Long-term evolution and transmission dynamics of swine influenza A virus. Percentage of samples. b 100

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1 doi:1.138/nture14 Long-term evolution nd trnsmission dynmics of swine influenz A virus Dhnsekrn Vijykrishn 1,2,3, Gvin J. D. Smith 1,2,3, Oliver G. Pybus 4, Huchen Zhu 1,2, Smir Bhtt 4, Leo L. M. Poon 1, Steven Riley 5, Justin Bhl 1,2,3, Siu K. M 1, Chung L. Cheung 1, Rnwk A. P. M. Perer 1, Honglin Chen 1,2, Kennedy F. Shortridge 1,2, Richrd J. Webby 6, Robert G. Webster 1,6, Yi Gun 1,2 & J. S. Mlik Peiris 1,7 Swine influenz A viruses (SwIV) cuse significnt economic losses in niml husbndry s well s instnces of humn disese 1 nd occsionlly give rise to humn pndemics 2, including tht cused by the H1N1/29 virus 3,4. The lck of systemtic nd longitudinl influenz surveillnce in pigs hs hmpered ttempts to reconstruct the origins of this pndemic 4. Most existing swine dt were derived from opportunistic smples collected from disesed pigs in disprte geogrphicl regions, not from prospective studies in defined loctions, hence the evolutionry nd trnsmission dynmics of SwIV re poorly understood. Here we quntify the epidemiologicl, genetic nd ntigenic dynmics of SwIV in Hong Kong using dt set of more thn 65 SwIV isoltes nd more thn 8 swine ser from 12 yers of systemtic surveillnce in this region, supplemented with dt stretching bck 34 yers. Intercontinentl virus movement hs led to ressortment nd linege replcement, creting n ntigeniclly nd geneticlly diverse virus popultion whose dynmics re quntittively different from those previously observed for humn influenz viruses. Our findings indicte tht incresed ntigenic drift is ssocited with ressortment events nd offer insights into the emergence of influenz viruses with epidemic potentil in swine nd humns. All mjor SwIV lineges of North Americn or Europen origin clssicl swine (CS), Europen or Eursin vin-like swine (EA) nd triple-ressortnt swine (TRIG) (see Supplementry Informtion for n overview) co-circulte in southern Chin 4,5, nd both humn (H3N2) nd vin (H5N1 nd H9N2) viruses hve been isolted from swine in the region 6 9. To ddress the criticl lck of structured swine influenz dt, we undertook virologicl nd serologicl nlysis of Hong Kong SwIV surveillnce smples. Most (8 95%) of the swine slughtered in Hong Kong originte from provinces in minlnd Chin (Supplementry Fig. 1 nd Supplementry Tble 1), the region with the world s lrgest swine popultion We isolted nd subtyped 573 H1N1 nd H1N2, 97 H3 nd 2 H9N2 viruses from fortnightly smpling of swine slughtered between My 1998 nd Jnury 21 (Fig. 1, b). We found no H5N1 viruses. From August 1998 to December 22, the isoltes were mostly CS H1N1 viruses. EA H1N1 viruses were detected only from 21 onwrds nd TRIG H1N2 from 22 onwrds. During 22 5, viruses clssified s CS, EA, TRIG nd H3N2 co-circulted nd fluctuted in reltive prevlence (Fig. 1b). After 25, EA H1N1 viruses becme dominnt nd H3N2 viruses disppered, lthough CS H1N2 nd TRIG H1N2 viruses continued to be isolted spordiclly (Fig. 1b). All three SwIV H1 lineges (CS, EA nd TRIG) hve co-circulted with H1N1/29 fter the introduction of the ltter virus into pigs 5. Comprehensive phylogenetic nlyses of the genes encoding surfce ntigens hemgglutinin (HA) nd neurminidse (NA) in ll H1N1 nd H1N2 isoltes (including 93 H1N1 viruses isolted during nd ) confirmed tht most isoltes belonged to the CS or EA lineges (Fig. 2 nd Supplementry Fig. 2 c). All pre-1998 viruses were CS except two pure vin viruses from 1993 (ref. 13); much greter HA diversity ws observed fter 1998 (Fig. 2). Notbly, our CS isoltes do not form single monophyletic group; rther, they re interspersed with North Americn CS viruses, indicting multiple introductions of CS into the study re. In contrst, EA viruses re monophyletic, indicting single introduction. All Hong Kong TRIG viruses form single group Percentge of smples testing positive for H1 or H3 b 1 Reltive frequency of SwIV Yer CS (n = 321) EA (n = 188) TRIG (n = 52) H1N1/29 (n = 1) H1N1 (n = 2) H3N2 (n = 97) Pigs smpled Yer Figure 1 Prevlence nd reltive frequency of swine influenz H1 nd H3 subtypes., b, Percentge prevlence () nd yer-verged reltive frequency (b) of the mjor HA vrints of SwIV. Colour codes nd numbers of isoltes (n) of H1 nd H3 subtype viruses detected from swine in Hong Kong between 1998 nd 21 re shown. The viruses detected include CS, EA, TRIG, H1N1/29 nd humn sesonl H1N1 nd H3N2 viruses. The ornge line indictes the number of pigs smpled during the surveillnce period Pigs smpled 1 Stte Key Lbortory of Emerging Infectious Diseses & Deprtment of Microbiology, Li K Shing Fculty of Medicine, The University of Hong Kong, 21 Sssoon Rod, Pokfulm, Hong Kong, Chin. 2 Interntionl Institute of Infection nd Immunity, Shntou University Medicl College, Shntou, Gungdong, Chin. 3 Lbortory of Virus Evolution, Progrm in Emerging Infectious Diseses, Duke-NUS Grdute Medicl School, 8 College Rd, , Singpore. 4 Deprtment of Zoology, University of Oxford, South Prks Rod, Oxford OX1 3PS, UK. 5 Deprtment of Community Medicine nd School of Public Helth, Li K Shing Fculty of Medicine, The University of Hong Kong, Pokfulm, Hong Kong, Chin. 6 Virology Division, Deprtment of Infectious Diseses, St Jude Children s Reserch Hospitl, Memphis, Tennessee 3815, USA. 7 HKU-Psteur Reserch Centre, The University of Hong Kong, Pokfulm, Hong Kong Specil Administrtive Region, Chin. 26 MAY 211 VOL 473 NATURE 519

2 RESEARCH LETTER b c H1 N1 N2 A Europen vin-like swine A Europen vin-like swine H1N1.7.4 H1N1.3 /swine Hong Kong surveillnce EA CS TRIG H1N1/29 swine Swine (sesonl influenz) GenBnk Swine Clssic swine Clssic swine Figure 2 Genetic reltionships of swine influenz A viruses for genes encoding surfce proteins. c, Hemgglutinin H1 (), neurminidse N1 (b), neurminidse N2 (c). Representtive vin, swine nd humn influenz A viruses obtined from GenBnk re represented by blck, drk grey nd light grey, respectively. Colour codes of H1N1 nd H1N2 subtype viruses detected except for isolte Sw/HK/78/23, indicting tht this virus ws introduced from North Americ seprtely. The Hong Kong TRIG viruses diverge from the North Americn TRIG nd H1N1/29 HA lineges soon fter the emergence of TRIG H1N2 viruses in North Americ nd thus constitute third distinct TRIG HA gene linege (Fig. 2) 14. Moleculr clock phylogenies of 221 whole genomes (33.2% of isoltes) reveled tht CS viruses isolted from 1976 to 1994 contined only CS genome segments: no ressortment ws detected during this period (Fig. 3 nd Supplementry Fig. 3). However, severl ressortnt SwIV were detected between 1998 nd 21. In ddition to the CS, EA, TRIG nd H1N1/29 viruses, we detected 14 genotypes generted by ressortment between circulting swine nd humn/vin lineges (Supplementry Figs 3 nd 4). Most of the newly identified ressortnts were observed only trnsiently nd usully contined genome segments from viruses tht were dominnt t tht time (Supplementry Fig. 4). Excepting the CS H1N2 virus, which cquired the humn H3N2 neurminidse gene repetedly, we detected no preferentil direction of horizontl gene trnsfer mong SwIV strins. Three of the 14 ressortnt genotypes were isolted repetedly (Supplementry Fig. 4); specificlly, (1) CS H1N2 viruses, (2) novel H1N2 ressortnts nd (3) Sw/HK/72/27-like (EA-ressortnt) strins, which hve cquired n NS gene from TRIG viruses nd which belong to divergent EA linege (Fig. 2). Since their initil detection in 27, EA-ressortnt viruses hve become the dominnt EA linege, constituting 12.5% of ll EA viruses in 27, 15.4% in 28 nd 41.4% in 29. Becuse most ressortnt genotypes were isolted only once, we hypothesize tht few re dpted for continuous circultion (lthough we cnnot exclude stochstic demogrphic effects or smpling bis s lterntive explntions). SwIV diversity in our popultion is probbly incresed by pig movements: breeding pigs constitute the bulk of live pigs imported into Chin nd dt indicte tht imports hve incresed since 199 (refs 11,12). For ll genome segments, moleculr clock phylogenies exhibited long brnches leding to severl ressortnt lineges (Fig. 3). This from swine during in Hong Kong re shown in the key. Arrow A indictes the ntigeniclly divergent EA-ressortnt viruses discussed in the text (for exmple, Sw/HK/72/27, Fig. 4). Scle brs represent substitutions per site. Fully detiled phylogenies including sequence nmes re provided in Supplementry Fig. 2 c. ws lso observed for the H1N1/29 virus 4 nd indictes long period of unsmpled diversity. Upon first detection, these ressortnt lineges tend to be more closely relted to viruses circulting in our popultion five to eleven yers previously, rther thn to co-circulting strins (tht is, they do not rise from the contemporneous prt of the phylogenetic bckbone ). We found extensive ntigenic crossrection mong CS, TRIG nd H1N1/29 viruses (Supplementry Tble 2 nd Supplementry Fig. 5). Ferret ntiser to these viruses lso crossrected with erly EA viruses (21 3) but rected poorly with more recent EA-ressortnt strins (Fig. 4). Interestingly, the six novel EA-ressortnt viruses tested (smpled between 27 nd 29) crossrected wekly with ll ferret ntiser used, including the ntiserum to Sw/HK/NS29/ 29. This group thus represents n ntigeniclly distinct SwIV linege. Excepting the erliest EA ressortnt (Sw/HK/72/27), ll remining EA ressortnts rected well ginst ntiser rised to the EA-ressortnt virus Sw/HK/1559/29 (Fig. 4), indicting progressive ntigenic chnge of EA-ressortnt viruses during our study. The erliest of the bove-mentioned EA ressortnts (Sw/HK/72/ 27) hd cquired two mino cid chnges in HA ntigenic sites nd lter EA ressortnts (for exmple, Sw/HK/1559/28 nd Sw/HK/ 1532/29) hd further five chnges t ntigenic sites (Supplementry Figs 3 nd 6). These findings support the hypothesis tht EAressortnt viruses hve ntigeniclly drifted wy from crossrecting ntibodies rising from CS, TRIG nd erly-ea virus infection. The observtion tht ntigenic chnge occurred in the ressortnt EA virus linege rther thn in the prentl linege indictes tht ressortment my fcilitte the genertion of SwIV ntigenic diversity. Although SwIV isoltion rtes declined fter EA viruses becme predominnt (Fig. 1), serologicl dt indicte tht overll SwIV seroprevlence hs not declined (Supplementry Tbles 3 nd 4). To test whether EA viruses hve competitive dvntge over CS strins, we intrnslly infected five-week-old, previously influenz-nive pigs with SwIV representtive of the lineges isolted here (Methods nd 52 NATURE VOL MAY 211

3 RESEARCH Segment Test ntigens Ferret ntiser 4167/ / / 26 Cl/4/ 29 NS29/ / 28 CS H1N1 CS H1N2 TRIG H1N2 H1N1/9 EA H1N1 EA* H1N1 Sw/HK/4167/1999 CS H1N1 1:2,48 1:32 1:1,24 1:2,56 1:2,56 <1:1 Sw/HK/134/23 CS H1N2 1:1,28 1:2,56 1:64 1:8 1:4 <1:1 Sw/HK/111/26 TRIG H1N2 1:4,96 1:1,28 1:1,24 1:64 1:5,12 <1:1 Cl/4/29 H1N1/29 1:64 1:64 1:2,56 1:1,28 16 <1:1 Sw/HK/8512/21 EA H1N1 1:1,24 1:1,28 1:5,12 1:2,56 1:1,24 1:32 Sw/HK/1669/22 EA H1N1 1:5,12 1:1,28 1:2,56 1:1,28 1:1,24 1:16 Sw/HK/NS129/23 EA H1N1 1:5,12 1:1,28 1:2,56 1:1,28 1:1,24 1:16 Sw/HK/1716/26 EA H1N1 1:2,56 1:64 1:1,28 1:64 1:2,56 <1:1 Sw/HK/NS952/28 EA H1N1 1:2,56 1:64 1:64 1:32 1:2,56 <1:1 Sw/HK/NS29/29 EA H1N1 1:64 1:16 1:1,28 1:8 1:1,24 <1:1 Sw/HK/72/27 EA* H1N1 <1:1 <1:1 <1:1 <1:1 1:4 <1:1 Sw/HK/1559/28 EA* H1N1 <1:1 <1:1 <1:1 <1:1 1:4 1:5,12 Sw/HK/247/29 EA* H1N1 <1:1 <1:1 <1:1 <1:1 1:4 1:2,56 Sw/HK/NS613/29 EA* H1N1 <1:1 <1:1 <1:1 <1:1 1:1 1:2,56 Sw/HK/2481/29 EA* H1N1 <1:1 <1:1 <1:1 <1:1 1:2 1:2,56 Sw/HK/NS186/29 EA* H1N1 <1:1 <1:1 <1:1 <1:1 1:4 1:2,56 b Figure 4 Antigenic chrcteriztion of SwIV mesured by hemgglutinin inhibition ssys. Titres re shded ccording to their respective mjor SwIV HA lineges (see Figs 1 3); low (1:2, 1:4) nd non-rective titres (,1:1) re shded in lighter colours. Underlined vlues represent homologous ntibody titres. EA-ressortnt viruses (indicted by sterisks) showed poor crossrectivity ginst ntiser rised towrds CS, TRIG, H1N1/29 nd lte (26 29) pure EA viruses. Excepting the erliest EA-ressortnt virus (Sw/ HK/72/27), ll remining EA-ressortnts rected well ginst ntiser rised towrds the EA-ressortnt virus Sw/HK/1559/29, indicting progressive ntigenic chnge of this novel ressortnt. CS EA TRIG H1N1/29 H3N Yer Figure 3 Phylogenies nd divergence times of the hemgglutinin genes of clssicl swine nd Europen vin-like SwIV., CS; b, EA. Coloured boxes djcent to tips show the linege clssifiction of ech gene segment of SwIV isolted in this study. Arrows indicte the long brnches tht led to newly detected ressortnt SwIV. Purple node brs represent 95% credible intervls of linege divergence times. A fully detiled HA phylogeny including sequence nmes is shown in Supplementry Fig. 3. Supplementry Fig. 7). EA viruses showed the highest nd most prolonged virus shedding, closely followed by TRIG viruses; CS viruses showed lower pek virl titres. Thus, the replictive dvntge of EA viruses, together with the low prevlence of crossrective ntibodies to EA in swine (15% in 2, 26% in 24; Supplementry Tble 4), my help to explin the replcement of other SwIV lineges with EA viruses. We trcked the evolution in our EA viruses of mino cids previously ssocited with dpttion of vin influenz to other species Purported vin residues were mintined t most of these sites (Supplementry Tble 5) despite the circultion of these viruses in swine for more thn 3 yers 18,19. However, the PDZ- (post-synptic density protein, Drosophil disc lrge tumor suppressor nd zonul occludens-1 protein) lignd t the 39 end of EA virus non-structurl (NS) 1 genes showed significnt host-specific evolution: erly Europen EA viruses hd the vin ESEV motif, with chnge to GSEV/GPEV motifs observed in severl hosts. By 1999, most viruses smpled hd the GPKV motif previously described from pigs 16. CS nd TRIG viruses tht contributed the NS gene to H1N1/29 hve truncted NS gene, s do the ntigeniclly vrint Sw/HK/72/27-like viruses. The role of the truncted NS gene in inter-species trnsmission clerly merits further study. Furthermore, modest but significnt (P,.1) chnge in selection pressure ws observed between Europen EA viruses isolted shortly fter cross-species trnsmission (non-synonymous to synonymous (d N /d S ) substitution rte rtio of.24; 95% confidence intervl ) nd those isolted lter (d N /d S 5.17; 95% confidence intervl ), consistent with the hypothesis tht hostspecific selection incresed virl dpttion fter the introduction of EA viruses into swine (Supplementry Tble 6). Our unique longitudinl study revels geneticlly nd ntigeniclly dynmic SwIV popultion within single region nd provides bseline for future studies of the virus elsewhere. The epidemiology nd evolution of SwIV seem to be strongly shped by gene flow mong continents nd species, fcilitting the ressortment of diverse lineges nd occsionlly resulting in ntigenic chnge. Although we confirm tht the H1N1/29 virus ws not generted within our study s ctchment, the processes of linege emergence, importtion, ressortment nd replcement described here re probbly representtive of the H1N1/29 source popultion. We show tht ressortments between EA nd TRIG viruses do occur, generting ressortnts tht estblish themselves s stble lineges in swine. SwIV ressortnts contining H1N1/29- like genome segments hve lso been trnsiently detected 5,2. Despite cler evidence of inter-continentl SwIV movement, gene flow is not so frequent tht the globl SwIV popultion cts s single gene pool (s observed for humn influenz A 21,22 ); insted higher diversity of mmmlin-dpted viruses in globl swine popultions is supported. Crucilly, the co-circultion of multiple SwIV lineges fcilittes the production of new genomic combintions. The evolutionry consequences of incresed SwIV movement re hrd to predict but require considertion given n incresingly globlized future. 26 MAY 211 VOL 473 NATURE 521

4 RESEARCH LETTER Our study revels frequent genertion of new ressortnts but the survivl nd persistence of only few, process we term recombinnt chtter 23. Our dt lso indicte tht ressortment nd ntigenic chnge re linked. This phenomenon ws described in North Americ CS viruses fter the events tht generted the TRIG viruses 24 ; it hs lso been observed in humn influenz 21. After ressortment, evolution in HA ntigenic domins my rise for severl resons: (1) becuse of herd-immune selection pressure; (2) becuse those residues re under wek selective constrint; or (3) to compenste for fitness costs of muttions ccruing elsewhere in the genome. The role of ressortment in driving genome-wide evolution requires detiled investigtion. We found tht the quntittive dynmics of SwIV genomic diversity nd linege turnover (Supplementry Fig. 8) re slower, less periodic nd less predictble thn the repeted nnul replcements typiclly seen for humn influenz A. The resons for SwIV linege chnge re uncler: previously, selection rising from herd immunity ws considered less importnt for pigs thn for humns becuse the short lifespn of frmed swine (,15 dys) lowers the chnce of re-infection, reducing the crossprotection tht probbly drives ntigenic drift. Furthermore, mternlly cquired swine immunity does not seem to interrupt SwIV infection or trnsmission, despite msking clinicl illness 25. Our surveillnce dt, niml infection experiments nd serologicl dt show tht one reson for linege chnge my be competitive dvntge of EA over CS nd TRIG viruses. The hypothesis tht pigs re importnt in pndemic emergence, s fcilittors of ressortment mong influenz viruses 26, hs regined fvour fter the emergence of H1N1/29. This virus represented subtype lredy endemic in humns, implying tht other H1 nd H3 viruses prevlent in swine re credible pndemic cndidtes, especilly when corresponding immunity in humns is bsent. Indeed, we found tht there re other swine viruses (for exmple, Sw/HK/NS29/9) to which humns lck herd immunity (Supplementry Tble 7). Hence, future ssessment of zoonotic potentil must combine the evlution of crossrective immunity in humns, the ssessment of trnsmissibility in niml models nd ongoing surveillnce of SwIV genetic diversity. The H1N1/29 virus hs lredy infected swine nd ressorted with other SwIV, indicting tht circulting SwIV will continue to cquire novel non-swiv genes 5 (notbly, vin viruses such s H9N2 nd H5N1 re occsionlly detected in swine in Asi 6,8,9,27 ). -to-swine nd swine-to-humn host dpttion of influenz viruses re both poorly understood in comprison to vin-to-humn dpttion nd re priority for future reserch. METHODS SUMMARY Systemtic swine influenz A virus surveillnce ws initited in My 1998 t centrl slughterhouse in Hong Kong. During , bout 15 2% of the pigs were frmed loclly in Hong Kong nd the reminder were imported from severl provinces in Chin; however, since 28 the proportion of loclly produced pigs fell to 5% (see Supporting Informtion). About 128 nsl nd trchel swbs were collected twice monthly from August 1998 to April 29; since My 29, smple numbers were doubled (Fig. 1). Genes encoding surfce proteins (HA nd NA) were sequenced for ll 573 H1N1 viruses isolted from 1998 to 21 nd for 93 swine H1 viruses from our repository, isolted during the periods nd Full genome sequencing ws crried out for 221 representtive viruses. To estimte the genetic diversity nd the level of gene ressortment, phylogenetic trees were constructed for ech genomic segment independently (Supplementry Fig. 2). On the bsis of the phylogenetic reltionships of ech gene segment, mjor swine virus lineges circulting in Hong Kong were identified nd more detiled Byesin phylogenetic nlysis for ech linege ws conducted, thereby estimting rtes of virl evolution nd dtes of divergence (Supplementry Fig. 3). Full Methods nd ny ssocited references re vilble in the online version of the pper t Received 22 September 21; ccepted 17 Mrch Shinde, V. et l. Triple-ressortnt swine influenz A (H1) in humns in the United Sttes, N. Engl. J. Med. 36, (29). 2. Smith, G. J. D. et l. Dting the emergence of pndemic influenz A viruses. Proc. Ntl Acd. Sci. USA 16, (29). 3. Grten, R. J. et l. Antigenic nd genetic chrcteristics of swine-origin 29 A(H1N1) influenz viruses circulting in humns. Science 325, (29). 4. Smith, G. J. D. et l. Origins nd evolutionry genomics of the 29 swine-origin H1N1 influenz A epidemic. Nture 459, (29). 5. Vijykrishn, D. et l. Ressortment of pndemic H1N1 viruses in swine. Science 328, 1529 (21). 6. Peiris, J. S. M. et l. Cocircultion of vin H9N2 nd contemporry humn H3N2 influenz A viruses in pigs in southestern Chin: potentil for genetic ressortment? J. Virol. 75, (21). 7. Yu, H. et l. Genetic evolution of swine influenz A (H3N2) viruses in Chin from J. Virol. 46, (28). 8. Cong, Y. L. et l. Antigenic nd genetic chrcteriztion of H9N2 swine influenz viruses in Chin. J. Gen. Virol. 88, (27). 9. Li, H. Y. et l. Isoltion nd chrcteriztion of H5N1 nd H9N2 influenz viruses from pigs in Chin. Chinese J. Vet. Prev. Med. 26, 1 6 (24). 1. Clements, A. C. A., Pfeiffer, D. U., Otte, M. J., Morteo, K. & Chen, L. A globl livestock production nd helth tls (GLiPHA) for interctive presenttion, integrtion nd nlysis of livestock dt. Prev. Vet. Med. 56, (22). 11. Zhng, J. & Beckmn, C. People s Republic of Chin: Agriculturl sitution: Livestock nd Products 28. (USDA Foreign Agriculture Service, 28). 12. Wng, R. Chin pork powerhouse of the world. Advnces Pork Prod. 17, (26). 13. Gun, Y. et l. Emergence of vin H1N1 influenz viruses in pigs in Chin. J. Virol. 7, (1996). 14. Lorusso, A. et l. Genetic nd ntigenic chrcteriztion of H1 influenz viruses from United Sttes swine from 28. J. Gen. Virol. 92, (211). 15. Finkelstein, D. B. et l. Persistent host mrkers in pndemic nd H5N1 influenz viruses. J. Virol. 81, (27). 16. Obenuer, J. C. et l. Lrge-scle sequence nlysis of vin influenz isoltes. Science 311, (26). 17. Tubenberger, J. K. et l. Chrcteriztion of the 1918 influenz virus polymerse genes. Nture 437, (25). 18. Pensert, M., Ottis, K., Vnderputte, J., Kpln, M. M. & Buchmnn, P. A. Evidence for the nturl trnsmission of influenz A virus from wild ducks to swine nd its potentil for mn. Bull. World Helth Orgn. 59, (1981). 19. Dunhm, E. et l. Different evolutionry trjectories of Europen vin-like nd clssicl swine H1N1 influenz A viruses. J. Virol. 83, (29). 2. Moreno, A. et l. Novel H1N2 swine influenz ressortnt strin in pigs derived from the pndemic H1N1/29 virus. Vet. Microbiol. 149, (211). 21. Rmbut, A. et l. The genomicndepidemiologicl dynmics of humn influenz A virus. Nture 453, (28). 22. Russell, C. A. et l. The globl circultion of sesonl influenz A (H3N2) viruses. Science 32, (28). 23. Wolfe, N. D., Dunvn, C. P. & Dimond, J. Origins of mjor humn infectious diseses. Nture 447, (27). 24. Webby, R. J. et l. Evolution of swine H3N2 influenz viruses in the United Sttes. J. Virol. 74, (2). 25. Loeffen, W. L. A., Heinen, P. P., Binchi, A. T. J., Hunnemn, W. A. & Verheijden, J. H. M. Effect of mternlly derived ntibodies on the clinicl signs nd immune response in pigs fter primry nd secondry infection with n influenz H1N1 virus. Vet. Immunol. Immunopthol. 92, (23). 26. Scholtissek, C., Hinshw, V. S. & Olsen, C. W. Influenz in pigs nd their role s the intermedite host. In Textbook of Influenz (eds Nicholson, K. G., Webster R. G. & Hy A. J.) (Blckwell Scientific, 1998). 27. Nidom, C. A. et l. Influenz A (H5N1) viruses from pigs, Indonesi. Emerg. Infect. Dis. 16, (21). Supplementry Informtion is linked to the online version of the pper t Acknowledgements This reserch ws supported in prt by the Ntionl Institute of Allergy nd InfectiousDiseses (NIAID) contrcthhsn26675cndthe Are of Excellence Scheme of the University Grnts Commission (grnt AoE/M-12/6) of the Hong Kong SAR Government. We cknowledge the Food nd Environmentl Hygiene Deprtment of Hong Kong for fcilitting the study. We cknowledge support from The Royl Society of London (O.G.P.), UK COSI (S.B.), NIAID (G.J.D.S.), the Agency for Science, Technology nd Reserch nd the Ministry of Helth, Singpore (D.V., G.J.D.S nd J.B.). We thnk C. Y. H. Leung for producing some of the ferret ntiser used in this study. Author Contributions J.S.M.P. nd Y.G. conceived the study, conducted surveillnce, performed nlyses nd co-wrote the pper. D.V., G.J.D.S. nd O.G.P. conceived the study, performed nlyses, co-wrote the pper nd contributed eqully to this work. H.Z., S.B., L.L.M.P., S.R., J.B., R.A.P.M.P. nd H.C. performed nlyses, S.K.M. conducted surveillnce, C.L.C. conducted sequencing, K.F.S. nd R.G.W. initited surveillnce in 1976 nd provided viruses nd R.J.W. provided viruses nd regents. All uthors commented on nd edited the pper. Author Informtion Sequences generted in this study hve been deposited with GenBnk under the ccession numbers CY8447 CY85121, CY8531 CY86876 nd CY8741 CY Reprints nd permissions informtion is vilble t The uthors declre no competing finncil interests. Reders re welcome to comment on the online version of this rticle t Correspondence nd requests for mterils should be ddressed to Y.G. (ygun@hkucc.hku.hk) or J.S.M.P. (mlik@hkucc.hku.hk). 522 NATURE VOL MAY 211

5 RESEARCH METHODS Surveillnce. Systemtic influenz surveillnce ws conducted from My 1998 until Jnury 21 in swine t n bttoir in Hong Kong, where trchel or nsl swbs were collected fortnightly from slughtered swine. During , bout 15 2% of the pigs were frmed loclly in Hong Kong nd the reminder were imported from severl provinces in Chin; however, since 28 the proportion of loclly produced pigs fell to 5% (see Supporting Informtion). Serum smples were collected from 5 slughtered pigs ech month. Routine virologicl surveys were lso conducted in Hong Kong in nd Swb mterils were inoculted into nine-to-ten-dy-old embryonted chicken eggs nd Mdin Drby cnine kidney (MDCK) cells; virus isoltes were identified nd subtyped by hemgglutintion inhibition ssys s previously described 24. Virus isoltion nd sequencing. Virl RNA extrction, complementry DNA synthesis, PCR nd sequencing were crried out s described 5,28. Virl RNA ws extrcted directly from infected llntoic fluid or cell culture using the QIAmp virl RNA minikit (Qigen). cdna ws synthesized by reverse trnscription; gene mplifiction by PCR ws performed using specific primers for ech gene segment. PCR products were purified with the QIAquick PCR purifiction kit (Qigen) nd sequenced using synthetic oligonucleotides. Rections were performed using the Big Dye-Termintor v3.1 Cycle Sequencing Rection Kit on n ABI PRISM 37 DNA Anlyser (Applied Biosystems) following the mnufcturer s instructions. All sequences were ssembled nd edited with Lsergene version 6.1 (DNASTAR). The HA nd NA genes were sequenced for ll viruses collected in this study nd full genome sequencing ws conducted for representtive viruses, selected on the bsis of HA nd NA gene diversity nd including representtive viruses smpled on ech positive smpling occsion. All novel ressortnts detected on the bsis of full genome sequencing were subjected to plque cloning nd full genome sequencing (of t lest six rndomly selected clones per virus) to confirm tht the ressortnt ws not n rtefct of mixed infection. Antigenic nlyses. The ntigenic chrcteristics of SwIV were compred using hemgglutintion inhibition ssy with ferret ntiser rised ginst representtive influenz A viruses. Ferret ntiser rised ginst Sw/HK/4167/1999 (CS H1N1), Sw/ HK/111/26 (TRIG H1N2), Sw/HK/NS29/29 (EA H1N1) nd A/Cliforni/4/ 29 were produced t the Deprtment of Infectious Diseses t St Jude Children s Reserch Hospitl, Memphis, Tennessee ndthe Deprtment of Microbiology, The University of Hong Kong. The hemgglutintion inhibition ssy strted t 1:4 dilutions for ferret ntiser. To detect ntibody prevlence towrds mjor SwIV lineges, we used the hemgglutintion inhibition ssy with five representtive viruses including the ntigeniclly divergent Sw/HK/72/27-like EA viruses. This llowed us to quntify chnges in seroprevlence in serum collected from swine during 2, 24, 29 nd 21. Experimentl infection of pigs. To chrcterize in vivo replictive behviour of viruses from the mjor SwIV lineges, we experimentlly infected locl domestic hybrid (Putin white nd Ninbin vrint) pigs (Sus scrof domesticus) obtined from commercil herd nd confirmed to be sero-negtive nd free of influenz virus by HI ssys nd virus isoltion in MDCK cells. Pigs were infected with representtive strins belonging to the CS (Sw/HK/4167/1999, Sw/HK/134/23), TRIG (Sw/HK/ 111/26), EA (Sw/HK/NS29/29) nd novel EA-ressortnt (Sw/HK/72/27) lineges isolted in this study. Two five-week-old pigs (one mle nd one femle) were intrnslly infected with 1 ml of Egle s miniml essentil medium (MEM) contining 1 6 5% tissue culture infectious doses (TCID 5 )ofvirusstrin.nsl swbs were collected for 14 d fter inocultion from ech piglet nd plced in.6 ml of virus trnsport medium. Virus shedding in the nsl swbs of pigs ws clculted in MDCK by the 5% end-point method 29 nd ws expressed s TCID 5 ml 21 of swb. Animl experiments were crried out in biosfety level three continment fcilities t 2 21 uc nd % reltive humidity. Experiments were pproved by the Shntou University Medicl College nd conducted in complince with university guidelines on niml ethics nd welfre. Moleculr evolution nd dpttion. Globl d N /d S rte rtios for ech Hong Kong swine linege nd the hemgglutinin gene of Europen EA viruses were estimted using the codon-bsed single likelihood ncestor counting method 3. To determine whether selection ws cting differentilly on mjor lineges, the d N /d S rte rtio estimte for linege ws enforced to other co-circulting lineges. A likelihood rtio test ws conducted to evlute whether this fit ws significntly worse thn unconstrined nlysis (nd vice vers), with criticl P vlue of.1. This test ws repeted using the upper nd lower limits of the confidence intervl. Phylogenetic nlyses. Phylogenetic trees were inferred using the neighbourjoining method, using genetic distnces clculted by mximum likelihood under the Hsegw, Kishino nd Yno (HKY) model with gmm-distributed mongsite rte vrition (HKY1C). The prmeters of this model were estimted using mximum likelihood on n initil tree. Temporl phylogenies nd rtes of evolution were inferred using relxed moleculr clock model tht llows evolutionry rtes to vry mong lineges in Byesin Mrkov chin Monte Crlo (MCMC) frmework 31 This ws used to smple phylogenies nd dtes of divergence while constrining ech sequence to its known dte of smpling. A model comprising codon-position-specific HKY1C substitution models ws used. For ll nlyses employing Byesin MCMC smpling, chin length of t lest 5 million steps ws used with 1% burn-in removed. At lest two independent runs of ech chin were performed nd compred to ensure dequte smpling. To estimte chnges in genetic diversity during our smpling period we used colescent-bsed flexible demogrphic model 32 to the bove MCMC pproch. An estimte of the reltive genetic diversity (N e t, where N e is the effective popultion size nd t is the genertion time) is obtined by integrting uncertinty cross the tree topologies. 28. Poon, L. L. M. et l. Rpid detection of ressortment of pndemic influenz H1N1. Clin. Chem. 56, (21). 29. Reed, L. J. & Muench, H. A. Simple method of estimting fifty percent endpoints. Am. J. Hyg. 27, (1938). 3. Koskovsky Pond, S. L. & Frost, S. D. W. Not so different fter ll: comprison of methods for detecting mino cid sites under selection. Mol. Biol. Evol. 22, (25). 31. Drummond, A. J., Ho, S. Y., Phillips, M. J. & Rmbut, A. Relxed phylogenetics nd dting with confidence. PLoS Biol. 4, e88 (26). 32. Drummond, A. J., Rmbut, A., Shpiro, B. & Pybus, O. G. Byesin colescent inference of pst popultion dynmics from moleculr sequences. Mol. Biol. Evol. 22, (25).