Brown, I H

Cheon, S-H

Cho, H-K

Heo, G-B

Kang, H-M

Kang, Y-M

Kim, Y-J

Kye, S-J

Lee, E-K

Lee, M

Lee, Y-J

Lee, Y-N

Lewis, N S

Park, B-K

Sagong, M

Emerging Microbes and Infections

English

Eun-Kyoung Lee

Yu-Na Lee

Soo-Jeong Kye

Nicola S. Lewis

Ian H. Brown

Mingeun Sagong

Gyeong-Beom Heo

Yong-Myung Kang

Hyun-Kyu Cho

Hyun-mi Kang

Sun-Ha Cheon

Myeongheon Lee

Bong-Kyun Park

Yong-Joo Kim

Youn-Jeong Lee

Crossref

Emerging Microbes & Infections

Characterization of a novel reassortant H5N6 highly pathogenic avian influenza virus clade 2.3.4.4 in Korea, 2017

RVC Repository (Royal Veterinary College)

Lee et al. Emerging Microbes & Infections  (2018) 7:103 Emerging Microbes & InfectionsDOI 10.1038/s41426-018-0104-3 www.nature.com/emiCORRESPONDENCE Open Ac ce s sCharacterization of a novel reassortantH5N6 highly pathogenic avian influenzavirus clade 2.3.4.4 in Korea, 2017Eun-Kyoung Lee1, Yu-Na Lee1, Soo-Jeong Kye1, Nicola S. Lewis2,3, Ian H. Brown2, Mingeun Sagong1,Gyeong-Beom Heo1, Yong-Myung Kang1, Hyun-Kyu Cho1, Hyun-mi Kang1, Sun-Ha Cheon1, Myeongheon Lee1,Bong-Kyun Park1, Yong-Joo Kim1 and Youn-Jeong Lee1Dear Editor,Highly pathogenic avian influenza (HPAI) viruses of theH5 subtype remain a serious concern for both poultry andhuman health. The clade 2.3.4.4 H5 viruses have evolvedinto four genetic groups (nominally designated A, B, C,and D). Clade 2.3.4.4.A and B H5N8 viruses appear tohave been reintroduced from poultry into wild birdpopulations, likely in Asia, and then spread globallythrough wild bird migration during 2014–2015 (2.3.4.4.A)and 2016–2017 (2.3.4.4.B)1, 2.Clade 2.3.4.4.C and D H5 HPAI viruses have circulatedexclusively in China and a few other countries in South-east Asia. Indeed, clade 2.3.4.4.C H5N6 HPAI viruses havebeen the predominant clade detected in poultry popula-tions in China3. Recently, clade 2.3.4.4 H5N6 subtypeshave also been associated with human infection in China(http://www.wpro.who.int/emerging_diseases/AvianInfluenza/en/).The clade 2.3.4.4.A H5N8 viruses were identified inChina in 2013 and were subsequently introduced intoKorea in early 20144, 5. Since then, the spread of clade2.3.4.4.A viruses mediated by wild bird migration hasresulted in intra- and intercontinental transmission.These viruses then reassorted with low pathogenic avianinfluenza (LPAI) of the Eurasian or North Americanlineage to result in novel H5Nx subtypes (such as N1, N2,N3, N5, and N8), which subsequently spread broadlythroughout Eurasia and North America6, 7.In mid-2016, a novel clade of 2.3.4.4.B H5N8 viruseswas detected in wild birds in Uvs-Nuur Lake in Siberia,Russia, near the border with Mongolia and in QinghaiLake in China. These H5N8 viruses were novel reassor-tants consisting of five segments (polymerase basic-2(PB2), polymerase basic-1 (PB1), polymerase acidic (PA),nucleoprotein (NP), and matrix (MP)) from EurasianLPAI viruses most similar to H5N8 viruses isolated inChina in 20138, 9. These Russia–Mongolia H5N8 virusesthen further reassorted with the NA genes from EurasianLPAI viruses to result in additional H5N5 andH5N6 subtypes. This 2.3.4.4.B lineage has been detectedin most European countries, Central Asia, the MiddleEast, and Africa during 2016–201710.In Korea, there have been four outbreaks of H5N1 HPAIfrom 2003 to 2011. However, since 2014, large epizooticsin poultry have been caused by clade 2.3.4.4.A H5N8viruses. Then, in late 2016, outbreaks in poultry resultedfrom clade 2.3.4.4.C H5N6 and clade 2.3.4.4.B H5N8 HPAIviruses, demonstrating the expanding diversity of thethreat to the poultry industry in Korea11, 12.In November 2017, we detected two novel H5N6 HPAIviruses, A/duck/Korea/HD1/2017(H5N6) (HD1) and A/mallard/Korea/Jeju-H24/2017(H5N6) (Jeju-H24), throughour active surveillance program for avian influenza. OnNovember 17, an H5N6 HPAI virus was detected in abroiler duck farm in Gochang of Jeonbuk Province(nominated HD1). The second H5N6 virus was isolatedfrom fecal samples taken from a wild mallard (Anas pla-tyrhynchos) collected in Jeju on November 27 (nominated© The Author(s) 2018OpenAccessThis article is licensedunder aCreativeCommonsAttribution 4.0 International License,whichpermits use, sharing, adaptation, distribution and reproductionin any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate ifchangesweremade. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to thematerial. Ifmaterial is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtainpermission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Correspondence: Youn-Jeong Lee (leeyj700@korea.kr) orYong-Joo Kim (kyjvet@korea.kr)1Avian Influenza Research and Diagnostic Division, Animal and PlantQuarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do39660, Republic of Korea2EU/OIE/FAO international reference laboratory for avian influenza, Animal andPlant Health Agency-Weybridge, Addlestone, Surrey KT15 3NB, UnitedKingdomFull list of author information is available at the end of the article.1234567890():,;1234567890():,;Jeju-H24). Virus isolation, sequencing, and phylogeneticanalysis were performed as described (SupplementaryMaterials and Methods 1). The two isolates shared99.69–100% nucleotide homology across all eight genes. ABLAST search (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) revealed that these novel H5N6 viruseswere reassortants with H5 genes most genetically similar toclade 2.3.4.4.B viruses derived from European H5N8 HPAIviruses circulating during the 2016–2017 epizootic and N6genes of a Eurasian LPAI virus lineage circulating in wildbirds (Fig. 1, Supplementary Figure S1 and Table S1).Phylogenetic analyses of all genes except for the NAgene showed that the closest genetic relatives to the twoviruses isolated in Korea were isolated from both wild anddomestic birds sampled in a number of European coun-tries in 2016/17. Furthermore, both the NP and NA geneswere most similar to the H5N6 reassortant virus isolatedfrom a chicken in Greece during this epizootic (Supple-mentary Figure S1 and S2).The H5N6 viruses had multiple polybasic amino acidmotifs at the HA cleavage site, characteristic of highlypathogenic influenza viruses (PLRERRRKR/GLF). Inter-estingly, the HPAI cleavage site motif differed between theGreek and the Korean clade 2.3.4.4.B strains. However, itis identical to the Japanese H5N6 HPAI virus (A/muteswan/Shimane/3211A001/2017) isolated from a wild bird‘13 China H5N8’16 EuropeH5N8’16.5. Russia, Mongolia H5N8’16.5. China H5N8’14.1. H5N8(Korea)’16.11. H5N8(Europe)‘13 H5N8(China)LPAI LPAI’16.5. H5N8(Russia, Mongolia, China) ’16.12. H5N8(Korea) 2013 2014 2015 2016 2017’17.11. Korea, JapanH5N6’14.1.  Korea H5N8’16.12.  Korea H5N8       ’17 GreeceH5N6’17.11. H5N6(Japan, Korea) Fig. 1 Evolutionary timeline of the genetic cassette of the current Korean 2.3.4.4B H5Nx highly pathogenic avian influenza viruses,generated through reassortment. Current reassortant H5N6 viruses consist of PB2, PB1, PA, HA, NP, M, and NS segments from European clade2.3.4.4.B H5N8 viruses isolated in 2016 and N6 gene segments from low pathogenic avian influenza viruses. Gray colors represent the lineage of thesegments originating from viruses of clade 2.3.4.4.B in 2013–2014, and the other colors denote the segments derived from low-pathogenic avianinfluenza virus through reassortment events. The eight horizontal bars indicate the gene segments in the order (top to bottom of the virion) PB2, PB1,PA, HA, NP, NA, M, and NS. HA hemagglutinin, M matrix, NA neuraminidase, NP nucleoprotein, NS nonstructural, PA polymerase acidic, PB1polymerase basic 1, PB2 polymerase basic 2Lee et al. Emerging Microbes & Infections  (2018) 7:103 Page 2 of 3on 5 November 2017, perhaps indicating heterogeneity inthe emerging 2.3.4.4.B H5N6 lineage that is potentiallybeing maintained in wild birds.The clade 2.3.4.4.B viruses have consistently evolved intonovel H5Nx subtypes with various gene constellations byreassortment with LPAI viruses since 2013. In particular,the internal gene segments derived from Eurasian LPAIviruses in wild birds that have reassorted with the clade2.3.4.4 ‘H5N8 backbone’ and the H5N5 and H5N8 HPAIsubtypes of various genotypes were detected in Germany,Italy, the Netherlands, Croatia and the Republic of Geor-gia13–15. Reassortant H5Nx viruses might diffuse amongwild bird species in Eurasian breeding or staging sites, suchas Central Asia and Siberia, and these viruses then dis-seminate into Eastern Asia (Korea and Japan). Conversely,there might be some degree of temporo-spatial segregationacross Eurasian wild bird populations, resulting in main-tenance of distinct variants and, therefore, heterogeneity inemerging strains in different geographic regions. Thesehypotheses are, as yet, difficult to test owing to significantsampling biases in wild bird influenza monitoring andwould require more strategically targeted surveillance inecologically relevant populations.The novel clade 2.3.4.4.B H5N6 viruses isolated inNovember 2017 were phylogenetically distinguishablefrom the clade 2.3.4.4 viruses (H5N6 and H5N8) pre-viously isolated in Korea during 2016–2017. In late 2016,clade 2.3.4.4.C H5N6 HPAI viruses that originated fromChina were introduced to Korea and Japan by wild birdsand caused enormous economic losses to the poultryindustry, particularly in Korea12. Recent H5N6 virusesfrom 2017 showed <95% homologies in eight gene seg-ments with previous Korean H5N6 viruses isolated in2016. In addition, novel H5N6 viruses showed <97% innucleotide identities with three genes (PB2, PA, and NP)with previous Korean H5N8 viruses of clade 2.3.4.4.B inthe 2016/2017 winter season. These results indicate thatnew H5N6 viruses are unlikely to have been circulatingundetected in Korea; therefore, they are more likely torepresent a new introduction mediated by migratory birds.In this study, we reported novel reassortant H5N6 HPAIviruses isolated from wild birds and poultry in November2017. Genetic analysis indicated that these isolates hadbeen generated through reassortment events betweenEuropean H5N8 HPAI viruses in 2016/2017, which weredescendants of Russia–Mongolia H5N8 viruses, andEurasian lineage LPAI viruses. Novel H5N6 viruses withsimilar gene constellations were founded in both Greeceand Korea, potentially derived by new reassortant eventsin Eurasian wild bird populations at breeding sites orstaging sites and then spread to multiple geographicregions through different migratory subpopulations. Moredetailed information on HPAI viruses identified throughactive surveillance programs throughout Eurasia is neededto better evaluate the ecology, evolution, dynamics anddiffusion of these viruses in wild birds, particularly inbreeding sites in Mongolia and Siberia where these spe-cies likely converge in the summer with the potential forreassortment-driven expansion in diversity, and to assessthe potential risks to animal and public health.AcknowledgementsWe thank Jung-Eui Lee, In-Kyung Kim, and Byung-suk Jeon for their excellenttechnical assistance. We also thank the Animal and Plant Quarantine Agency(APQA), Ministry of Agriculture, Food and Rural Affairs (MAFRA) and theRegional Office for Animal Disease Control for their efforts to control HPAI. Thisstudy was supported by a grant from the Animal and Plant Quarantine Agency(APQA) (no. B-1541781-2008-2019-01).Author details1Avian Influenza Research and Diagnostic Division, Animal and PlantQuarantine Agency, 177, Hyeoksin 8-ro, Gimcheon-si, Gyeongsangbuk-do39660, Republic of Korea. 2EU/OIE/FAO international reference laboratory foravian influenza, Animal and Plant Health Agency-Weybridge, Addlestone,Surrey KT15 3NB, United Kingdom. 3Department of Zoology, University ofCambridge, Downing Street, Cambridge CB2 #EJ, United KingdomReceived: 22 December 2017 Revised: 22 April 2018 Accepted: 25 April2018References1. Global Consortium for H5N8 and Related Influenza Viruses. Role for migratorywild birds in the global spread of avian influenza H5N8. Science 354, 213–217(2016).2. Lee, D. H., Bertran, K. & Kwon, J. H. et al. Evolution, global spread, andpathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4. J. Vet.Sci. 18(S1), 269–280 (2017).3. Bi, Y., Chen, Q. & Wang, Q. et al. Genesis, evolution and prevalence of H5N6avian influenza viruses in China. Cell Host Microbe 20, 810–821 (2016).4. Wu, H., Peng, X. & Xu, L. et al. Novel reassortant influenza A(H5N8) viruses indomestic ducks, eastern China. Emerg. Infect. Dis. 20, 1315–1318 (2014).5. Lee, Y. J., Kang, H. M. & Lee, E. K. et al. Novel reassortant influenza A(H5N8)viruses, South Korea, 2014. Emerg. Infect. Dis. 20, 1087–1089 (2014).6. Lee, D. H., Bahl, J. & Torchetti, M. K. et al. Highly pathogenic avian influenzaviruses and generation of novel reassortants, United States, 2014-5. Emerg.Infect. Dis. 22, 1283–1285 (2016).7. Lee, M. S., Chen, L. H. & Chen, Y. P. et al. Highly pathogenic avian influenzaviruses H5N2, H5N3, and H5N8 in Taiwan in 2015. Vet. Microbiol. 187, 50–57(2016).8. Lee, D. H., Sharshov, K. & Swayne, D. E. et al. Novel reassortant clade 2.3.4.4avian influenza A(H5N8) virus in wild aquatic birds, Russia, 2016. Emerg. Infect.Dis. 23, 359–360 (2017).9. Li, M., Liu, H. & Bi, Y. et al. Highly pathogenic avian influenza A(H5N8) virus inwild migratory birds, Qinghai Lake, China. Emerg. Infect. Dis. 23, 637–641 (2017).10. World Organization for Animal Health. Update on avian influenza in animals(type H5 and H7). www.oie.int/animal-health-in-the-world/update-on-avian-influenza/11. Kim, Y. I., Park, S. J. & Kwon, H. I. et al. Genetic and phylogenetic character-ization of a novel genotype of highly pathogenic avian influenza (HPAI) H5N8viruses in 2016/2017 in South Korea. Infect. Genet. Evol. 53, 56–67 (2017).12. Lee, E. K., Song, B. M. & Lee, Y. N. et al. Multiple novel H5N6 highly pathogenicavian influenza viruses, South Korea, 2016. Infect. Genet. Evol. 51, 21–23 (2017).13. Beerens, N., Heutink, R. & Bergervoet, S. A. et al. Multiple reassorted viruses ascause of highly pathogenic avian influenza A(H5N8) virus epidemic, theNetherlands, 2016. Emerg. Infect. Dis. 23, 1974–1981 (2017).14. Pohlmann, A., Starick, E. & Harder, T. et al. Outbreaks among wild birds anddomestic poultry caused by reassorted influenza A(H5N8) clade 2.3.4.4 viruses,Germany, 2016. Emerg. Infect. Dis. 23, 633–636 (2017).15. Fusaro, A., Monne, I. & Mulatti, P. et al. Genetic diversity of highly pathogenicavian influenza A (H5N8/H5N5) viruses in Italy, 2016-17. Emerg. Infect. Dis. 23,1543–1547 (2017).Lee et al. Emerging Microbes & Infections  (2018) 7:103 Page 3 of 3

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Characterization of a novel reassortant H5N6 highly pathogenic avian influenza virus clade 2.3.4.4 in Korea, 2017

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