Several studies have shown that reassortment events between H5N1 and VHDVRQDO KXPDQ LQÀXHQ]D YLUXVHV GR QRW \LHOG YLUXVHV WKDW DUH UHDGLO\ transmitted between ferrets (89-92). In our work, we investigated whether H5N1 virus could change its transmissibility characteristics without any UHTXLUHPHQWIRUUHDVVRUWPHQW :H FKRVH LQÀXHQ]D YLUXV $,QGRQHVLD IRU RXU VWXG\ EHFDXVH WKH incidence of human H5N1 virus infections and fatalities in Indonesia remains IDLUO\KLJK

(150) DQGWKHUHDUHFRQFHUQVWKDWWKLVYLUXVFRXOGDFTXLUHPROHFXODU characteristics that would allow it to become more readily transmissible between humans and initiate a pandemic. Because no reassortants between +1YLUXVHVDQGVHDVRQDORUSDQGHPLFKXPDQLQÀXHQ]DYLUXVHVKDYHEHHQ detected in nature and because our goal was to understand the biological SURSHUWLHV QHHGHG IRU DQ LQÀXHQ]D YLUXV WR EHFRPH DLUERUQH WUDQVPLVVLEOH LQPDPPDOVZHGHFLGHGWRXVHWKHFRPSOHWH$,QGRQHVLDYLUXVWKDW ZDVLVRODWHGIURPDKXPDQFDVHRI+3$,+1LQIHFWLRQ We chose the ferret (Mustela putorius furo) as the animal model for our VWXGLHV)HUUHWVKDYHEHHQXVHGLQLQÀXHQ]DUHVHDUFKVLQFHEHFDXVH WKH\ DUH VXVFHSWLEOH WR LQIHFWLRQ ZLWK KXPDQ DQG DYLDQ LQÀXHQ]D YLUXVHV

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(154) DQGWKHĮ±OLQNHG6$UHFHSWRUVDUH mainly present in the lower respiratory tract. In chickens and other birds, Į±OLQNHG 6$V SUHGRPLQDWH EXW ERWK Į±OLQNHG DQG Į±OLQNHG 6$ DUH SUHVHQW WKURXJKRXW WKH UHVSLUDWRU\ DQG HQWHULF WUDFWV )LJXUH 6

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(156) The differences in receptor distribution between humans and avian species DUHWKRXJKWWRGHWHUPLQHWKHKRVWUHVWULFWLRQRILQÀXHQ]D$YLUXVHV$VZLWFK LQ UHFHSWRU VSHFL¿FLW\ IURP DYLDQ Į6$ WR KXPDQ Į6$ UHFHSWRUV. 30.

(157) chapter p 2: Generation of airborne A/H5N1. ZKLFK FDQ EH DFTXLUHG E\ VSHFL¿F PXWDWLRQV LQ WKH UHFHSWRU ELQGLQJ VLWH 5%6

(158) RIWKH+$LVH[SHFWHGWREHQHFHVVDU\IRUDQDYLDQYLUXVWREHFRPH transmissible and, thus, gain the potential to become pandemic in humans. %HVLGHV D VZLWFK LQ UHFHSWRU VSHFL¿FLW\ WR IDFLOLWDWH LQIHFWLRQ RI FHOOV LQ WKH 857 LQFUHDVHG YLUXV SURGXFWLRQ LQ WKH 857 DQG HI¿FLHQW UHOHDVH RI YLUXV SDUWLFOHVIURPWKHUHVSLUDWRU\WUDFWWR\LHOGDLUERUQHYLUXVPD\DOVREHUHTXLUHG (1). Such traits are likely to be determined by the viral surface glycoproteins DQG WKH SURWHLQV WKDW IRUP WKH YLUDO SRO\PHUDVH FRPSOH[ $PLQR DFLG substitutions in the polymerase proteins have already been shown to be major determinants of host range and transmission, including for pandemic LQÀXHQ]D YLUXVHV

(159) :KHUHDV DYLDQ YLUXVHV LQ SULQFLSOH UHSOLFDWH DW temperatures around 41°C (the temperature in the intestinal tract of birds), for replication in humans the viruses need to adapt to 33°C (the temperature RIWKHKXPDQ857

(160) 7KHDPLQRDFLGVXEVWLWXWLRQ*OXĺ/\V (.

(161) LQWKHSRO\PHUDVHFRPSOH[SURWHLQ3%KDVEHHQDVVRFLDWHGZLWKLQFUHDVHG virus replication in mammalian cells at such lower temperatures (13, 88, 100) In addition, when newly formed virus particles bud from the host cell membrane DIWHUYLUXVUHSOLFDWLRQWKH1$SUHVHQWRQWKHYLUXVPHPEUDQHIDFLOLWDWHVWKH UHOHDVHRISDUWLFOHV)RU+1WKLVSURFHVVLVUDWKHULQHI¿FLHQWDQGUHOHDVHG particles tend to form virus aggregates (1). Therefore, a balance between WKHSURSHUWLHVHQGRZHGE\+$DQG1$PD\EHUHTXLUHGWRJHQHUDWHVLQJOH particles. These established effects were thus used as the basis for the initial substitutions chosen in the current study. +XPDQWRKXPDQWUDQVPLVVLRQRILQÀXHQ]DYLUXVHVFDQRFFXUWKURXJKGLUHFW contact, indirect contact via fomites (contaminated environmental surfaces), DQGRUDLUERUQHWUDQVPLVVLRQYLDVPDOODHURVROVRUODUJHUHVSLUDWRU\GURSOHWV 7KHSDQGHPLFDQGHSLGHPLFLQÀXHQ]DYLUXVHVWKDWKDYHFLUFXODWHGLQKXPDQV throughout the past century were all transmitted via the airborne route, in FRQWUDVWWRPDQ\RWKHUUHVSLUDWRU\YLUXVHVWKDWDUHH[FOXVLYHO\WUDQVPLWWHGYLD FRQWDFW7KHUHLVQRH[DFWSDUWLFOHVL]HFXWRIIDWZKLFKWUDQVPLVVLRQFKDQJHV IURPH[FOXVLYHO\ODUJHGURSOHWVWRDHURVROV+RZHYHULWLVJHQHUDOO\DFFHSWHG that for infectious particles with a diameter of 5 mm or less, transmission occurs via aerosols. Because we did not measure particle size during our H[SHULPHQWV ZH ZLOO XVH WKH WHUP ³DLUERUQH WUDQVPLVVLRQ´ WKURXJKRXW WKLV Report. Biosafety and biosecurity concerns have remained foremost in our planning IRU WKLV UHVHDUFK SURJUDP7KH GHWDLOV DUH H[SODLQHG LQ WKH VXSSOHPHQWDU\ PDWHULDOV DQG DUH VXPPDUL]HG KHUH 7KH HQKDQFHG $QLPDO %LRVDIHW\ /DERUDWRU\ OHYHO $%6/

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(163) 5RWWHUGDP WKH 1HWKHUODQGV ZDV FRQVWUXFWHG IRU WKH VSHFL¿F SXUSRVH RI FRQWDLQLQJ SDWKRJHQLF DQG WUDQVPLVVLEOH LQÀXHQ]D YLUXVHV DQG RWKHU pathogens of concern. The facility consists of a negatively pressurized ODERUDWRU\ZLWKDQLQWHUORFNURRP$OOLQYLYRDQGLQYLWURH[SHULPHQWDOZRUN. 31.

(164) chapter p 2: Generation of airborne A/H5N1. is carried out in negatively pressurized class 3 isolators or class 3 biosafety cabinets, respectively. The facility is secured by procedures recognized as DSSURSULDWHE\WKHLQVWLWXWLRQDOELRVDIHW\RI¿FHUVDQGIDFLOLW\PDQDJHPHQWDW Erasmus MC, as well as Dutch and U.S. government inspectors. %HIRUH DQG GXULQJ WKH UHVHDUFK ELRVDIHW\ RI¿FHUV RI (UDVPXV 0& DQG inspectors from the Dutch government, as well as from the U.S. Centers for Disease Control and Prevention, approved the facilities and procedures. ([SOLFLWSHUPLWVIRUUHVHDUFKRQJHQHWLFDOO\PRGL¿HGDLUERUQHWUDQVPLVVLEOH H5N1 virus were obtained from the Dutch government. The research was performed strictly in accordance with the Dutch Code of Conduct for %LRVHFXULW\

(165) $OOSHUVRQQHOZHUHLQVWUXFWHGDQGWUDLQHGH[WHQVLYHO\IRU ZRUNLQJLQWKH$%6/IDFLOLW\KDQGOLQJ KLJKO\SDWKRJHQLF

(166) LQÀXHQ]DYLUXV and controlling incidents (such as spills). To further prevent occupational ULVNV UHVHDUFK SHUVRQQHO XVHG SURWHFWLYH HTXLSPHQW DQG ZHUH RIIHUHG VHDVRQDODQG+1LQÀXHQ]DYDFFLQHV)RUHPHUJHQF\SXUSRVHV(UDVPXV 0&KROGVVXSSOLHVRIRVHOWDPLYLUDQGKDVTXDUDQWLQHKRVSLWDOURRPV Using a combination of targeted mutagenesis followed by serial virus passage LQ IHUUHWV ZH LQYHVWLJDWHG ZKHWKHU +1 YLUXV FDQ DFTXLUH PXWDWLRQV WKDW would increase the risk of mammalian transmission (102). We have previously VKRZQ WKDW VHYHUDO DPLQR DFLG VXEVWLWXWLRQV LQ WKH 5%6 RI WKH +$ VXUIDFH JO\FRSURWHLQRI$,QGRQHVLDFKDQJHWKHELQGLQJSUHIHUHQFHIURPWKH DYLDQ Į±OLQNHG 6$ UHFHSWRUV WR WKH KXPDQ Į±OLQNHG 6$ UHFHSWRUV

(167) $,QGRQHVLDYLUXVZLWKDPLQRDFLGVXEVWLWXWLRQV1.4/ *6RU1.4/*6 QXPEHUVUHIHUWRDPLQRDFLGSRVLWLRQVLQ WKH PDWXUH + +$ SURWHLQ 1$VQ 4 *OQ / /HX * *O\ 6 6HU

(168) LQ +$ display attachment patterns similar to those of human viruses to cells of the UHVSLUDWRU\WUDFWRIIHUUHWVDQGKXPDQV

(169) 2IWKHVHFKDQJHVZHNQRZWKDW WRJHWKHU4/DQG*6VZLWFKWKHUHFHSWRUELQGLQJVSHFL¿FLW\RI+DQG +VXEW\SHLQÀXHQ]DYLUXVHVDVWKLVVZLWFKFRQWULEXWHGWRWKHHPHUJHQFH of the 1957 and 1968 pandemics (20). N182K has been found in a human FDVH RI +1 YLUXV LQIHFWLRQ

(170) 2XU H[SHULPHQWDO UDWLRQDOH WR REWDLQ transmissible H5N1 viruses was to select a mutant H5N1 virus with receptor VSHFL¿FLW\ IRU Į±OLQNHG 6$ VKHG DW KLJK WLWHUV IURP WKH 857 RI IHUUHWV Therefore, we used the QuickChange multisite-directed mutagenesis kit $JLOHQW7HFKQRORJLHV$PVWHOYHHQWKH1HWKHUODQGV

(171) WRLQWURGXFHDPLQRDFLG VXEVWLWXWLRQV1.4/*6RU1.4/*6LQWKH+$RI ZLOGW\SH :7

(172) $,QGRQHVLDUHVXOWLQJLQ+1+$1., H5N1+$4/*6, and H5N1+$ 1.4/*6 ([SHULPHQWDO GHWDLOV IRU H[SHULPHQWV WR DUH SURYLGHG LQ WKH VXSSOHPHQWDU\ PDWHULDOV )RU H[SHULPHQW ZH LQRFXODWHG these mutant viruses and the H5N1wildtypeYLUXVLQWUDQDVDOO\LQWRJURXSVRIVL[ IHUUHWVIRUHDFKYLUXV )LJXUH6

(173) 7KURDWDQGQDVDOVZDEVZHUHFROOHFWHGGDLO\ and virus titers were determined by end-point dilution in Madin Darby canine NLGQH\ 0'&.

(174) FHOOVWRTXDQWLI\YLUXVVKHGGLQJIURPWKHIHUUHW8577KUHH. 32.

(175) chapter p 2: Generation of airborne A/H5N1. DQLPDOVZHUHHXWKDQL]HGDIWHUGD\WRHQDEOHWLVVXHVDPSOHFROOHFWLRQ$OO remaining animals were euthanized by day 7 when the same tissue samples were taken. Virus titers were determined in the nasal turbinates, trachea, and lungs collected postmortem from the euthanized ferrets. Throughout the GXUDWLRQRIH[SHULPHQWIHUUHWVLQRFXODWHGLQWUDQDVDOO\ZLWK+1wildtype virus produced high titers in nose and throat swabs - up to 10 times more than H5N1+$4/*6, which yielded the highest virus titers of all three mutants GXULQJ WKH GD\ SHULRG )LJXUH

(176) +RZHYHU QR VLJQL¿FDQW GLIIHUHQFH ZDV observed between the virus shedding of ferrets inoculated with H5N1+$4/ or H5N1+$1.GXULQJWKH¿UVWGD\VZKHQVL[DQLPDOVSHUJURXSZHUH G224S SUHVHQW 7KXV RI WKH YLUXVHV ZLWK VSHFL¿FLW\ IRU ĮOLQNHG 6$ +1+$ \LHOGHGWKHKLJKHVWYLUXVWLWHUVLQWKHIHUUHW857 )LJXUH

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(179) ZH LQWURGXFHG (. LQWR WKH 3% JHQH RI $,QGRQHVLD E\ VLWHGLUHFWHG PXWDJHQHVLV DQG SURGXFHG WKH recombinant virus H5N1+$4/*63%(.. The introduction of E627K in PB2 GLGQRWVLJQL¿FDQWO\DIIHFWYLUXVVKHGGLQJLQIHUUHWVEHFDXVHYLUXVWLWHUVLQWKH URT were similar to those seen in H5N1+$4/*6-inoculated animals [up WRî4WLVVXHFXOWXUHLQIHFWLRXVGRVHV 7&,'

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(182) :KHQIRXUQDwYHIHUUHWV were housed in cages adjacent to those with four inoculated animals to test for airborne transmission as described previously (95), H5N1+$4/*6 ZDVQRWWUDQVPLWWHG )LJXUH6

(183) PB2E627K Because the mutant virus harboring the E627K mutation in PB2 and Q222L DQG*6LQ+$GLGQRWWUDQVPLWLQH[SHULPHQWZHGHVLJQHGDQH[SHULPHQW to force the virus to adapt to replication in the mammalian respiratory tract and to select virus variants by repeated passage (10 passages in total) of the constructed H5N1+$ 4/*6 3% (. virus and H5N1wildtype virus in WKH IHUUHW 857 )LJXUH DQG )LJXUH 6

(184) ,Q H[SHULPHQW RQH IHUUHW ZDV inoculated intranasally with H5N1wildtype and one ferret with H5N1+$4/*6 . Throat and nose swabs were collected daily from live animals until PB2 E627K 4 days postinoculation (dpi), at which time the animals were euthanized to collect samples from nasal turbinates and lungs. The nasal turbinates were homogenized in 3 ml of virus-transport medium, tissue debris was pelleted E\ FHQWULIXJDWLRQ DQG PO RI WKH VXSHUQDWDQW ZDV VXEVHTXHQWO\ XVHG WR LQRFXODWH WKH QH[W IHUUHW LQWUDQDVDOO\ SDVVDJH

(185) 7KLV SURFHGXUH ZDV repeated until passage 6. )URPSDVVDJHRQZDUGLQDGGLWLRQWRWKHVDPSOHVGHVFULEHGDERYHDQDVDO wash was also collected at 3 dpi. To this end, 1 ml of phosphate-buffered saline (PBS) was delivered dropwise to the nostrils of the ferrets to induce VQHH]LQJ$SSUR[LPDWHO\PORIWKH³VQHH]H´ZDVFROOHFWHGLQD3HWULGLVK DQG3%6ZDVDGGHGWRD¿QDOYROXPHRIPO7KHQDVDOZDVKVDPSOHVZHUH. 33.

(186) chapter p 2: Generation of airborne A/H5N1. 7DEOH Lethality of WT and airborne-transmissible H5N1 virus in ferrets upon inoculation via different URXWHVQQXPEHURIDQLPDOV1$QRWDSSOLFDEOH ,QRFXODWLRQURXWH. 9LUXV. 'HDGRUPRULEXQG QRGHDGQRWHVWHG

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(188). Intratracheal. $+1wildtype. . 2 (n = 2), 3 (n = 4). $+1). . 3 (n = 6). $+1ZLOGW\SH3. . 6 (n = 2). $+1+$4/*63%(.3. . 1$. $+1). . 1$. $+1). . 6 (n = 1). $+1wildtype. 1$. 1$. $+1+$ $4/*63%(.3. . 1$. $+1). . 1$. $+1). . 1$. Intranasal. $LUERUQH. 7KHVHGDWDUHIHUWRDSXEOLVKHGVWXG\

(189) 7KHVHIHUUHWVZHUHLQRFXODWHGZLWK3+1wildtype virus, but data are consistent with previous studies that used larger groups of animals inoculated with the original strain (106, 107).. 7DEOH 5HFHSWRU VSHFL¿FLW\ RI WKH GLIIHUHQW PXWDQW +1 YLUXVHV DV GHWHUPLQHG E\ D PRGL¿HG 75%&KHPDJJOXWLQDWLRQDVVD\,QWURGXFWLRQRI4/DQG*6LQWKH+1+$UHVXOWHG LQDUHFHSWRUELQGLQJSUHIHUHQFHVZLWFKIURPWKHDYLDQĮWRWKHKXPDQĮOLQNHG6$ UHFHSWRU6XEVHTXHQWVXEVWLWXWLRQRI+<DQG7$UHVXOWHGLQDQLQFUHDVHGDI¿QLW\IRU Į DQG ĮOLQNHG 6$ LQ DJUHHPHQW ZLWK JO\FDQ DUUD\ VWXGLHV

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(191) 9LUXV. 34. 6XEW\SH. 75%&. Į±OLQNHG 75%&. Į±OLQNHG 75%&. $1HWKHUODQGV. H3N2. 64. 0. 64. $9LHWQDP. H5N1. 64. 64. 0. $+1PB2 E627K. H5N1. 64. 16. 0. $+1+$+<7$3%(.. H5N1. 64. 48. 0. $+1+$4/*63%(.. H5N1. 64. 0. 24. $+1+$+<7$4/*63%(.. H5N1. 64. 4. 32.

(192) chapter p 2: Generation of airborne A/H5N1. XVHG IRU LQWUDQDVDO LQRFXODWLRQ RI WKH IHUUHWV IRU WKH VXEVHTXHQW SDVVDJHV 7 through 10. We changed the source of inoculum during the course of the H[SHULPHQWEHFDXVHSDVVDJLQJQDVDOZDVKHVPD\IDFLOLWDWHWKHVHOHFWLRQRI YLUXVHVWKDWZHUHVHFUHWHGIURPWKH857%HFDXVHLQÀXHQ]DYLUXVHVPXWDWH UDSLGO\ZHDQWLFLSDWHGWKDWSDVVDJHVZRXOGEHVXI¿FLHQWIRUWKHYLUXVWR DGDSWWRHI¿FLHQWUHSOLFDWLRQLQPDPPDOV Virus titers in the nasal turbinates of ferrets inoculated with H5N1wildtype ranged IURPaî5WRî77&,'JUDPWLVVXHWKURXJKRXWVHULDOSDVVDJHV )LJXUH$DQG)LJXUH6

(193) ,QIHUUHWVLQRFXODWHGZLWK+1+$4/*63%(. virus, a moderate increase in virus titers in the nasal turbinates was observed DVWKHSDVVDJHQXPEHULQFUHDVHG7KHVHWLWHUVUDQJHGIURPî47&,' JUDPWLVVXHDWWKHVWDUWRIWKHH[SHULPHQWWRî5WRî67&,' JUDPWLVVXHLQWKH¿QDOSDVVDJHV )LJXUH$DQG)LJXUH6

(194) 1RWDEO\YLUXV titers in the nose swabs of animals inoculated with H5N1+$4/*63%(. also increased during the successive passages, with peak virus shedding of î5 7&,'DWGSLDIWHUSDVVDJHV )LJXUH%

(195) 7KHVHGDWDLQGLFDWH that H5N1+$4/*63%(. was developing greater capacity to replicate in the ferret URT after repeated passage, with evidence for such adaptation becoming apparent by passage number 4. In contrast, virus titers in the nose swabs of the ferrets collected at 1 to 4 dpi throughout 10 serial passages with H5N1wildtype revealed no changes in patterns of virus shedding. 3DVVDJLQJRILQÀXHQ]DYLUXVHVLQIHUUHWVVKRXOGUHVXOWLQWKHQDWXUDOVHOHFWLRQ RI KHWHURJHQHRXV PL[WXUHV RI YLUXVHV LQ HDFK DQLPDO ZLWK D YDULHW\ RI PXWDWLRQVVRFDOOHGYLUDOTXDVLVSHFLHV

(196) 7KHJHQHWLFFRPSRVLWLRQRIWKH YLUDOTXDVLVSHFLHVSUHVHQWLQWKHQDVDOZDVKHVRIIHUUHWVDIWHUSDVVDJHV of H5N1wildtype and H5N1+$ 4/*6 3% (. ZDV GHWHUPLQHG E\ VHTXHQFH DQDO\VLV XVLQJ WKH 5RFKH *6)/; VHTXHQFLQJ SODWIRUP 5RFKH Woerden, the Netherlands) (Tables S1 and S2). The mutations introduced in H5N1+$4/*63%(. by reverse genetics remained present in the virus SRSXODWLRQDIWHUFRQVHFXWLYHSDVVDJHVDWDIUHTXHQF\! )LJXUH and Table S1). Numerous additional nucleotide substitutions were detected in all viral gene segments of H5N1wildtype and H5N1+$ 4/*6 3% (. after SDVVDJLQJH[FHSWLQVHJPHQW 7DEOHV6DQG6

(197) 2IWKHQXFOHRWLGH substitutions selected during serial passage, 53% resulted in amino acid substitutions. The only amino acid substitution detected upon repeated passage of both H5N1wildtype and H5N1+$4/*63%(.ZDV7$ 77KU $$OD

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(199) LQ+$DQGZDVGHWHFWHGLQRI the H5N1wildtypeVHTXHQFHVDIWHUSDVVDJHV7$ZDVGHWHFWHGLQRI the H5N1+$4/*63%(. VHTXHQFHVDIWHUSDVVDJHVDQGWKHRWKHU RIVHTXHQFHVSRVVHVVHGWKHVXEVWLWXWLRQ1.ZKLFKUHPRYHVWKHVDPH SRWHQWLDO1OLQNHGJO\FRV\ODWLRQVLWHLQ+$ ,Q H[SHULPHQW VHH VXSSOHPHQWDU\ PDWHULDOV

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(201) chapter p 2: Generation of airborne A/H5N1. airborne-transmissible viruses were present in the heterogeneous virus SRSXODWLRQ JHQHUDWHG GXULQJ YLUXV SDVVDJLQJ LQ IHUUHWV )LJXUH 6

(202) 1DVDO wash samples, collected at 3 dpi from ferrets at passage 10, were used in WUDQVPLVVLRQ H[SHULPHQWV WR WHVW ZKHWKHU DLUERUQHWUDQVPLVVLEOH YLUXV ZDV SUHVHQW LQ WKH YLUXV TXDVLVSHFLHV )RU WKLV SXUSRVH QDVDOZDVK VDPSOHV ZHUH GLOXWHG LQ 3%6 DQG VXEVHTXHQWO\ XVHG WR LQRFXODWH VL[ QDwYH ferrets intranasally: two for passage 10 H5N1wildtype and four for passage 10 H5N1+$4/*63%(. virus. The following day, a naïve recipient ferret was placed in a cage adjacent to each inoculated donor ferret. These cages are designed to prevent direct FRQWDFWEHWZHHQDQLPDOVEXWDOORZDLUÀRZIURPDGRQRUIHUUHWWRDQHLJKERULQJ UHFLSLHQW IHUUHW )LJXUH 6

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(204) $OWKRXJK PXWDWLRQV KDG DFFXPXODWHG LQ the viral genome after passaging of H5N1wildtype in ferrets, we did not detect replicating virus upon inoculation of MDCK cells with swabs collected from naïve recipient ferrets after they were paired with donor ferrets inoculated with passage 10 H5N1wildtype YLUXV )LJXUH $ DQG %

(205) ,Q FRQWUDVW ZH GLG detect virus in recipient ferrets paired with those inoculated with passage 10 H5N1+$ 4/*6 3% (. YLUXV 7KUHH ) WR )

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