The first H9N2 avian influenza outbreak caused by Ck/BJ/1/94-like viruses was reported in eastern China in 1994. In late 1998, most chicken farms throughout eastern China suffered serious economic losses due to H9N2 avian influenza viruses. In the present study, our findings reveal that H9N2 influenza A viruses isolated during 1996 to 2008 in eastern China have evolved continuously and have undergone wide reassortment with aquatic avian influenza A viruses, generating six genotypes in eastern China during the past 10 years. Furthermore, it is noteworthy that all six virus genotypes identified in this study have different phenotypic characteristics. Most of the H9N2 viruses isolated before 2000 can replicate mainly in inoculated chickens but have failed to be transmitted efficiently to aerosol contact groups. In contrast, most of the viruses isolated from 2001 onwards not only replicated efficiently in the infected chickens but also were transmitted efficiently among aerosol contact chickens. Therefore, the H9N2 viruses circulating in eastern China appear to have undergone phenotypic changes.
Chickens are generally considered aberrant hosts of influenza viruses because the mutation rates of many chicken viruses are higher than those for viruses isolated from aquatic birds (29
). However, previous studies have shown that H9N2 influenza viruses have circulated widely among chicken flocks for more than 10 years since they were first detected in mainland China in 1994 (4
). Furthermore, it is worth noting that chickens not only are able to harbor genotype A (Ck/BJ/1/94-like) viruses but may have also carried genotype H (Ck/SH/F/98-like) viruses over the past 10 years. Zhang et al. (36
) recently demonstrated that Ck/SH/F/98-like H9N2 influenza viruses have been circulating in chicken flocks from the same integrated chicken operation for at least 5 years. Ito et al. (12
) also demonstrated that chickens have molecular characterizations classifying them as potential intermediate hosts for transmission of avian influenza viruses to humans. Furthermore, the recent transmission of avian H5N1 and H9N2 influenza viruses from chickens and/or quails to humans indicates that avian influenza viruses can directly infect humans without an intermediate host (17
). All available evidence suggests that H9N2 influenza viruses have adapted to chickens. Furthermore, chickens have played an important role in the evolution of influenza viruses by acting as mixing vessels or disseminators of avian/mammalian reassortant influenza A viruses.
Although many novel genotypes have been generated in eastern China during the past 10 years, only two genotypes (genotypes A and H) have been well adapted to chickens and have been circulating in chickens for some time. Genetic and antigenic studies demonstrated that genotype A was the predominant genotype circulating in poultry before 2000. However, from 2000 onward, the genotype A viruses were replaced by a novel genotype, genotype H (Ck/SH/F/98-like), which has become the predominant genotype in poultry in eastern China. Furthermore, the genotype H viruses appear to be better adapted to poultry and more easily reassorted with other subtype avian influenza A viruses. The other H9N2 virus genotypes were very short-lived reassortants, none of which had become established in poultry, suggesting that those viruses were not well adapted to chickens. This finding provides potentially useful information regarding key molecular determinants for such selection.
Because pigs contain both Neu5Acα2-3Gal and Neu5Acα2-6Gal linkages to support replication of both avian and human influenza viruses, pigs have been recognized as “mixing vessels” for avian and human viruses (12
). During the past 5 years, H9N2 influenza viruses have been isolated repeatedly from pigs in eastern China (6
). However, phylogenetic analysis of the internal genes of those swine H9N2 viruses showed that almost all swine H9N2 viruses recently isolated from eastern China also possess Ck/SH/F/98-like lineage RNP genes (6
). Furthermore, the mutation Glu (Q)→Leu (L) at position 226 found in human H2 and H3 isolates occurred in those of genotype H H9N2 viruses. A key amino acid mutation from His to Ser at position 183 (H3 numbering) in the HA receptor binding site has never before been documented. These phenomena increased the chances of generating new influenza viruses with pandemic potential in this region. Therefore, surveillance of H9N2 viruses in this region for the next possible influenza pandemic should be given a high priority.
Donors of internal genes present in H5N1 variants circulating throughout the world have not been well established. However, based on the phylogenetic relationships of NP genes, the present study demonstrated that Ck/SH/F/-98-like H9N2 viruses may be a possible donor of H5N1/01-like internal genes to currently circulating viruses. Furthermore, based on the sequence of reassortment events, Ck/SH/F/98-like internal genes were first incorporated into H5N1/01-like viruses in 2001 or before. In addition, it was noted that Ck/BJ/1/94-like viruses and Ck/SH/F/98-like viruses first isolated in chickens have also been recognized in domestic ducks in this region. This raises the possibility of bidirectional transmission between chickens and domestic ducks. However, the possibility of gene flow from chickens to ducks could not be excluded in Ck/BJ/1/94-like and Ck/SH/F/98-like H9N2 viruses. Domestic ducks may serve as mixing vessels to facilitate reassortment events for H9N2 and H5N1 viruses and the emergence of novel genotypes in this region.
The receptor specificity of influenza viruses is one factor that allows avian influenza viruses to cross the species barrier. Some H9N2 viruses isolated since 2001 contained G1-like receptor HA signatures described by Gambarian et al. (8
). Wan and Perez (31
) also demonstrated that the Glu-to-Leu mutation at amino acid position 226 in HA allows H9N2 viruses to replicate more efficiently in human airway epithelial cells cultured in vitro. Substitution of Gln for Leu at residue 226 and the change of Ala to Thr at position 190 that occurred in the receptor binding site of these H9N2 variants have been previously reported to be involved in binding specificity to receptors in the host cell (23
). In this study, we found that H9N2 viruses isolated during the past 10 years differed from each other in their ability to agglutinate erythrocytes from goat and buffalo, suggesting differences in receptor specificities in H9N2 influenza viruses. Ito et al. (12
) demonstrated that the α2,3 linkage and NeuGc, but not NeuAc, recognition appear essential for agglutination of bovine and equine erythrocytes. Therefore, additional studies are needed to understand the role of mutations in the receptor binding site in restricting the host range of these H9N2 variants.
The current study demonstrates the continued circulation of H9N2 influenza viruses from poultry in eastern China and shows that the genotypes of H9N2 viruses circulating in eastern China are different from those isolated in southern China. The Ck/SH/F/98-like lineage has replaced the Ck/BJ/1/94-like lineage and has become a predominant genotype circulating in poultry in eastern China. Furthermore, reassortment between H9N2 and H5N1 subtype viruses has contributed to the generation of novel genotypes of both subtypes. It is clear that Ck/SH/F/98-like viruses could provide internal genes for H5N1 viruses currently circulating throughout the world. Therefore, it is imperative that particular attention be paid to H9N2 viruses of avian origin to avert any future pandemic in humans in this region.