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Since the 2000s, the Y439 lineage of H9N2 avian influenza virus (AIV) has been the predominant strain circulating in poultry in Korea; however, in 2020, the Y280 lineage emerged and spread rapidly nationwide, causing large economic losses. To prevent further spread and circulation of such viruses, rapid detection and diagnosis through active surveillance programs are crucial. Here, we developed a novel H9 rRT-PCR assay that can detect a broad range of H9Nx viruses in situations in which multiple lineages of H9 AIVs are co-circulating. We then evaluated its efficacy using a large number of clinical samples. The assay, named the Uni Kor-H9 assay, showed high sensitivity for Y280 lineage viruses, as well as for the Y439 lineage originating in Korean poultry and wild birds. In addition, the assay showed no cross-reactivity with other subtypes of AIV or other avian pathogens. Furthermore, the Uni Kor-H9 assay was more sensitive, and had higher detection rates, than reference H9 rRT-PCR methods when tested against a panel of domestically isolated H9 AIVs. In conclusion, the novel Uni Kor-H9 assay enables more rapid and efficient diagnosis than the "traditional" method of virus isolation followed by subtyping RT-PCR. Application of the new H9 rRT-PCR assay to AI active surveillance programs will help to control and manage Korean H9 AIVs more efficiently., (© 2023. The Author(s), under exclusive licence to Microbiological Society of Korea.)

The H9N2 avian influenza (AI) has become endemic in poultry in many countries since the 1990s, which has caused considerable economic losses in the poultry industry. Considering the long history of the low pathogenicity H9N2 AI in many countries, once H9N2 AI is introduced, it is more difficult to eradicate than high pathogenicity AI. Various preventive measures and strategies, including vaccination and active national surveillance, have been used to control the Y439 lineage of H9N2 AI in South Korea, but it took a long time for the H9N2 virus to disappear from the fields. By contrast, the novel Y280 lineage of H9N2 AI was introduced in June 2020 and has spread nationwide. This study reviews the history, genetic and pathogenic characteristics, and control strategies for Korean H9N2 AI. This review may provide some clues for establishing control strategies for endemic AIV and a newly introduced Y280 lineage of H9N2 AI in South Korea., Competing Interests: The authors declare no conflicts of interest., (© 2023 The Korean Society of Veterinary Science.)

We report the first detection of Y280-lineage H9N2 avian influenza viruses in live bird markets in Korea during July 2020. The viruses were isolated from domestic ducks and chickens traded in three markets in two different provinces, indicating dispersal of the newly introduced viruses. Complete genome sequencing and comparative phylogenetic analyses of all eight gene segments of the viruses showed high nucleotide homology to a Y280-lineage H9N2 avian influenza virus isolated in a chicken farm in China, which belongs to one of the most prevalent H9N2 genotypes in China. Increasing human cases of the same genotype H9N2 infection in China and the mammalian specific markers present in the viruses isolated suggest potential implications for public health., (© 2021 Wiley-VCH GmbH.)

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health., Competing Interests: The authors declare no conflicts of interest., (© 2021 The Korean Society of Veterinary Science.)

Wild aquatic birds, a natural reservoir of avian influenza viruses (AIVs), transmit AIVs to poultry farms, causing huge economic losses. Therefore, the prevalence and genetic characteristics of AIVs isolated from wild birds in South Korea from October 2019 to March 2020 were investigated and analyzed. Fresh avian fecal samples (3256) were collected by active monitoring of 11 wild bird habitats. Twenty-eight AIVs were isolated. Seven HA and eight NA subtypes were identified. All AIV hosts were Anseriformes species. The HA cleavage site of 20 representative AIVs was encoded by non-multi-basic amino acid sequences. Phylogenetic analysis of the eight segment genes of the AIVs showed that most genes clustered within the Eurasian lineage. However, the HA gene of H10 viruses and NS gene of four viruses clustered within the American lineage, indicating intercontinental reassortment of AIVs. Representative viruses likely to infect mammals were selected and evaluated for pathogenicity in mice. JB21-58 (H5N3), JB42-93 (H9N2), and JB32-81 (H11N2) were isolated from the lungs, but JB31-69 (H11N9) was not isolated from the lungs until the end of the experiment at 14 dpi. None of infected mice showed clinical sign and histopathological change in the lung. In addition, viral antigens were not detected in lungs of all mice at 14 dpi. These data suggest that LPAIVs derived from wild birds are unlikely to be transmitted to mammals. However, because LPAIVs can reportedly infect mammals, including humans, continuous surveillance and monitoring of AIVs are necessary, despite their low pathogenicity.

The present study was conducted to monitor sales activity and immunogenicity of commercial H9N2 vaccines produced in Korea from 2007 to 2017. Recorded sales of H9N2 vaccine were around 671 million doses, with 10 million doses sold in 2007, rising to a peak of 93 million doses in 2016, with a slight fall in 2017. Multivalent combined vaccines made up around 90% of all vaccine sales, and around 30% of all vaccines were distributed by regional governments for free. The regional vaccination rate was the highest in Gyeonggi and Chungnam, respectively with proportional to the population of layer and breeder chickens. There have been no cases of field infection since 2009. The mean antibody titer was 5.82 log 2 across the study period. Our results suggest that continuous genetic monitoring of H9N2 viruses circulating in the field and updating the vaccine seed strain periodically are necessary in order to control H9N2 outbreaks., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Live bird markets (LBMs) in Korea have been recognized as a reservoir, amplifier, and source of avian influenza viruses (AIVs); however, little was known about the role of LBMs in the epidemiology of AIVs in Korea until recently. Through 10 years of surveillance (2006-2016) we have isolated and sequenced H9N2 viruses in Korean LBMs. To understand how H9N2 evolves and spreads in Korea, a statistical Bayesian phylogenetic model was used. Phylogenetic analysis suggests that three separate introductions of progenitor gene pools, Korean domestic duck-origin and two wild aquatic bird-origin AIVs, contributed to the generation of the five genotypes of H9N2 viruses in Korea. Phylogenetic reconstruction of ecological states infer that the LBMs are where chickens become infected with the virus, with domestic ducks playing a major role in the transmission and evolution of the H9N2 viruses. Three increases in the genetic diversity of H9N2 viruses were observed and coincided with transitions in host species and the locations (domestic farm, LBM, slaughterhouse, and wild aquatic bird habitat) where the viruses were isolated, accompanying genetic reassortment. Following the introduction of a wild aquatic bird-origin AIVs in 2008, six genes of the Korean lineage H9N2 virus were replaced with genes originating from wild aquatic birds, and viruses with this new genotype became predominant in Korean LBMs.

We isolated a serotype H3N2 influenza virus from a dog with severe respiratory distress in an animal clinic in South Korea in 2015 and characterized the sequences of its eight genes. The following seven genes were derived from canine influenza virus: PB2, PB1, HA, NP, NA, M, and NS. However, the PA gene was derived from avian H9N2 influenza virus that is circulating in poultry in Korea. These findings suggest that the continued surveillance of the influenza virus in dogs is warranted because humans have close contact with dogs, which may promote viral transmission.

Avian influenza A virus (AIV) is commonly isolated from domestic poultry and wild migratory birds, and the H9N2 subtype is the most prevalent and the major cause of severe disease in poultry in Korea. In addition to the veterinary concerns regarding the H9N2 subtype, it is also considered to be the next potential human pandemic strain due to its rapid evolution and interspecies transmission. In this study, we utilize serial lung-to-lung passage of a low pathogenic avian influenza virus (LPAI) H9N2 (A/Ck/Korea/163/04, WT163) (Y439-lineage) in mice to increase pathogenicity and investigate the potential virulence marker. Mouse-adapted H9N2 virus obtained high virulence (100% mortality) in mice after 98 serial passages. Sequence results show that the mouse adaptation (ma163) possesses several mutations within seven gene segments (PB2, PA, HA, NP, NA, M, and NS) relative to the wild-type strain. The HA gene showed the most mutations (at least 11) with one resulting in the loss of an N-glycosylation site (at amino acid 166). Moreover, reverse genetic studies established that an E627K substitution in PB2 and the loss of the N-glycosylation site in the HA protein (aa166) are critical virulence markers in the mouse-adapted H9N2 virus. Thus, these results add to the increasing body of mutational analysis data defining the function of the viral polymerase and HA genes and their roles in mammalian host adaptation. To our knowledge, this is first report of the generation of a mammalian-adapted Korea H9N2 virus (Y493-lineages). Therefore, this study offers valuable insights into the molecular evolution of the LPAI Korean H9N2 in a new host and adds to the current knowledge of the molecular markers associated with increased virulence.

In 2011, we isolated a natural recombinant H9N2 avian influenza virus from fecal droppings of bean goose (Anser fabalis) in Korea. Phylogenetic analyses showed that the A/bean goose/Korea/220/2011(H9N2) isolate is a reassortant of Eurasian and North American lineages of avian influenza virus. In addition, the complete genome sequence, including all 8 gene segments, was associated with Chinese H9N2 viruses isolated from wild birds in the Hunan East Dongting Lake National Nature Reserve. These data provide direct evidence for the exchange of avian influenza viruses between Korea and China via wild birds., (Copyright © 2014 Elsevier B.V. All rights reserved.)

We performed whole genome sequencing of 22 H9N2 avian influenza viruses (AIV) isolated from domestic laying hens on farms between 2005 and 2008, and compared the sequences with viruses previously reported in Asia. A previous study revealed that two antigenically distinct sublineages were established within the MS96 lineage by antigenic drift since the first H9N2 AIV outbreak in South Korea. We designated them as the 01310-like lineage and the 116/04-like lineage. Since late 2004, most identified isolates in Korea have belonged to the 116/04-like lineage, however, in this study we found that six among twenty-two isolates were belonged to 01310-like lineage, indicating that the genetic divergence is still occurring after 2004. Furthermore, it is noteworthy that five isolates among the defined 01310-like lineage had a 24 amino acid deletion in the neuraminidase stalk region, which were not in any other H9N2 isolates previously reported. The internal genes analysis demonstrated extensive reassortment events among isolates from poultry farms, live bird markets, and wild birds, and multiple new genotypes were identified. We identified several features of gene evolution in H9N2 AIV suggesting that the long-term H9N2 AIV surveillance study should be continued in South Korea.

An outbreak of avian influenza, caused by an H9N2 low-pathogenic avian influenza virus (AIV), occurred in a chicken farm and caused severe economic losses due to mortality and diarrhea. AIV was isolated and identified in a sample from an affected native Korean chicken. Genetic analysis of the isolate revealed a high sequence similarity to genes of novel reassortant H9N2 viruses isolated from slaughterhouses and live bird markets in Korea in 2008 and 2009. Animal challenge studies demonstrated that the replication kinetics and pathogenicity of the isolate were considerably altered due to adaptation in chickens. Vaccine protection studies indicated that commercial vaccine was not able to prevent virus shedding and clinical disease when chickens were challenged with the isolate. These results suggest that the novel H9N2 virus possesses the capacity to replicate efficiently in the respiratory system against vaccination and to cause severe disease in domestic chickens. The results also highlight the importance of appropriate updating of vaccine strains, based on continuous surveillance data, to prevent the possibility of a new H9N2 epidemic in Korea.

To investigate whether currently circulating H9N2 avian influenza viruses (AIVs) in domestic poultry have evolved in Korean poultry since 2007, genetic and serological comparisons were conducted of H9N2 isolates from poultry slaughterhouses from January 2008 to December 2009. The isolation rate was relatively low in 2008 but increased gradually from January 2009 onwards. Genetic and phylogenetic analyses revealed that reassortant viruses had emerged, generating at least five novel genotypes, mostly containing segments of a previously prevalent domestic H9N2 virus lineage (Ck/Korea/04116/04-like). It was noteworthy that the N2 genes of some H9N2 isolates (genotypes D, E and F) were derived from those of H3N2-like viruses commonly isolated among domestic ducks in live-poultry markets. Animal challenge studies demonstrated that the pathogenicity of Ck/Korea/SH0906/09 (genotype B) and Ck/Korea/SH0912/09 (genotype F) in domestic avian species was altered due to reassortment. Furthermore, serological analysis revealed that the isolates were antigenically distinct from previous Korean H9N2 viruses including Ck/Korea/01310/01. Such antigenic diversity was illustrated further in experiments using H9N2-immunized chickens, which could not inhibit the replication and transmission of challenge viruses from each genotype. These results suggest that H9N2 viruses from domestic poultry have undergone substantial evolution since 2007 by immune selection as a result of vaccinal and natural immunity, coupled with reassortment. Taken together, this study demonstrates that periodical updating of vaccine strains, based on continuous surveillance data, is an important issue in order to provide sufficient protectivity against AIV infections.

We characterized low pathogenic avian influenza (LPAI) H5N2 and H9N2 viruses isolated in South Korea from 2008 to 2009. Genetic analysis of the H5N2 viruses isolated from wild birds and domestic ducks demonstrated that they were related to the recently isolated southern Chinese LPAI H5 viruses and various influenza viruses circulating in Eurasia. Three H9N2 viruses obtained at live bird markets and duck farms were reassortant viruses generated from the H5N2 viruses of domestic ducks and the H9N2 virus endemic in Korean chickens. The H5N2 viruses did not replicate well in experimentally infected chickens and mice, but novel H9N2 viruses, without pre-adaptation, were recovered at high titres in chickens. Our results show that reassortment between H5N2 and H9N2 viruses must have occurred in domestic ducks and may have contributed to the diversity expansion of the gene pool, which has potential to alter the pathogenicity and host range of the influenza virus.

In order to control the H9N2 subtype low pathogenic avian influenza (LPAI), an inactivated vaccine has been used in Korea since 2007. The Korean veterinary authority permitted the use of a single H9N2 LPAI vaccine strain to simplify the evolution of the circulating virus due to the immune pressure caused by the vaccine use. It is therefore important to determine the suitability of the vaccine strain in the final inactivated oil emulsion LPAI vaccine. In this study, we applied molecular rather than biological methods to verify the suitability of the vaccine strain used in commercial vaccines and successfully identified the strain by comparing the nucleotide sequences of the hemagglutinin and neuraminidase genes with that of the permitted Korean LPAI vaccine strain. It is thought that the method used in this study might be successfully applied to other viral genes of the LPAI vaccine strain and perhaps to other veterinary oil emulsion vaccines.

Surveillance of H9 avian influenza viruses in Korean live-poultry markets from September 2004 through October 2007 was carried out to investigate active reassortment between wild migratory birds and domestic poultry in Korea. Antigenic and phylogenetic analyses showed that most of the isolates belong to the previous Korean H9N2-like lineage and differ from the southeastern Chinese strains. Interestingly, the Ck/Korea/LPM77/06 group (genotype B) and Dk/Korea/LPM248/07 group (genotype C) showed unique properties distinct from those of other Korean H9N2 strains. Although the HA genes of these two groups belong to Korean H9N2-like lineage, the PA genes closely resemble those of the Chinese Y280-like lineage. In addition, the PB2 genes of the Dk/Korea/LPM248/07 group were closely related to those isolated from migratory birds. Several other isolates also clustered within the H9N2 B genotype, an indication that there are at least two predominant H9N2 influenza genotypes in Korea. Another isolate, Dk/Korea/LPM71/06, was identified as an H9N1 subtype, the first ever discovered in Korean live-poultry markets. These findings reveal that reassortment of Korean H9 influenza viruses has occurred frequently in live-poultry markets and may have been mediated by introduction of genetic material from viruses circulating among migratory wild birds to domestic birds. Consequently, the new dominant H9N2 genotypes have become established in Korean live-poultry markets through continued reassortment.

We report the genetic characterization of H6 avian influenza (AI) viruses isolated from domestic ducks and wild birds in Korea between April 2008 and April 2009. A phylogenetic analysis showed that the H6N1 viruses of wild birds and domestic ducks were of the same genotype (K-1) and were similar to the H6N1 virus isolated from a live poultry market in 2003, as six of the eight gene segments of those viruses had a common source. However, the H6N2 viruses of domestic poultry were separated into four genotypes (K-2a, K-2b, K-2c and K-2d) by at least a triple reassortment between influenza viruses of low pathogenicity from Korean poultry (H9N2 and H3N2) and viruses from aquatic birds. In an experimental infection of animals, certain H6 AI viruses replicated well in chickens and mice without pre-adaptation, indicating that H6 virus pathogenicity has the potential to be altered due to multiple reassortments, and that these reassortments could result in interspecies transmission to mammals.