H9N2 Viruses In Poultry Research Articles

Reported human infections of H9N2 avian influenza virus in China in 2021.

The continued emergence of human infections of H9N2 avian influenza virus (AIV) poses a serious threat to public health. The prevalent Y280/G9 lineage of H9N2 AIV in Chinese poultry can directly bind to human receptors, increasing the risk of spillover infections to humans. Since 2013, the number of human cases of H9N2 avian influenza has been increasing continuously, and in 2021, China reported the highest number of human cases, at 25. In this study, we analyzed the age, geographic, temporal, and sex distributions of humans with H9N2 avian influenza in 2021 using data from the National Influenza Center (Beijing, China). We also conducted evolutionary, gene homology, and molecular characterization analyses of the H9N2 AIVs infecting humans. Our findings show that children under the age of 12 accounted for 80% of human cases in 2021, and females were more frequently affected than males. More cases occurred in winter than in summer, and most cases were concentrated in southern China. Human-infecting H9N2 viruses showed a high level of genetic homology and belonged to the prevalent G57 genotype. Several additional α2,6-SA-binding sites and sites of mammalian adaptation were also identified in the genomes of human-infecting H9N2 viruses. Therefore, continuous monitoring of H9N2 AIV and the implementation of further measures to control the H9N2 virus in poultry are essential to reduce the interspecies transmission of the virus.

Rapid emergence of a PB2 D701N substitution during adaptation of an H9N2 avian influenza virus in mice.

H9N2 avian influenza viruses (AIVs) have been isolated frequently from multiple avian species and, occasionally, from humans. To explore the potential molecular basis of cross-species transmission of H9N2 AIVs, an H9N2 AIV (A/chicken/Zhejiang/221/2016) was serially passaged in mouse lung. The results showed that the mouse-adapted H9N2 virus exhibited higher virulence and replicated more efficiently in mouse lung and liver. Whole-genome sequencing showed an amino acid substitution, D701N, in the PB2 protein, which is likely associated with the increased replicative ability of H9N2 virus in mice. The rapid emergence of adaptive substitutions indicates the necessity of continuous monitoring of H9N2 virus in poultry.

Emerging threats and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for the influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into a new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that the next human pandemic variant will be the avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccine updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Emerging threat and vaccination strategies of H9N2 viruses in poultry in Indonesia: A review.

Avian influenza virus subtype H9N2 was first documented in Indonesia in 2017. It has become prevalent in chickens in many provinces of Indonesia as a result of reassortment in live bird markets. Low pathogenic avian influenza subtype H9N2 virus-infected poultry provides a new direction for influenza virus. According to the latest research, the Indonesian H9N2 viruses may have developed through antigenic drift into new genotype, posing a significant hazard to poultry and public health. The latest proof of interspecies transmission proposes that, the next human pandemic variant will be avian influenza virus subtype H9N2. Manipulation and elimination of H9N2 viruses in Indonesia, constant surveillance of viral mutation, and vaccines updates are required to achieve effectiveness. The current review examines should be investigates/assesses/report on the development and evolution of newly identified H9N2 viruses in Indonesia and their vaccination strategy.

Molecular characterization, receptor binding property and replication in chickens and mice of H9N2 avian influenza viruses isolated from chickens, peafowls and wild birds in eastern China

H9N2 avian influenza viruses are widely prevalent in birds and pose an increasing threat to humans because of their enhanced virulence and transmissibility in mammals. Active surveillance on the prevalence and evolution of H9N2 viruses in different avian hosts will help develop eradication measures. We isolated 16 H9N2 viruses from chickens, green peafowls, and wild birds in eastern China from 2017 to 2019 and characterized their comparative genetic evolution, receptor-binding specificity, antigenic diversity, replication, and transmission in chickens and mice. The phylogenetic analysis indicated that the green peafowl viruses and swan reassortant shared the same ancestor with the poultry H9N2 viruses prevalent in eastern China, while the seven wild bird viruses belonged to wild bird lineage. The chicken, peafowl, and swan H9N2 viruses that belonged to the poultry lineage preferentially recognized α-2, 6-linked sialic acids (human-like receptor), but the wild bird lineage viruses can bind both α-2, 3 (avian-like receptor) and human-like receptor similarly. Interestingly, the H9N2 viruses of poultry lineage replicated well and transmitted efficiently, but the viruses of wild bird lineage replicated and transmitted with low efficiency. Importantly, the H9N2 viruses of poultry lineage replicated in higher titer in mammal cells and mice than the viruses of wild birds lineage. Altogether, our study indicates that co-circulation of the H9N2 viruses in poultry, wild birds, and ornamental birds increased their cross-transmission risk in different birds because of their widespread dissemination.

Evolution of H9N2 avian influenza viruses in Iran, 2017-2019.

Since its first detection in 1998, avian influenza virus (AIV) subtype H9N2 has been enzootic in Iran. To better understand the evolutionary history of H9N2 viruses in Iran, we sequenced 15 currently circulating H9N2 viruses from domestic poultry during 2017-2019 and performed phylogenetic analysis of complete genome sequences. Phylogenetic analyses indicated that the Iranian H9N2 viruses formed multiple well-supported monophyletic groups within the G1-lineage of H9N2 virus. Our analysis of viral population dynamics revealed an increase in genetic diversity until 2007, corresponding to the multiple introductions and diversification of H9N2 viruses into multiple genetic groups (named Iran 1-4 subgroups), followed by a sudden decrease after 2008. Only the Iran 4 subgroup has survived, expanded, and currently circulates in Iran. The H9N2 viruses possessed many molecular markers associated with mammalian adaption in all gene segments, except neuraminidase gene. Considering the presence of mammalian host-specific markers, the public health threat of H9N2 viruses continues. Molecular analysis showed that Iranian H9N2 strains have continued to evolve and recent strains have multiple amino acid changes and addition of potential N-glycosylation on the antigenic sites of haemagglutinin. Continued antigenic and molecular surveillance of H9N2 viruses in poultry and mammals would be required to monitor further increments in viral evolution and their potential threat to public health.

Non-basic amino acids in the hemagglutinin proteolytic cleavage site of a European H9N2 avian influenza virus modulate virulence in turkeys

H9N2 avian influenza virus (AIV) is the most widespread low pathogenic (LP) AIV in poultry and poses a serious zoonotic risk. Vaccination is used extensively to mitigate the economic impact of the virus. However, mutations were acquired after long-term circulation of H9N2 virus in poultry, particularly in the hemagglutinin (HA) proteolytic cleavage site (CS), a main virulence determinant of AIV. Compared to chickens, little is known about the genetic determinants for adaptation of H9N2 AIV to turkeys. Here, we describe 36 different CS motifs in Eurasian H9N2 viruses identified from 1966 to 2019. The European H9N2 viruses specify unique HACS with particular polymorphism by insertion of non-basic amino acids at position 319. Recombinant viruses carrying single HACS mutations resembling field viruses were constructed (designated G319, A319, N319, S319, D319 and K319). Several viruses replicated to significantly higher titers in turkey cells than in chicken cells. Serine proteases were more efficient than trypsin to support multicycle replication in mammalian cells. Mutations affected cell-to-cell spread and pH-dependent HA fusion activity. In contrast to chickens, mutations in the HACS modulated clinical signs in inoculated and co-housed turkeys. G319 exhibited the lowest virulence, however, it replicated to significantly higher titers in contact-turkeys and in vitro. Interestingly, H9N2 viruses, particularly G319, replicated in brain cells of turkeys and to a lesser extent in mammalian brain cells independent of trypsin. Therefore, the silent circulation of potentially zoonotic H9N2 viruses in poultry should be monitored carefully. These results are important for understanding the adaptation of H9N2 in poultry and replication in mammalian cells.

Retraction: Emerging threat of H9N2 viruses in poultry of Pakistan and vaccination strategy

Retraction: Emerging threat of H9N2 viruses in poultry of Pakistan and vaccination strategy

Emerging threat of H9N2 viruses in poultry of Pakistan and vaccination strategy

Emerging threat of H9N2 viruses in poultry of Pakistan and vaccination strategy

Development of a dual-protective live attenuated vaccine against H5N1 and H9N2 avian influenza viruses by modifying the NS1 gene.

An increasing number of outbreaks of avian influenza H5N1 and H9N2 viruses in poultry have caused serious economic losses and raised concerns for human health due to the risk of zoonotic transmission. However, licensed H5N1 and H9N2 vaccines for animals and humans have not been developed. Thus, to develop a dual H5N1 and H9N2 live-attenuated influenza vaccine (LAIV), the HA and NA genes from a virulent mouse-adapted avian H5N2 (A/WB/Korea/ma81/06) virus and a recently isolated chicken H9N2 (A/CK/Korea/116/06) virus, respectively, were introduced into the A/Puerto Rico/8/34 backbone expressing truncated NS1 proteins (NS1-73, NS1-86, NS1-101, NS1-122) but still possessing a full-length NS gene. Two H5N2/NS1-LAIV viruses (H5N2/NS1-86 and H5N2/NS1-101) were highly attenuated compared with the full-length and remaining H5N2/NS-LAIV viruses in a mouse model. Furthermore, viruses containing NS1 modifications were found to induce more IFN-β activation than viruses with full-length NS1 proteins and were correspondingly attenuated in mice. Intranasal vaccination with a single dose (10(4.0) PFU/ml) of these viruses completely protected mice from a lethal challenge with the homologous A/WB/Korea/ma81/06 (H5N2), heterologous highly pathogenic A/EM/Korea/W149/06 (H5N1), and heterosubtypic highly virulent mouse-adapted H9N2 viruses. This study clearly demonstrates that the modified H5N2/NS1-LAIV viruses attenuated through the introduction of mutations in the NS1 coding region display characteristics that are desirable for live attenuated vaccines and hold potential as vaccine candidates for mammalian hosts.

Residues 41V and/or 210D in the NP protein enhance polymerase activities and potential replication of novel influenza (H7N9) viruses at low temperature.

BackgroundThe influenza A (H7N9) virus emerged in the spring of 2013 in China. It contained six internal genes from Y280-like H9N2 viruses, which have co-circulated with G1-like lineage viruses throughout poultry in China. Accompanied with continuous reassortment among H7N9 and H9N2 viruses in poultry, it is possible for H7N9 viruses to acquire internal genes of G1-lineage viruses. Thus, it is important to evaluate potential impact of G1-like viruses on the H7N9 viruses.FindingsWe used in vitro assays of polymerase activities and growth kinetics to evaluate the potential contribution of G1-like virus genes to the replication abilities of H7N9 viruses. Two mutations in the NP protein (41V and/or 210D) could enhance H7N9 RNP activities, especially at low temperature (33°C, which is similar to the temperature of human upper respiratory tract). Meanwhile, G1 viruses with V41I or D210E substitutions exhibited poor growth ability in the early infection stage at low temperature. The D210E substitution also reduced the replication ability of G1 virus at 12 and 24 hour post infection at 37°C. In both tested temperatures, V41I could compensate for the defective virus replication induced by the D210E mutation.ConclusionsMutations 41V and/or 210D in the NP protein conferred improved RNP activity in H7N9 viruses and promoted the replication ability of H9N2 viruses, particularly at lower temperature. Substitutions at these two positions may promote the replication ability of H7N9 viruses in low temperature and thus might contribute to viral transmissibility. While these two residues have not yet been observed in H7N9 viruses, attention should be devoted to these two residues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-015-0304-6) contains supplementary material, which is available to authorized users.

Characterization of an H9N2 avian influenza virus from a Fringilla montifringilla brambling in northern China

Avian H9N2 influenza viruses circulating in domestic poultry populations are occasionally transmitted to humans. We report the genomic characterization of an H9N2 avian influenza virus (A/Brambling/Beijing/16/2012) first isolated from a healthy Fringilla montifringilla brambling in northern China in 2012. Phylogenetic analyses revealed that this H9N2 virus belongs to the BJ/94-like sublineage. This virus had a low pathogenicity for chickens and was able to replicate at a low level in mouse lung tissue. Transmission studies in ferrets showed that this H9N2 strain shed high levels of the virus in nasal and throat swabs. In vitro receptor binding assays, the virus bound only to α-2,6 linkage receptors and not to the avian-type α-2,3 linkage receptors, suggesting that H9N2 influenza viruses present potential public health risks. Therefore, attention should be paid to H9N2 influenza viruses and the close surveillance of H9N2 viruses in poultry.

Genetic diversity of early (1998) and recent (2010) avian influenza H9N2 virus strains isolated from poultry in Iran

Infection with avian influenza H9N2 virus is widespread in the Asian poultry industry, resulting in great economic losses due to mortality and a severe decline in egg production. To obtain more-comprehensive genomic data from circulating H9N2 viruses in Iran, we sequenced the whole genomes of early (Ck/IR/ZMT-101/98) and recent (Ck/IR/EBGV-88/10) isolates of this virus in Iran. The M and NS genes of Ck/IR/EBGV-88/10 shared a high level of similarity with a highly pathogenic H7N3 virus isolated from Pakistan. The cleavage site within the HA protein of these viruses contained two different motifs, RSSR and KSSR, which are similar to those found in low-pathogenic viruses. The deduced amino acid sequence of the new isolate contained the mutation Q226L, which is a characteristic of human-type sialic acid influenza receptor binding. An analysis of the viral amino acid sequence of the M2 protein of the recent strain revealed a V27A mutation, which is associated with amantadine resistance in avian influenza virus. The present results emphasize the need for continuous surveillance of H9N2 viruses in poultry and the human population to obtain more information about the nature and evolution of future pandemic influenza viruses.

Susceptibility and intra-species transmission of the H9N2 G1 prototype lineage virus in Japanese quail and turkeys

Avian influenza viruses of the H9N2 subtype have circulated in the poultry population in Asia, Far and Middle East since the mid-1990 s. One of the most widespread lineages established in poultry is the G1 lineage. This lineage has undergone further evolution and reassortment since its first detection in 1997 and G1-like H9N2 viruses still circulate. In this study we have investigated the susceptibility of quail and turkeys to the H9N2 G1-lineage prototype strain (A/quail/Hong Kong/G1/97). Contact transmission experiments were carried out in both avian species. Animals were infected oro-nasally with increasing doses of the virus (10(3)-10(6) EID 50/0.1 ml) and sentinel birds were introduced 4 days post infection (pi) in each experimental group. Quail were more susceptible than turkeys, as they were readily infected with lower challenge doses. Interestingly, infection of turkeys was associated with worse clinical condition. Transmission was detected in both species. Quail infected with a dose less than or equal to 10(4) EID50 transmitted the virus to the sentinels without showing any signs of disease. These findings reinforce the hypothesis that quail may ensure the perpetuation of H9N2 viruses in poultry, acting as a silent reservoir.

Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications.

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.

A Novel Genotype H9N2 Influenza Virus Possessing Human H5N1 Internal Genomes Has Been Circulating in Poultry in Eastern China since 1998

Many novel reassortant influenza viruses of the H9N2 genotype have emerged in aquatic birds in southern China since their initial isolation in this region in 1994. However, the genesis and evolution of H9N2 viruses in poultry in eastern China have not been investigated systematically. In the current study, H9N2 influenza viruses isolated from poultry in eastern China during the past 10 years were characterized genetically and antigenically. Phylogenetic analysis revealed that these H9N2 viruses have undergone extensive reassortment to generate multiple novel genotypes, including four genotypes (J, F, K, and L) that have never been recognized before. The major H9N2 influenza viruses represented by A/Chicken/Beijing/1/1994 (Ck/BJ/1/94)-like viruses circulating in poultry in eastern China before 1998 have been gradually replaced by A/Chicken/Shanghai/F/1998 (Ck/SH/F/98)-like viruses, which have a genotype different from that of viruses isolated in southern China. The similarity of the internal genes of these H9N2 viruses to those of the H5N1 influenza viruses isolated from 2001 onwards suggests that the Ck/SH/F/98-like virus may have been the donor of internal genes of human and poultry H5N1 influenza viruses circulating in Eurasia. Experimental studies showed that some of these H9N2 viruses could be efficiently transmitted by the respiratory tract in chicken flocks. Our study provides new insight into the genesis and evolution of H9N2 influenza viruses and supports the notion that some of these viruses may have been the donors of internal genes found in H5N1 viruses.

Is H9N2 Avian Influenza Virus a Pandemic Potential?

To the Editor: H1N1 and H3N2 influenza virus subtypes continue to cause human disease (1,2), while avian influenza A H5 and H7 subtypes spread globally among birds with limited an inefficient transmission to humans (3,4). Results from recent ferret research on the H9N2 subtype has demonstrated an increased theoretical threat to humans from the potential emergence of novel subtypes of avian influenza. In North America, there is no evidence of fixed lineages of the H9N2 avian influenza virus in land-based poultry; it is found in wild ducks and shore birds (5). In contrast, H9N2 remains endemic across Asia, mainly limited to outbreaks in domestic land-based poultry, but overshadowed as a pandemic threat by H5N1 bird flu, which has spread from Asia into Africa and Europe (6–9). However, there is evidence of interspecies transmission of H9N2 from land-based poultry to mammals, such as pigs and swine (10–13). Further evidence of an expanded mammalian host range includes efficient replication of H9N2 in mice without adaptation (14). H9N2 has already caused mild respiratory disease in humans in Hong Kong and mainland China in 1999 and 2003 (10,13,15,16). The six genes encoding the internal components of the H9N2 virus are similar to those found in a previous 1997 outbreak of H5N1 that caused several human deaths in Hong Kong (15). Furthermore, circulating H9N2 strains show human-like receptor specificity with amino acid leucine at position 226 at the receptor-binding site of human airway epithelial cells cultured in vitro (17). H9N2 isolated from live bird markets in Hong Kong possessed receptor specificity similar to human H3N2 viruses (18) and mutations similar to human H2N2 and H3N2 viruses, so the glycoproteins of the Hong Kong H9N2 viruses may potentially promote human infection. In a ferret model of transmission (9), the H9N2 avian reassortment subtype appears to be evolving. The H9N2 virus replicates in the respiratory tract of ferrets and can spread to noninfected ferrets (9). The amino acid leucine residue located at position 226 in the hemagglutinin receptor-binding site (instead of glutamine), plays a key role in human virus-like receptor specificity, and promotes transmission of the H9N2 virus in ferrets. Airborne transmission has not been detected. Mixing the H9N2 viral genes containing the surface glycoprotein and the six internal genes of a human H3N2 virus resulted in increased transmissibility. The model and reassortment mixing results raises concern about viral evolution as well as efficient pandemic transmission, and suggests that the H9N2 avian virus could be of pandemic importance (19). To conclude that H9N2 is the next human pandemic strain is premature at this time given the unfolding evidence. Yet, the possibility for competent nonavian intermediate reassorters generating novel and virulent pandemic strains of H9N2 (or other avain influenza strains) has increased given the recent raccoon influenza transmission findings (20). Additional study and timely surveillance of H9N2 is needed to identify any increments in viral adaptation to human beings. Studies should consider the widespread prevalence of the H9N2 virus in poultry, and co-circulation and mixing of avian H9N2 with human H3N2, H5, H7 and other avian and mammalian viruses.

Replication and Transmission of H9N2 Influenza Viruses in Ferrets: Evaluation of Pandemic Potential

H9N2 avian influenza A viruses are endemic in poultry of many Eurasian countries and have caused repeated human infections in Asia since 1998. To evaluate the potential threat of H9N2 viruses to humans, we investigated the replication and transmission efficiency of H9N2 viruses in the ferret model. Five wild-type (WT) H9N2 viruses, isolated from different avian species from 1988 through 2003, were tested in vivo and found to replicate in ferrets. However these viruses achieved mild peak viral titers in nasal washes when compared to those observed with a human H3N2 virus. Two of these H9N2 viruses transmitted to direct contact ferrets, however no aerosol transmission was detected in the virus displaying the most efficient direct contact transmission. A leucine (Leu) residue at amino acid position 226 in the hemagglutinin (HA) receptor-binding site (RBS), responsible for human virus-like receptor specificity, was found to be important for the transmission of the H9N2 viruses in ferrets. In addition, an H9N2 avian-human reassortant virus, which contains the surface glycoprotein genes from an H9N2 virus and the six internal genes of a human H3N2 virus, showed enhanced replication and efficient transmission to direct contacts. Although no aerosol transmission was observed, the virus replicated in multiple respiratory tissues and induced clinical signs similar to those observed with the parental human H3N2 virus. Our results suggest that the establishment and prevalence of H9N2 viruses in poultry pose a significant threat for humans.

Two lineages of H9N2 influenza viruses continue to circulate in land-based poultry in southeastern China

Background: The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1/97-like) internal genome complex is still present in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Methods: Virus isolation, serological identification and genetic analysis. Results: Two lineages of H9N2 influenza viruses were isolated in the live poultry markets and are represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97) and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97). No reassortment between the two H9N2 virus lineages was detected despite their co-circulation in the poultry markets. The Qa/HK/G1/97-like viruses were isolated most frequently from quail and Dk/HK/Y280/97-like viruses from chicken. Conclusion: The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to H5N1/97-like virus emphasizes the need for continued surveillance of mammals including humans for the H9N2 virus.

H9N2 Influenza Viruses Possessing H5N1-Like Internal Genomes Continue To Circulate in Poultry in Southeastern China

The transmission of H9N2 influenza viruses to humans and the realization that the A/Hong Kong/156/97-like (H5N1) (abbreviated HK/156/97) genome complex may be present in H9N2 viruses in southeastern China necessitated a study of the distribution and characterization of H9N2 viruses in poultry in the Hong Kong SAR in 1999. Serological studies indicated that H9N2 influenza viruses had infected a high proportion of chickens and other land-based birds (pigeon, pheasant, quail, guinea fowl, and chukka) from southeastern China. Two lineages of H9N2 influenza viruses present in the live-poultry markets were represented by A/Quail/Hong Kong/G1/97 (Qa/HK/G1/97)-like and A/Duck/Hong Kong/Y280/97 (Dk/HK/Y280/97)-like viruses. Up to 16% of cages of quail in the poultry markets contained Qa/HK/G1/97-like viruses, while about 5% of cages of other land-based birds were infected with Dk/HK/Y280/97-like viruses. No reassortant between the two H9N2 virus lineages was detected despite their cocirculation in the poultry markets. Reassortant viruses represented by A/Chicken/Hong Kong/G9/97 (H9N2) were the major H9N2 influenza viruses circulating in the Hong Kong markets in 1997 but have not been detected since the chicken slaughter in 1997. The Qa/HK/G1/97-like viruses were frequently isolated from quail, while Dk/HK/Y280/97-like viruses were predominately associated with chickens. The Qa/HK/G1/97-like viruses were evolving relatively rapidly, especially in their PB2, HA, NP, and NA genes, suggesting that they are in the process of adapting to a new host. Experimental studies showed that both H9N2 lineages were primarily spread by the aerosol route and that neither quail nor chickens showed evidence of disease. The high prevalence of quail infected with Qa/HK/G1/97-like virus that contains six gene segments genetically highly related to HK/156/97 (H5N1) virus emphasizes the need for surveillance of mammals including humans.