Phylogenetic and antigenic characterization of reassortant H9N2 avian influenza viruses isolated from wild waterfowl in the East Dongting Lake wetland in 2011–2012

Influenza A viruses can be divided into different subtypes based upon the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) [1, 2]. Wild waterfowl are recognized as the natural reservoir of influenza A viruses, especially low pathogenic avian influenza virus (LPAIV) [3]. To date, all of the HA (H1–H16) and NA (N1–N9) subtype avian influenza viruses (AIVs) have been identified in wild waterfowl, with the exceptions of H17N10 and H18N11, which were isolated from bats [2, 4, 5]. Previous studies indicated that migratory birds play an important role in the emergence of epidemics in birds, pigs, horses, and humans [1]. Generally, AIVs are nonpathogenic in wild birds, although they sometimes cause significant morbidity and mortality when transmitted to domestic poultry [6‐8].

The H9N2 subtype influenza virus was first isolated from a turkey in Wisconsin in 1966. It has been most prevalent in wild ducks and shorebirds and has shown no evidence of establishing a stable lineage in land-based poultry in North America [9]. Since the l990s, H9N2 subtype influenza viruses have become prevalent in land-based poultry across East Asia, Middle Asia, Europe and Africa. Epidemiological and phylogenetic studies indicate that three distinct sublineages of H9N2 subtype influenza viruses have been established: Ck/Bj/94-like (A/chicken/Beijing/1/94 or A/duck/Hongkong/Y280/1997), G1-like (A/Quail/Hong Kong/G1/1997) and Korea-like (A/chicken/Y439/1997 or A/chicken/Korea/383490-p96323/1996) [10‐12].

Poultry infected with H9N2 subtype AIVs have reduced egg production and moderate morbidity when co-infected with other viruses or bacteria [13]. Since 1999, some H9N2 viruses have been identified with potential human virus receptor specificity, and have been occasionally transmitted to human and swine [14‐16]. Moreover, H9N2 viruses may have contributed internal genes to the highly pathogenic avian influenza (HPAI) H5N1 virus in Hong Kong in 1997 and the novel H7N9 avian influenza virus in mainland China in 2013 [17, 18]. Therefore, the H9N2 subtype avian influenza viruses have been classified as candidate viruses with pandemic potential [19, 20]. The threat to the poultry industry and public health posed by H9N2 subtype avian influenza viruses should not be ignored.

Hunan East Dongting Lake Nature Reserve is one of the largest wetlands in mainland China and is an important overwintering area and staging site for migratory birds that fly along the East Asia–Australia flyway [21]. Moreover, numerous duck farms are found around the lake [22, 23]. Every migrating season, wild birds congregate at the lake where they share a common habitat with domestic ducks, which provides an opportunity for virus reassortment. During active surveillance of AIVs between 2011 and 2012, we isolated H9N2 viruses from wild waterfowl in East Dongting Lake wetlands in November 2011 (7 isolates) and March 2012 (15 isolates). The whole genome sequences of all 22 isolates were obtained, and phylogenetic trees for each gene segment were generated to analyze the relationship of these isolates with other circulating viruses in wild birds or poultry. Furthermore, we performed antigenic analyses to investigate the antigenic characteristics of the isolates.

It is generally accepted that wild waterfowl are the natural reservoir for AIVs, and play important roles in the perpetuation and dissemination of AIVs, especially LPAIV. Previous surveillance studies indicated that AIVs circulate in diverse bird species, including Anseriforms (ducks, geese, and swans) and Charadriiforms (gulls and terns). AIVs preferentially infect cells that line the intestinal tract and numerous viruses can be excreted at high concentrations in wild waterfowl feces. Influenza viruses remain infectious in lake water for up to 4 days at 22°C and for over 30 days at 0°C. Contaminated lake water may result in efficient transmission to naïve birds by the fecal-oral route [32‐34]. Migrating birds annually travel between breeding and overwintering sites, so AIVs harbored by migrating birds can be distributed along the migration flyway. The Hunan East Dongting Lake Nature Reserve is located along the East Asia–Australia flyway and is a major staging and overwintering site for migratory birds. Moreover, many domestic duck farms are located in this region and employ a free-range style to raise domestic ducks [22]. Because wild waterfowl and domestic ducks may share common habitats, water, and food, genetic exchange between different subtypes of AIVs circulating in wild waterfowl and domestic poultry may be possible in this location [35].

In this study, we obtained 22 H9N2 subtype AIV isolates from wild waterfowl in East Dongting Lake wetland in November 2011 and March 2012. Genetic analyses showed that the 22 H9N2 viruses shared over 99% nucleotide sequence homology with the strain A/Egret/Hunan/1/2012 (H9N2) in all eight gene segments, which was isolated from an egret in the same region in January 2012. These findings indicated that these viruses had a common origin. Long-term surveillance studies in America and Eurasia demonstrated that the most prevalent AIVs in wild birds could be separated into Eurasian and American lineages. While intercontinental virus exchange existed in migrating birds, its frequency was limited and invasions by whole viruses were not observed [36]. Instances of invasion by the American lineage H9N2 virus into the Eurasian continent have been reported [24]. Notably, the 22 isolates reported in this study were novel recombinant H9N2 subtype AIVs, which encoded genes derived from both the American and Eurasian gene pools circulating in wild birds. Therefore, genes that originated from the American lineage may have been carried by migratory birds from North America, leading to the generation of multiple AIV reassortants circulating in wild birds in Eurasia. Moreover, this recombinant genotype was detected both by us and Chen and colleagues in different wild birds in three months during 2011–2012 (November 2011, January 2012, and March 2012), suggesting that this virus has been circulating in wild birds in this region [24]. Although H9N2 viruses of this genotype have not been reported in other regions, they may have been distributed to other places by migratory birds along their flyways.

H9N2 AIVs have been endemic in domestic poultry across Eurasia and Africa since the late 1990s. The wide circulation of H9N2 viruses provides more opportunities for reassortment with other AIV subtypes in poultry. In the last few decades, H9N2 viruses may have provided internal genes to the HPAI H5N1 and the novel H7N9 avian influenza viruses [17, 18]. Additionally, H9N2 viruses have been occasionally transmitted from poultry to mammals (including humans and swine), and some of the H9N2 viruses showed the ability to bind efficiently to α2, 6-linked sialic acid, which can indicate human virus receptor specificity [15, 26]. Further studies indicated that H9N2 viruses that encoded Leu226 could replicate in ferrets and be transmitted by direct contact [37]. Therefore, many researchers believe that the H9N2 viruses have the potential to cause a future pandemic. Even if they do not directly cause a pandemic, they could indirectly contribute to an influenza pandemic by contributing internal genes to a reassortant virus [17, 18, 38]. Our findings demonstrated that all 22 isolates were LPAIVs, and we observed neither G226L substitutions in HA proteins nor any other pathogenicity-associated mutations in other viral proteins. Nevertheless, the pathogenicity of these viruses should be further accessed in poultry and mammalian animal models.

Antigenic analyses suggested that the antigenicity of the isolates were significantly different from the G1- and Y280-like viruses, which have been dominant in domestic poultry in Eurasia and Africa, and the “America” virus, which is closely related to the Korea-like viruses. The antigenic and phylogenetic analyses were generally consistent with each other. Importantly, poultry in mainland China have been widely vaccinated with commercial inactivated vaccines that contain representative prevalent influenza strains circulating in poultry in Eurasia, but not strains of the American lineage. Therefore, the currently used commercial vaccines may not protect poultry or prevent the transmission of the novel H9N2 subtype viruses in China.

In summary, the 22 H9N2 viruses isolated from wild waterfowl in 2011–2012 were novel reassorted H9N2 subtype AIVs with similar genotypes. All isolates encoded genes for proteins with low pathogenic characteristics. Determining whether these H9N2 AIVs can transmit to poultry or other animals or further adapt to new hosts will require continuous monitoring in the future. Our findings extend our knowledge of the ecology of AIVs circulating in wild birds in the Dongting Lake region and highlight the importance of intercontinental AIV gene flow in migratory birds. Therefore, we emphasize the vital need for continued surveillance of AIVs in wild birds and poultry to prepare for and respond to potential influenza pandemics.