Characterization of recombinant H9N2 influenza viruses isolated from wild ducks in China

Abstract

Wild birds are considered to be the natural reservoirs for avian influenza A viruses (AIV). During active influenza surveillance in Poyang Lake of southeast China, we isolated and characterized 11 H9N2 viruses from two species of wild ducks. Phylogenetic analysis showed that the 11 isolates were almost identical with 99.3–100% nucleotide homology in their entire genome, and they all closely related in whole eight genes (95.6–99.4% homology) to human H9N2 isolates (HK/33982/2009) and clustered in the same sublineage. The isolates belonged to triple reassortant H9N2 genotype viruses containing Ck/Bei-like NA genes, Y439-like PA genes and six other G1-like genes. We also found that the subtype of virus replicated efficiently in the lungs and tracheas of BALB/c mice and caused mortality in 20–40% of infected groups after 3–6 days, which indicates that the subtype of virus is capable of establishing lethal mammalian infections. However, whether or not the virus has features transmittable from wild ducks to humans is not known. This study showed H9N2 subtype avian influenza virus for the first time in wild birds, and suggests that wild birds may carry the virus along migratory routes, highlighting the need for continued surveillance of wild birds.

Introduction

The H9N2 avian influenza viruses have been endemic in poultry in the Eurasian continent since the mid-1990s (Guo et al., 2000). Some avian-origin H9N2 viruses can replicate in mice and ferrets without prior adaptation (Choi et al., 2004, Li et al., 2005, Bi et al., 2010), and occasionally are able to expand their host range to mammals, including pigs and humans (Lin et al., 2000, Cong et al., 2007). Several human cases of H9N2 infection have been reported since 1999 (Peiris et al., 1999, Butt et al., 2005). In addition, H9N2 viruses show a high genetic compatibility with other subtypes, providing their “internal” genes to other viruses. Genetic analysis shows that the H9N2 viruses might be the donors of the “internal” genes of the lethal H5N1 viruses that prevailed in Hong Kong in 1997 (Guan et al., 1999), and the human H7N9 viruses that emerged in China in March 2013 (Gao et al., 2013). It is also noteworthy that the acquisition of a polybasic HA cleavage site through reassortment with other subtypes of influenza viruses probably enhanced the pandemic potential of H9N2 (Gohrbandt et al., 2011, Veits et al., 2012). These findings show that the H9N2 viruses pose potential hazards to human populations. Thus, longitudinal surveillance on the prevalence of H9N2 might be critical in limiting or preventing the source of the transmission of AIV in humans.

In China, H9N2 subtype viruses have become the principal reported strains in poultry following their first report in 1992 (Liu et al., 2003), with at least 74 different genotypes identified (Dong et al., 2011). New lineages and genotypes are being discovered throughout China. Three main lineages of H9N2 influenza viruses, Chicken/Beijing/1/94 (Ck/Bei-like), Quail/Hong Kong/G1/97 (G1-like) and Duck/Hong Kong/Y439/97 (Y439-like or Korean-like), have been responsible for influenza outbreak events in China since the mid-1990s (Li et al., 2005, Xu et al., 2007a, Xu et al., 2007b, Sun et al., 2010). The G1-like viruses are primarily detected in quails and humans, and rarely detected in domestic poultry and wild birds (Guan et al., 1999, Xu et al., 2007a). Ck/Bei-like H9N2 viruses are mainly prevalent in chickens, and the lineage was gradually replaced by the F98-like or G9-like viruses (represented by A/Ck/Shanghai/F/98 or CK/HK/G9/97, respectively) in eastern and southeastern China (Chen et al., 2003a, Zhang et al., 2009). Y439-like (Korean-like) H9N2 viruses were isolated from domestic ducks in Hong Kong and chickens in South Korea (Choi et al., 2005).

Previous surveillance of H9N2 viruses in China has focused mainly on domestic poultry, such as chickens, ducks, geese and other minor poultry species (Li et al., 2003, Xu et al., 2007a, Bi et al., 2010). Although H9N2 viruses isolated from humans are likely of avian origin, they are unable to persist for a long period in avian species due to the birds’ relatively short life span and public health control efforts (Suarez, 2000). Wild aquatic birds are considered the natural hosts for avian influenza A virus and they are the reservoirs of all known subtypes H1–H16 and N1–N9 (Webster et al., 1992). Avian influenza infections in wild aquatic birds are usually asymptomatic and control of AIV outbreaks in wild birds is both difficult and costly (Webster et al., 1992). Wild birds, especially wild ducks, are very common in southern China and their populations appear to have increased recently. Some wild ducks (such as mallard) have been domesticated and farmed, which may alter the interspecies transmission cycle of influenza viruses in southern China. However, surveillance of H9N2 influenza virus in wild aquatic birds and viral genomic information is limited. In order to explore the role of wild birds and the disease ecology of H9N2 virus, we characterized 11 strains of virus isolated from wild ducks in Jiangxi, China in 2011.