Characterization of Avian Influenza Viruses A (H5N1) from Wild Birds, Hong Kong, 2004–2008

Repeated detection of subclade 2.3.2 viruses in nonpasserine birds from different regions suggests possible establishment of this lineage in wild bird species.

H ighly pathogenic avian infl uenza (HPAI) viruses (H5N1) derived from the goose/Guangdong/1/96 (Gs/ GD) lineage have spread to more than 60 countries across Eurasia and Africa (1)(2)(3). The unprecedented panzootic caused by the HPAI viruses (H5N1) has been mediated by the movement of poultry and poultry products and, in some instances (e.g., clade 2.2 viruses), by wild bird migration (4)(5)(6). After introduction, the viruses became endemic in some countries, causing repeated poultry outbreaks and spilling over to cause zoonotic infection in humans, thus posing a persistent potential pandemic threat (7)(8)(9). However, in some affected countries with substantial resources (e.g., Japan and South Korea), despite the repeated introduction of subtype H5N1 viruses that have occasionally led to associated outbreaks in poultry, early and aggressive intervention measures prevented these viruses from becoming endemic in poultry, and no human cases were detected (2,(10)(11)(12)(13).
HPAI viruses (H5N1) were fi rst observed to cause outbreaks of disease in wild and captive birds in Penfold and Kowloon Parks, Hong Kong, in late 2002 and in 2003 (14). The Kowloon Park outbreak was concurrent with outbreaks caused by this virus in several live poultry markets and on some chicken farms in Hong Kong (14). Measures to improve biosecurity on farms, changes in the poultry marketing system, the introduction of rest days in poultry markets, and vaccination for all poultry entering Hong Kong markets have prevented subsequent HPAI (H5N1) outbreaks in farmed poultry in Hong Kong (15). No further cases of infection in live poultry markets were detected from November 2003 through June 2008, when live bird market surveillance detected incursion of a new HPAI (H5N1) virus (2).
The avian infl uenza control program in Hong Kong includes intensive active surveillance of live poultry markets, aviary bird markets, poultry farms, and migratory birds at several wetland sites in Hong Kong (16). In addition, avian infl uenza surveillance has been conducted on wild birds found dead (wild birds and caged birds released for ceremonial purposes are collectively referred to as wild birds in this article) (17). Until the recent incursion of HPAI virus (H5N1) in June 2008, no viruses of subtype H5N1 had been detected on poultry farms or in markets in Hong Kong since November 2003, although 2 HPAI viruses (H5N1) were detected in chickens smuggled into Hong Kong in 2006 (18). However, HPAI viruses (H5N1) have been detected every year in a variety of dead wild birds, including falcons, egrets, herons, and various passerine species (1,4,7,14,18,19).
In this study, we antigenically and genetically characterized all HPAI (H5N1) viruses isolated from the dead bird surveillance program in Hong Kong to gain insights into the evolutionary history and possible transmission pathways of the viruses. Our research shows that viruses isolated each winter from 2004 through 2007 were genetically distinct, belonging to different subtype H5N1 clades. These different clades suggest multiple introductions of HPAI virus (H5N1) reassortments into Hong Kong through wild birds. This study also demonstrates that wild birds can disseminate the HPAI virus (H5N1) and have the potential to seed areas otherwise free from the virus.

Virus Isolation and Characterization
Viruses were isolated from specimens obtained from dead wild birds (online Appendix Table, available from www.cdc.gov/EID/content/15/3/402-appT.htm) by inoculating embryonated eggs at the laboratory of the Agriculture, Fisheries and Conservation Department of the Hong Kong SAR Government. Viruses were identifi ed by real time-PCR and by standard hemagglutination-inhibition (HI) tests using a panel of World Organization for Animal Health's Avian Infl uenza Reference Laboratory antisera (Veterinary Laboratory Agency, Weybridge, UK) as previously described (14,23,24). All virus isolation was conducted in biosafety level 3 facilities. Details of the avian infl uenza (H5N1) surveillance program in Hong Kong for dead wild birds, including pathologic fi ndings and diagnostic testing, are reported separately (17).

Antigenic Analysis
Antigenic characterization of the infl uenza viruses (H5N1) was carried out by HI assay using 5 ferret polyclonal antisera, as previously described (24

Phylogenetic and Molecular Analysis
To understand the evolutionary history of avian infl uenza viruses (H5N1) isolated from wild birds in Hong Kong, we conducted whole genome sequencing of 29 avian infl uenza viruses (H5N1) that were isolated from dead wild birds in 2006-2008. All 8 gene segments of these viruses were characterized and phylogenetically analyzed. These data were compared with the virus sequence data for an additional 18 infl uenza viruses (H5N1) isolated from dead wild birds in Hong Kong in 2004-2008, with virus sequence data for the 2 viruses obtained from chickens smuggled into Hong Kong in 2006, and with all other available sequence data from the NCBI Infl uenza Virus Resource (25).
Viral RNA extraction, cDNA synthesis, PCR, and sequencing were carried out as described previously (19). Sequences were assembled and edited with Lasergene version 7.2 (DNASTAR, Madison, WI, USA). Se-Al version 2.0 was used for alignment and residue analysis (http://tree.bio.ed.ac.uk/software/seal). The program MrModeltest version 2.2 was used to determine the appropriate DNA substitution model and rate heterogeneity (26). The generated model was used in all subsequent analyses. Neighbor-joining trees were constructed with PAUP* version 4.0b (27), and Bayesian analysis was conducted with MrBayes version 3.1.2 (28) by using 2 replicates of 1 million generations with 6 chains, sampling every 100 generations. The convergence of chains and the estimation of burn-in were assessed using Tracer version 1.4 (http://beast.bio.ed.ac.uk). Estimates of the phylogenies were calculated by performing 1,000 neighbor-joining bootstrap replicates, and Bayesian posterior probabilities were calculated from the consensus of 18,000 trees after excluding the fi rst 2,000 trees as burn-in. The full-genome sequences of 29 infl uenza viruses (H5N1) obtained in this study are available from GenBank under accession nos. CY036042-CY036273.

Virus Isolation
From early 2004 through June 2008, most isolates of infl uenza virus (H5N1) from dead wild birds were detected during the cooler months (i.e., from December to the following February) (online Appendix Table). Almost all positive samples of infl uenza virus subtype H5N1 were isolated from a variety of dead wild birds, including falcons, egrets, herons, and various passerine species. On 2 occasions, infl uenza virus (H5N1) was isolated from smuggled chickens (online Appendix Table).
To understand the molecular epidemiology of the viruses isolated from the dead birds, we conducted phylogenetic analysis of the hemagglutinin (HA), neuramindase (NA), and each of the 6 internal gene segments of the viruses, along with the Gs/GD-like HPAI viruses (H5N1) isolated from different regions of Hong Kong. In the HA gene tree, the wild bird viruses fell into 2 main groups, either clade 2.3.2 or 2.3.4, with the exception of 1 virus in clade 9 ( Figure). The phylogenetic placement of these viruses corresponds well with the known evolution of the infl uenza virus subtype H5N1 that has been documented in Asia.
The Phylogenetic analysis of the NA of these isolates showed a similar phylogenetic relationship to that observed for the HA (data not shown). These fi ndings show that infl uenza viruses (H5N1) detected each winter from 2004 through 2007 were genetically distinct and belonged to different sublineages or clades, suggesting that multiple introductions occurred during the past 4 years.
Phylogenetic analyses of the internal gene complex showed that the viruses from dead wild birds in Hong Kong belonged to different subtype H5N1 genotypes (online Appendix  Table). In contrast, 3 (11%) of 28 viruses in clade 2.3.4 were isolated from nonpasserine hosts, excluding the viruses from the 2 chickens. Although inconclusive, this pattern suggests that clade 2.3.2 viruses may have an adaptation that enables them to infect and cause disease in nonpasserine species more easily than in other bird species.

Molecular Characterization
All 29 viruses characterized were highly pathogenic with variations of the multibasic cleavage site in the HA molecule. However, all clade 2.3.4 viruses had a Gln→Leu substitution at position -9 from the cleavage site (LRERRRK-RG), a factor consistent with previous reports (18). The receptorbinding pocket of the HA1 retains amino acid residues 222-Gln and 224-Gly (H5 numbering used throughout) that preferentially bind to α-2,3-NeuAcGal receptors (29)(30)(31). Other amino acid residues relevant to receptor-binding sites were identical to those of HK/156/1997 and Gs/GD-like viruses in most isolates. However, all clade 2.3.2 viruses characterized had an HA Ser129Leu substitution, a factor previously observed in both clade 1 and 2 viruses (8,32). The clade 2.3.2 virus grey heron/HK/3088/2007 also had a Lys212Arg substitution (30).
In the NA amino acid sequences, all isolates characterized had 274-His, indicating sensitivity to oseltamivir (33). One virus (common buzzard/HK/9213/2007) had a Ser31Asn substitution in the M2 protein, a change that may confer resistance to the adamantanes and that has been present in all Clade 1 viruses characterized to date. This substitution has also been sporadically detected in other H5N1 lineages (34). No amantadine-resistant mutations were observed in the remaining isolates. None of these viruses have the Lys627 residue commonly found in Qinghai-like (clade 2.2) viruses (6).

Discussion
Genetic and antigenic characterization of HPAI wild bird viruses (H5N1) suggests that they are closely related to viruses isolated in Asia during the same time (1,7,18). During this period, an intensive avian infl uenza (H5N1) surveillance program was conducted concurrently on poultry farms and at markets in Hong Kong, and no subtype H5N1 viruses were detected from late 2003 until June 2008, when it was detected in fecal droppings in retail poultry markets (2). Thus, the repeated fi nding of infl uenza virus (H5N1) from dead wild birds in the absence of local poultry infection demonstrates the potential of wild birds to disperse the virus over at least moderate distances (i.e., tens or hundreds of kilometers).
The present study also demonstrates the role of the Hong Kong SAR as a sentinel for detecting emerging infectious diseases in Asia. It further demonstrates that surveillance of avian infl uenza virus (H5N1) in dead wild birds can play a key role as an early warning system for the introduction of this virus, a factor consistent with experience elsewhere (e.g., in Germany, United Kingdom, Russia). A similar strategy of conducting surveillance on wild birds would be useful for other regions in monitoring for these viruses that have the potential to infect a wide range of hosts, including humans (2,35).
Viruses  (2,6,37). This potential for spread, along with the fact that some clade 2.3.2 viruses are antigenically distant from current avian infl uenza vaccine candidates, highlights why a clade 2.3.2 virus, common magpie/HK/5052/2007, has been recently recommended as an avian infl uenza (H5N1) vaccine candidate by the World Health Organization (38). These developments indicate a need for more intensive surveillance in the region and may also have implications for vaccination programs for poultry.  (20). †Clade designations according to the World Health Organization influenza (H5N1) nomenclature system (21). ‡Genotypes according to Duan et al. (22). §Characterized in this study.