Veterinary Microbiology 173 (2014) 249–257

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Highly pathogenic avian influenza virus (H5N8) in domestic poultry and its relationship with migratory birds in South Korea during 2014 Jipseol Jeong, Hyun-Mi Kang, Eun-Kyoung Lee, Byung-Min Song, Yong-Kuk Kwon, Hye-Ryoung Kim, Kang-Seuk Choi, Ji-Ye Kim, Hyun-Jeong Lee, Oun-Kyong Moon, Wooseog Jeong, Jida Choi, Jong-Ho Baek, Yi-Seok Joo, Yong Ho Park, Hee-Soo Lee, Youn-Jeong Lee * Animal and Plant Quarantine Agency, 175 Anyangro, Anyangsi, Gyeonggido 430-757, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 3 June 2014 Received in revised form 3 August 2014 Accepted 4 August 2014

Highly pathogenic H5N8 avian influenza viruses (HPAIVs) were introduced into South Korea during 2014, thereby caused outbreaks in wild birds and poultry farms. During the 2014 outbreak, H5N8 HPAIVs were isolated from 38 wild birds and 200 poultry farms (up to May 8, 2014). To better understand the introduction of these viruses and their relationships with wild birds and poultry farm, we analyzed the genetic sequences and available epidemiological data related to the viruses. Genetic analysis of 37 viruses isolated from wild birds and poultry farms showed that all of the isolates belonged to clade 2.3.4.6 of the hemagglutinin (HA) gene, but comprised two distinct groups. During the initial stage of the outbreak, identical isolates from each group were found in wild birds and poultry farms near Donglim Reservoir, which is a resting site for migratory birds, thereby indicating that two types of H5N8 HPAIVs were introduced into the lake at the same time. Interestingly, the one group of H5N8 HPAIV predominated around Donglim Reservoir, and the predominant virus was dispersed by wild birds among the migratory bird habitats in the western region of South Korea as time passed, and it was also detected in nearby poultry farms. Furthermore, compared with the results of the annual AIV surveillance of captured wild birds, which has been performed since 2008, more HPAIVs were isolated and H5 sero-prevalence was also detected during the 2014 outbreak. Overall, our results strongly suggest that migratory birds played a key role in the introduction and spread of viruses during the initial stage of the 2014 outbreak. ß 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

Keywords: HPAI H5N8 Wild birds South Korea

1. Introduction Highly pathogenic avian influenza viruses (HPAIVs) have caused considerable economic damage to the global * Corresponding author at: Avian Disease Division, Animal and Plant Quarantine Agency, 175 Anyangro, Anyangsi, Gyeonggido 430-757, Republic of Korea. Tel.: +82 31 467 1807; fax: +82 31 467 1814. E-mail address: [email protected] (Y.-J. Lee).

poultry industry and pose a major public health hazard worldwide. (FAO, 2005, http://www.fao.org/avianflu/ documents/Economic-and-social-impacts-of-avian-influenza-Geneva.pdf/, 2005; OIE, 2014, http://www.oie.int/ animal-health-in-the-world/web-portal-on-avian-influenza/, 2014; WHO, 2014, http://www.who.int/influenza/ human_animal_interface/en/, 2014). In Hong Kong, an H5N1 HPAI outbreak occurred during 1997 in chicken farms and live bird markets, which also resulted in the first

http://dx.doi.org/10.1016/j.vetmic.2014.08.002 0378-1135/ß 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/3.0/).

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case report of direct chicken to human transmission of H5 HPAIV (de Jong et al., 1997). Officially, between 2003 and March 2014, H5N1 HPAIVs have emerged in 53 countries, including China, Vietnam, and Thailand, and they have human infections in 15 countries (OIE, 2014, http:// www.oie.int/animal-health-in-the-world/web-portal-onavian-influenza/, 2014; WHO, 2014, http://www.who.int/ influenza/human_animal_interface/en/, 2014). In general, the poultry trade and mechanical movements of infected materials are the most likely modes that facilitate the spread of HPAIVs (Alexander, 2000). However, the identification of HPAIV-infected wild birds has prompted concerns about their potential roles in the spread of HPAIVs (Alexander, 2000; Olsen et al., 2006). The first H5 HPAIV reported to cause the death of aquatic wild birds was A/tern/South Africa/61(H5N3) (Becker, 1966). After that, H5N1 HPAIV infections of many wild bird species were reported from two waterfowl parks in Hong Kong during 2002 (Ellis et al., 2004) and more significant H5N1 HPAI outbreaks occurred in wild birds at Lake Qinghai in western China during 2005 (Liu et al., 2005). Subsequently, HPAIVs have appeared throughout Asia, Europe, the Middle East, and in several African countries, where they have been linked to migratory bird movements (Olsen et al., 2006). Wild bird infections have been reported in several of these countries, suggesting that HPAIVs could be spread by migratory bird (Liu et al., 2005; Olsen et al., 2006). Furthermore, experimental infection studies have shown that several bird species, including mallard, can survive HPAIV infections and shed the virus without exhibiting apparent disease symptoms (HulsePost et al., 2005; Perkins and Swayne, 2003; SturmRamirez et al., 2004). Despite numerous strategic efforts to control the spread of H5N1 HPAIVs, these emerging viruses continue to survive and evolve (OIE, 2014, http://www.oie.int/animalhealth-in-the-world/web-portal-on-avian-influenza/, 2014). In particular, H5N1 HPAIVs have become endemic in China since 1996, which has led to the emergence of multiple genotypes or sublineages in this region (Li et al., 2010). Recently, various neuraminidase (NA) subtypes of H5 HPAIVs (H5N2, H5N5, and H5N8) with the genetic backbone of clade 2.3.4 viruses have been detected in ducks, geese, quail, and chickens (Gu et al., 2011; Liu et al., 2013; Zhao et al., 2012, 2013). Among the reassortant viruses, H5N8 HPAIVs from clade 2.3.4 were first isolated from ducks in China during 2010, but they were not reported in other countries until January 2014 (Zhao et al., 2013). During January 2014, however, two types of H5N8 HPAIVs were isolated from a poultry farm in South Korea. These were the first reported cases of H5N8 HPAIVs from the Gs/GS–lineage outside of China (Lee et al., 2014). A subsequent study (Wu et al., 2014) showed that one of the two H5N8 HPAIVs isolated from South Korea in 2014 had a high nucleotide sequence identity (>99%) with the H5N8 HPAIVs detected in domestic ducks in Eastern China during 2013. During 2014, H5N8 HPAIVs were isolated from 38 wild birds and 200 poultry farms in South Korea (up to May 8, 2014). Although the number of outbreak cases has declined significantly, H5N8 HPAIVs continue to be detected in

poultry farms in a sporadic manner. However, few genetic data have been published from this epidemic (Lee et al., 2014). In this study, we analyzed the genetic sequences of HPAIVs isolated from wild birds and poultry farms to determine whether additional H5N8 HPAIV types were present or if re-assortment events had occurred between previously identified viruses. We also analyzed the geographical and chronological distributions of the viruses isolated from wild birds and poultry farms to elucidate how the viruses were introduced and determine whether the HPAI outbreak in domestic poultry farms was related to wild bird movements. 2. Result 2.1. Isolation and identification of HPAI H5N8 In the present study, we isolated 238 H5N8 viruses between January 16 and May 8, 2014. Among the 238 viruses, whole genome sequencing was performed on 19 and 18 viruses from wild birds and poultry farms, respectively, including three previously identified viruses, as shown in Table 1 (Lee et al., 2014). 2.2. Epidemiological features of the outbreak Throughout the outbreak, eight Korean provinces were affected by H5N8 HPAI infections up to May 8, 2014. As shown in Fig. 1, many outbreaks occurred on poultry farms and viruses were isolated from wild birds around Donglim Reservoir in Jeonbuk province between January 16 and 21, 2014, which is referred to as phase I. During phase I, eight H5N8 HPAIVs were isolated from the carcasses of wilds birds, including Baikal teal (five cases) and bean goose (three cases) around Donglim Reservoir. The bean goose carcasses were found individually whereas there were over a hundred Baikal teal carcasses. Donglim Reservoir is one of the most important resting sites for migratory Baikal teal, which overwinter in South Korea, Japan, and northern and Eastern China (BLI, 2014, http://www.birdlife.org/, 2014). During phase I, the H5N8 HPAI outbreak emerged in poultry farms (10 cases in ducks) at the same time as the virus was isolated from wild birds around Donglim Reservoir. Between January 22 and 31, 2014, H5N8 HPAIVs isolates were obtained from wild birds that had spread to north (CN: 1.22–1.27 and GG: 1.24–1.28) and south (JN: 1.27–1.29) in the migratory bird habitats of western Korea, which is referred to as phase II (Fig. 1B). H5N8 HPAIVs were isolated from the carcasses of Baikal teal (five cases), mallard (two cases), bean goose (one case), and coot (one case). The viruses were also detected in the feces of wild birds from a lake (one case). During phase II, ducks (26 cases) and chickens (three cases), were confirmed to be H5N8 HPAI positive in poultry farms in six provinces. In each province, wild bird isolations were geographically close to poultry farms that had infected birds during the initial stage. As shown in Fig. 1C, the HPAI outbreak continued to spread widely, and on May 8, 2014, further 161 poultry farms and 20 wild birds were confirmed to be infected with H5N8 HPAIVs, which we refer to as phase III (Fig. 1C). In CB,

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Table 1 Influenza viruses with complete genome sequences isolated from Korea in 2014. Avian species Domestic

Breeder duck Broiler duck Broiler duck Broiler duck Broiler duck Broiler duck Broiler duck Broiler duck Broiler duck Breeder chicken Breeder duck Broiler duck Broiler duck Breeder duck Breeder duck Breeder duck Breeder chicken Korean native chicken

Wild birds Baikal teal Bean goose Baikal teal Baikal teal Bean goose Baikal teal Baikal teal Baikal teal Baikal teal Coot Baikal teal Baikal teal Mallard White-fronted goose Mallard Bean goose Tundra swan Common teal Spot-billed duck

Virus name (H5N8)

Date Region Sample (2014)

A/breeder duck/Korea/Gochang1/2014a A/broiler duck/Korea/Buan2/2014a A/broiler duck/Korea/H29/2014 A/broiler duck/Korea/H31/2014 A/broiler duck/Korea/H32/2014 A/broiler duck/Korea/H47/2014 A/broiler duck/Korea/H48/2014 A/broiler duck/Korea/H49/2014 A/broiler duck/Korea/H65/2014 A/breeder chicken/Korea/H122/2014 A/breeder duck/Korea/H128/2014 A/broiler duck/Korea/H133/2014 A/broiler duck/Korea/H145/2014 A/breeder duck/Korea/H158/2014 A/breeder duck/Korea/H200/2014 A/breeder duck/Korea/H249/2014 A/breeder chicken/Korea/H250/2014 A/Korean native chicken/Korea/H257/2014

1.16. 1.17. 1.18. 1.19. 1.19. 1.20. 1.20. 1.20. 1.21. 1.24. 1.25. 1.25. 1.25. 1.26. 1.27. 1.28. 1.28. 1.28.

JB JB JB JB JB JB JB JB JB CN JN JB JN CN CB JN GG GN

Carcass, feces Carcass, op Carcass, feces, Carcass, feces Carcass, op Carcass, op Carcass, feces, feces Carcass, feces, Carcass Carcass, feces Carcass, feces, Carcass, feces feces, op, cl Carcass, feces Carcass, feces Carcass, feces Carcass

A/Baikal teal/Korea/Donglim3/2014a A/Bean goose/Korea/H40/2014 A/Baikal teal/Korea/H41/2014 A/Baikal teal/Korea/H52/2014 A/Bean goose/Korea/H53/2014 A/Baikal teal/Korea/H62/2014 A/Baikal teal/Korea/H66/2014 A/Baikal teal/Korea/H68/2014 A/Baikal teal/Korea/H80/2014 A/Coot/Korea/H81/2014 A/Baikal teal/Korea/H84/2014 A/Baikal teal/Korea/H96/2014 A/Mallard/Korea/H207/2014 A/White-fronted Goose/Korea/H231/2014 A/Mallard/Korea/H297/2014 A/Bean goose/Korea/H328/2014 A/Tundra swan/Korea/H411/2014 A/Common teal/Korea/H455-30/2014 A/Spot-billed duck/Korea/H455-42/2014

1.17. 1.19. 1.19. 1.20. 1.20. 1.21. 1.21. 1.22. 1.22. 1.22. 1.22 1.23. 1.27 1.28 1.29 2.1. 2.6. 2.8. 2.8.

JB JB JB JB JB JB JB CN JB JB CN CN JN GG JN GG JB CN CN

Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass op, cl op, cl

Genetic GenBank or group references B A op A A A A op A A op, cl A A A op A A A A A A A

Lee et al. (2014) Lee et al. (2014) KJ508897–KJ508904 KJ508905–KJ508912 KJ508913–KJ508920 KJ508937–KJ508944 KJ508945–KJ508952 KJ508953–KJ508960 KJ508985–KJ508992 KJ509041–KJ509048 KJ509049–KJ509056 KJ509057–KJ509064 KJ509065–KJ509072 KJ509073–KJ509080 KJ509081–KJ509088 KJ509105–KJ509112 KJ509113–KJ509120 KJ509121–KJ509128

A A A B A A A A A A A A A A A A A A A

Lee et al. (2014) KJ508921–KJ508928 KJ508929–KJ508936 KJ508961–KJ508968 KJ508969–KJ508976 KJ508977–KJ508984 KJ508993–KJ509000 KJ509001–KJ509008 KJ509009–KJ509016 KJ509017–KJ509024 KJ509025–KJ509032 KJ509033–KJ509040 KJ509089–KJ509096 KJ509097–KJ509104 KJ509129–KJ509136 KJ509137–KJ509144 KJ509145–KJ509152 KJ509153–KJ509160 KJ509161–KJ509168

op,oropharayngeal; cl,cloacal swabs; JB, Jeonbuk; JN, Jeonnam; CB, Chungbuk; CN, Chungnam; GG, Gyeonggi; and GN,Gyeongnam. a Indicates viruses reported by Lee et al. (2014).

JB, and JN provinces, the number of HPAI outbreak cases increased rapidly in poultry farms that were located close to each other. In the poultry-dense areas of those provinces, human-mediated operations such as poultry trade or the movement of contaminated equipment (including clothing, boots, and vehicles) occurred during the epidemic and the viruses were spread via the network of contacts. 2.3. Phylogenetic analysis Phylogenetic trees were constructed for each gene to understand the evolutionary relationships between the H5N8 HPAIVs and other influenza viruses (Fig. 2 and Sup. Fig.). The phylogenetic analysis of the hemagglutinin (HA) gene showed that all the viruses were closely related to the H5 HPAIVs that circulated in Eastern China during 2010– 2013 and they belonged to clade 2.3.4.6 (Fig. 2). The phylogenetic analysis of the NA genes showed that all the viruses belonged to the Eurasian lineage and they were closely related to A/duck/Jiangsu/k1203/2010 (H5N8) and A/duck/Zhejiang/W24/2013(H5N8). The genes for each of

the polymerase subunits (PB1, PB2, and PA), nucleoprotein (NP), matrix (M), and non-structural (NS) protein from the isolates were allocated to previously identified groups (Lee et al., 2014), which were designated as group A and group B. No further re-assortment events had occurred between the two groups. Supplementary Fig. I related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vetmic. 2014.08.002. It should be noted that the dominant viruses belonged to group A during this outbreak. In particular, 35 viruses (18 from wild birds and17 from poultry farms) belonged to group A and two isolates (one from a wild bird and one from a poultry farm) belonged to group B. All of the H5N8 HPAIVs from wild birds were confirmed as belonging to group A, except for A/Baikal teal/Korea/H52/2014(H5N8) (H52), which was isolated from Donglim Reservoir. In the poultry farms, a group B virus was detected only from the first breeder farm outbreak, whereas the subsequent H5N8 HPAIVs found in poultry farms belonged to group A, which was similar to the virus isolation pattern in wild birds.

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Fig. 1. Progress of the South Korea HPAI outbreak between January 16 and May 8, 2014. Red and green circles indicate locations where HPAI viruses were isolated from poultry farms and wild birds, respectively. A. HPAI-positive cases identified from samples collected during January 16–21, 2014 (phase I). The arrows indicate the first cases identified in wild birds and poultry farms, respectively. B. HPAIV-positive cases identified by January 31, 2014 (phase II). The shaded arrows indicate the spread of viruses to northern and southern areas after phase I. C. HPAIV-positive cases identified by May 8, 2014 (phase III). The shaded circles indicate the spread of viruses to nearby regions. GG, Gyeonggi; GW, Gangwon; CN, Chungnam; CB, Chungbuk; JB, Jeonbuk; JN, Jeonnam; GB, Gyeongbuk; GN, Gyeongnam.

2.4. Surveillance of HPAI in captured wild birds

3. Discussion

As shown in Table 2, the surveillance of AIVs in captured wild bird species has been conducted annually since 2008 in South Korea. Among the subtypes found in captured wild birds, the H5 HPAIV subtype was only isolated during 2010 and 2014, which is agreement with the result of HPAI outbreaks in poultry farms (Kim et al., 2012). In addition to the numbers of viral isolates, the proportion of H5 seropositive cases also increased during the HPAI outbreak seasons: 2008 = 0.28%, 2009 = 0%, 2010 = 0.3%, 2011 = 3.4%, 2012 = 0.84%, and 2014 = 16.6%. During the 2014 HPAI outbreak, 643 captured wild birds were tested from nine species (Table 3). The overall seropositive rate for H5 was 16.6% in wild birds but the rate varied from 0% to 53% among species. The captured Baikal teal had a seropositive rate of 53%, which was the highest among the wild bird species and some of their sera were also confirmed N8 antibody based on the NA inhibition assay (data not shown). The other seropositive wild bird species were as follows: Eurasian wigeon (50%), spot-billed duck (18%), mallard (14%), and common teal (10%), all of which belong to the family Anatidae. As shown in Fig. 3 and Table 3, after the second week in February, H5N8 HPAIVs were isolated from spot-billed duck (two cases), mallard (three cases), and common teal (four cases). In addition, the number of seropositive cases increased significantly from the fourth week of February until the second week of March. The migratory birds moved north after the third week of March; however, the number of captured wild birds decreased (Fig. 3), as well as the number of seropositive cases.

An outbreak of H5N8 HPAI affected wild birds and domestic poultry in South Korea from January 16, 2014. This was the first official report of a H5N8 HPAI outbreak with the HA gene derived from A/goose/Guandong/1/96like (H5N1) (Gs/GS–lineage) (OIE, 2014, http://www.oie. int/animal-health-in-the-world/web-portal-on-avianinfluenza/, 2014;Lee et al., 2014). Previously, the only official outbreak of H5N8 HPAIVs among poultry was in a turkey from Ireland in 1983 (Murphy, 1986). Recently, Zhao et al. (2013) reported H5N8 virus in a mallard duck, which was isolated via the active surveillance of live bird markets (LBM) in China during 2010. Furthermore, identical Group B viruses isolated from South Korea were obtained from LBMs in Eastern China during 2013 (Wu et al., 2014). However, the Chinese H5N8 outbreaks were not reported to OIE as is required. South Korea has experienced four HPAI outbreaks caused by H5N1. Three different clades of H5 gene were detected during the four different HPAI outbreaks in South Korea, i.e., A/chicken/Korea/ES/2003 (clade 2.5), A/chicken/ Korea/IS/2006 (clade 2.2), A/chicken/Korea/Gimje/2008 and A/duck/Korea/Cheonan/2010 (clade 2.3.2.1). During the 2014 outbreak in South Korea, the HA gene of the H5N8 HPAIVs belonged to the proposed clade 2.3.4.6 in the phylogenetic tree (Gu et al., 2013). H5 HPAIVs that belong to clade 2.3.4 were first identified in Southern China and they have been prevalent in poultry since 2005 (Li et al., 2010). In addition, clade 2.3.4 has continued to circulate by evolving into new subclades. During 2009–2012, new fourth-order clades, i.e., 2.3.4.4, 2.3.4.5, and 2.3.4.6, have

J. Jeong et al. / Veterinary Microbiology 173 (2014) 249–257 Fig. 2. Maximum-likelihood phylogenetic trees for the hemagglutinin (HA) and neuraminidase (NA) gene segments of H5N8 HPAIVs from South Korea in 2014 compared with other viruses. HPAI isolates from wild birds and poultry farms during 2014 are shown in green and red, respectively. Other viruses detected in South Korea are indicated in boldface. The vertical lines indicate HA gene clades or groups of viruses. The HA (A, >1600 nt) and NA (B, >1300 nt) genes were multiply aligned. The phylogenetic distances were calculated using phyML 3.1 with the maximum-likelihood algorithm and the trees were visualized using FigTree 1.3.1. The numbers at the nodes represent bootstrap values. HA, hemagglutinin 5; NA, neuraminidase; Gs/Gd, Goose/Guangdong; LPAI, low pathogenic avian influenza; HPAI, highly pathogenic avian influenza. The scale bar indicates the nucleotide substitutions per site.

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Table 2 HPAIV isolates and H5 antibodies detected in captured wild birds from South Korea during 2010–2014.

No. of captured birds No. of H5HPAI virus No. of H5 antibody

2008

2009

2010

2011

2012

2013

2014*

1035

1531

1700 1 0/6

2008

2013

1054

47/22

17/0

0/0

643 9 107/0

3/0

**

0/0

* Data collected up to May 2014. ** No. H5 antibody detected cases of first/second halves of the year.

Table 3 H5N8 HPAIV isolates and H5 antibodies (Ab) detected in captured wild birds from Korea up to May during 2014. Species

No. of samples

Baikal teal Eurasian wigeon Spot-billed duck Mallard Common teal Pintail Mandarin duck Red-crested pochard Gadwall

30 12 146 379 50 16 8 1 1

16 6 26 54 5 0 0 0 0

Total

643

107

H5Ab

Ab positive rate (%)

H5N8 HPAIVs

53 50 18 14 10 0 0 0 0

0 0 2 3 4 0 0 0 0

16.6

9

emerged in poultry in Eastern China. In particular, clade 2.3.4.6 H5 HPAIVs with various NA subtypes (H5N1, H5N5, and H5N8) were reported in Eastern China (Gu et al., 2011; Liu et al., 2013; Zhao et al., 2012, 2013). In a previous study, we identified two distinct genetic groups among three H5N8 HPAIVs, which were generated independently by re-assortment events among the influenza viruses circulating in China during 2009–2012, and the two groups fell into A and B groups in this study (Lee et al., 2014). In the present study, we isolated 238 viruses,

including three previously known viruses, during the H5N8 HPAI outbreak of 2014. The whole genomes of the 37 viruses were sequenced, which were classified into two previously identified groups (Fig. 2) and no further reassortant viruses had exchanged internal segments. Interestingly, there was a difference in the prevalence of the two distinct groups in South Korea. The isolates from group A (35 cases) were detected throughout the country, whereas those from group B (two isolates) were only detected around Donglim Reservoir. It should be noted that genetically closely related isolates (>99%) from each group were found in wild birds and poultry, and their first isolation sites were around Donglim reservoir, thereby suggesting that two distinct viruses were initially introduced to the lake and that the predominant virus group A spread to other regions. We could not exclude the possibility that the viruses were introduced via the movements of people or goods, but it is more reasonable to assume that wild birds were involved in viral transmission to South Korea. The discovery of carcasses of Baikal teal around Donglim Reservoir was the first indicator of the presence of H5N8 HPAIVs in wild birds. Furthermore, antibodies were first detected in Baikal teal on January 29, 2014. Baikal teal are migratory birds, which spend every winter in western habitat sites throughout South Korea such as

Fig. 3. H5N8 HPAIVs isolates and detection of H5 antibodies in captured wild birds between February and April 2014. The closed bars, open bars and dash lines indicated the No. of H5 antibodies, No. of H5N8 viruses and No. of captured birds, respectively. Left and right Y axis indicated the No. of H5N8 viruses or of H5 antibodies, and No. of captured birds, respectively. X axis indicated weeks in each month.

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Donglim Reservoir, Youngam Lake, and Geumgang Lake (unpublished data, Ministry of Environment, Korea). Over 200,000 Baikal teal arrived at Donglim Reservoir in January 2014 and with the passage of time, a subpopulation moved to other habitat sites in the west of South Korea. The flyways of the Baikal teal flock were similar to the pattern of viral spread between the regions in South Korea. Therefore, it is likely that Baikal teal participated in the transmission of HPAIVs between regions during the initial stage of the outbreak. However, it is unclear whether Baikal teal played a primary role as a vector among the wild birds that came to South Korea from other countries. The H5N8 HPAIVs were isolated from healthy captured mallard, spot-billed duck, and common teal, thereby suggesting that these species may also be candidates for the introduction of H5N8 HPAIVs. In particular, mallard are considered to be one of the major vector birds that do not exhibit clinical signs despite shedding the virus, although other studies showed pathogenic to mallard infected with H5 HPAI (Sturm-Ramirez et al., 2004; Hulse-Post et al., 2005; Phuong do et al., 2011; Tang et al., 2009). Indeed, H5N1 HPAIVs were isolated from healthy captured mallard and from subsequent outbreaks in poultry farms during the 2010–2011 outbreak in South Korea (Kim et al., 2012). South Korea has performed annual active surveillance of captured wild birds since 2008 to obtain early warnings of the introduction of HPAIVs. As shown in Table 2, the number of H5 seropositive cases was usually very low in captured wild birds. These results are consistent with previous reports, which demonstrated the low prevalence of H5 AI viruses (0.13%) in fecal samples from wild bird habitats in South Korea (Kang et al., 2010). A H5N1 HPAI outbreak affected wild birds and poultry farms in the winter seasons of 2010–2011 and the number of H5 seropositive cases also increased significantly among wild birds. The high rate of sero-prevalence among wild birds continued until March 2012. The number of H5 seropositive cases was zero in December 2013, but a high rate of H5 sero-prevalence reappeared because the HPAI outbreak emerged in wild birds and poultry farms in January 2014. These results demonstrate that fluctuations in H5 seroprevalence among wild birds were closely related to the HPAI outbreak in poultry in South Korea. Furthermore, during the 2014 H5N8 HPAI outbreak, a significant increase in the number of seropositive cases was observed after the fourth week of February (Fig. 3), indicating that the HPAIVs had spread among wild birds, including Baikal teal during January around Donglim Reservoir, as well dispersing to other regions of South Korea. Among the wild bird species, members of the family Anatidae, including Baikal teal, Eurasian wigeon, spot-billed duck, mallard, and common teal, accounted for many seropositive cases (Table 3), thereby indicating that these species experienced asymptomatic infections with H5N8 HPAIVs, and they may play roles in the spread of H5N8 HPAIVs. The present study suggests that HPAIVs were introduced into South Korea via wild bird movements in 2014 and were associated with viral transmission throughout South Korea. During phase III, however, it is likely that the spread of the poultry farm outbreaks within each region of South Korea was caused mainly by the poultry trade or the

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mechanical movement of infected materials between farms. In the winter season of 2010–2011, South Korea and Japan both experienced H5N1 HPAI outbreaks initiated by migratory birds (Kim et al., 2012; Sakoda et al., 2012). However, the subsequent outbreaks in the poultry farms of South Korea were much more severe than those in Japan. The different poultry populations may explain the variation in prevalence between the two countries. The domestic duck populations are low in Japan, whereas the domestic duck industry has grown rapidly in South Korea since 2000 (KDA, 2014, http://www.koreaduck.org/ index.asp, 2014). Ducks are considered to be a silent reservoir species for avian influenza, including HPAI (WHO, 2007, http://www.who.int/water_sanitation_ health/emerging/h5n1background.pdf, 2007) because of their mild clinical symptoms and their recovery from HPAI infection (Perkins and Swayne, 2003). Furthermore, densely populated duck farms are located in the western region of South Korea, where the major resting and wintering sites of many migratory birds are also located. Therefore, the biosafety or sanitation procedures in domestic duck farms should be strictly enforced to prevent the introduction of viruses and inter-farm transmission. However, considering that the outbreaks in duck farm accounted for 75.5% of those in poultry farms during the 2014 HPAI outbreak in South Korea, it is likely that there was poor compliance with biosafety measures. Geographically, HPAIVs are endemic in Asian countries, including China and Vietnam, and neighboring countries experience repeated epidemics due to migratory birds or movements of goods. The Korean peninsula is also situated in a high-risk region for HPAIV introduction. Therefore, better enforcement and more stringent biosafety instructions or training should be implemented in domestic duck farms to reduce the economic losses in the poultry industry in South Korea due to HPAIVs. 4. Materials and methods 4.1. Virus isolation and identification The suspected samples from domestic poultry farms and wild birds were diagnosed using both RT-PCR and virus isolation techniques, as described previously (Lee et al., 2014). To isolate viruses, samples that comprised oropharayngeal and cloacal swabs, feces, and the tissues of dead birds were inoculated into 9- to 11-day-old embryonated specific pathogen-free chicken eggs. The presence of viruses in allantoic fluids was screened using a hemagglutination (HA) assay and RT-PCR, and they were confirmed by sequencing analysis. Serological tests, i.e., HA inhibition (HI) and NA inhibition (NI) test, were performed as described OIE terrestrial manuals (OIE, 2009, http:// web.oie.int/fr/normes/mmanual/2008/pdf/2.03.04_AI.pdf, 2009). The antigen used in the HI test was 8 hemagglutination units (HAU) of inactivated A/broiler duck/Korea/ Buan2/2014 (H5N8) (Buan2). Samples with HI titers over log23 (23) were considered positive. Some sera of index cases and captured wild birds were performed NI test. As reference or control, antisera of the N1–N9 of previous study (Lee et al., 2013) were used. The antigen used in the

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NI test was A/broiler duck/Korea/Buan2/2014 (H5N8) (Buan2). NA inhibition activity was determined by the absence or significant reduction of colors. 4.2. Wild bird surveillance Since 2003, nationwide surveillance for AIV from feces of wild birds in major migratory bird habitats has been conducted in South Korea. Since 2008, additionally, 1000– 2000 of healthy migratory birds per years have been captured, and samples of swabs (oropharyngeal and cloacal) and serum were taken as part of active surveillance. Samples of swabs and serum were assayed for virus isolation and HI tests as described above, respectively. 4.3. Viral sequencing Viral RNA was extracted from allantoic fluid using a Viral Gene-SpinTM kit (Intron Biotechnology, Korea). All eight viral RNA segments were amplified using segmentspecific primers with a One Step RT-PCR premix kit (Qiagen, USA) (Hoffmann et al., 2001). The PCR products were purified using a Qiagen gel extraction kit (Qiagen, USA) and the products were sequenced directly (Macrogen, Korea) with an ABI 3730XL Analyzer (Applied Biosystems, USA). The GenBank accession numbers of the gene segments from the isolates obtained between January 16 and May 8, 2014 are KJ508897–KJ509168. 4.4. Phylogenetic analysis The representative influenza virus sequences from other countries, which were used in the phylogenetic comparison, were obtained from NCBI Influenza Virus Resource (IVR, 2014, http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU. html, 2014) and GISAID (http://platform.gisaid.org/epi3/ frontend#2af1d0, 2014). Editing and sequence analyses were performed using BioEdit version 7.2.5. Multiple nucleotide and amino acid sequence alignments of all eight gene segment were performed using CLUSTALW2. The multiple alignments were used to infer the phylogenies with the maximum-likelihood (ML) method implemented in phyML version 3.0 (Guindon et al., 2010). To obtain the ML tree topologies, 100 bootstrap replicates were performed for each dataset. The inferred tree topologies were inspected visually using FigTree version 1.3.1. Acknowledgments We thank the Animal and Plant Quarantine Agency (QIA), Ministry of Agriculture, Food, and Rural Affairs (MAFRA), and the Regional Office for Animal Disease Control for their efforts in the control of HPAI. This research was supported by a grant from the QIA (No. N-15417812008-19-01) of the Republic of Korea. The funders had no roles in the study design, data collection and analysis, decision to publish, or the preparation of the manuscript. References Alexander, D.J., 2000. A review of avian influenza in different bird species. Vet. Microbiol. 74, 3–13.

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