Concurrent Infection with Clade 2.3.4.4b Highly Pathogenic Avian Influenza H5N6 and H5N1 Viruses, South Korea, 2023.

Highly pathogenic avian influenza H5N6 and H5N1 viruses of clade 2.3.4.4b were simultaneously introduced into South Korea at the end of 2023. An outbreak at a broiler duck farm consisted of concurrent infection by both viruses. Sharing genetic information and international surveillance of such viruses in wild birds and poultry is critical.

Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

Highly Pathogenic Avian Influenza A(H5N6) in Domestic Cats, South Korea.

In December 2016, highly pathogenic avian influenza (HPAI) infection with systemic pathologic lesions was found in cats in South Korea. Genetic analyses indicated that the feline isolates were similar to HPAI H5N6 viruses isolated in chicken farms nearby. This finding highlights the need for monitoring of domestic mammals during HPAI outbreaks.

Evaluation of the Immune Responses to and Cross-Protective Efficacy of Eurasian H7 Avian Influenza Viruses.

Due to increasing concerns about human infection by various H7 influenza viruses, including recent H7N9 viruses, we evaluated the genetic relationships and cross-protective efficacies of three different Eurasian H7 avian influenza viruses. Phylogenic and molecular analyses revealed that recent Eurasian H7 viruses can be separated into two different lineages, with relatively high amino acid identities within groups (94.8 to 98.8%) and low amino acid identities between groups (90.3 to 92.6%). In vivo immunization with representatives of each group revealed that while group-specific cross-reactivity was induced, cross-reactive hemagglutination inhibition (HI) titers were approximately 4-fold lower against heterologous group viruses than against homologous group viruses. Moreover, the group I (RgW109/06) vaccine protected 100% of immunized mice from various group I viruses, while only 20 to 40% of immunized mice survived lethal challenge with heterologous group II viruses and exhibited high viral titers in the lung. Moreover, while the group II (RgW478/14) vaccine also protected mice from lethal challenge with group II viruses, it failed to elicit cross-protection against group I viruses. However, it is noteworthy that vaccination with RgAnhui1/13, a virus of a sublineage of group I, cross-protected immunized mice against lethal challenge with both group I and II viruses and significantly attenuated lung viral titers. Interestingly, immune sera from RgAnhui1/13-vaccinated mice showed a broad neutralizing spectrum rather than the group-specific pattern observed with the other viruses. These results suggest that the recent human-infective H7N9 strain may be a candidate broad cross-protective vaccine for Eurasian H7 viruses.IMPORTANCE Genetic and phylogenic analyses have demonstrated that the Eurasian H7 viruses can be separated into at least two different lineages, both of which contain human-infective fatal H7 viruses, including the recent novel H7N9 viruses isolated in China since 2013. Due to the increasing concerns regarding the global public health risk posed by H7 viruses, we evaluated the genetic relationships between Eurasian H7 avian influenza viruses and the cross-protective efficacies of three different H7 viruses: W109/06 (group I), W478/14 (group II), and Anhui1/13 (a sublineage of group I). While each vaccine induced group-specific antibody responses and cross-protective efficacy, only Anhui1/13 was able to cross-protect immunized hosts against lethal challenge across groups. In fact, the Anhui1/13 virus induced not only cross-protection but also broad serum neutralizing antibody responses against both groups of viruses. This suggests that Anhui1/13-like H7N9 viruses may be viable vaccine candidates for broad protection against Eurasian H7 viruses.

H13 influenza viruses in wild birds have undergone genetic and antigenic diversification in nature.

Among 16 haemagglutinin (HA) subtypes of avian influenza viruses (AIVs), H13 AIVs have rarely been isolated in wild waterfowl. H13 AIVs cause asymptomatic infection and are maintained mainly in gull and tern populations; however, the recorded antigenic information relating to the viruses has been limited. In this study, 2 H13 AIVs, A/duck/Hokkaido/W345/2012 (H13N2) and A/duck/Hokkaido/WZ68/2012 (H13N2), isolated from the same area in the same year in our surveillance, were genetically and antigenically analyzed with 10 representative H13 strains including a prototype strain, A/gull/Maryland/704/1977 (H13N6). The HA genes of H13 AIVs were phylogenetically divided into 3 groups (I, II, and III). A/duck/Hokkaido/W345/2012 (H13N2) was genetically classified into Group III. This virus was distinct from a prototype strain, A/gull/Maryland/704/1977 (H13N6), and the virus, A/duck/Hokkaido/WZ68/2012 (H13N2), both belonging to Group I. Antigenic analysis indicated that the viruses of Group I were antigenically closely related to those of Group II, but distinct from those of Group III, including A/duck/Hokkaido/W345/2012 (H13N2). In summary, our study indicates that H13 AIVs have undergone antigenic diversification in nature.

Pathogenicity and genetic characterisation of a novel reassortant, highly pathogenic avian influenza (HPAI) H5N6 virus isolated in Korea, 2017.

We investigated influenza A(H5N6) viruses from migratory birds in Chungnam and Gyeonggi Provinces, South Korea following a reported die-off of poultry in nearby provinces in November 2017. Genetic analysis and virulence studies in chickens and ducks identified our isolate from December 2017 as a novel highly pathogenic avian influenza virus. It resulted from reassortment between the highly virulent H5N8 strain from Korea with the N6 gene from a low-pathogenic H3N6 virus from the Netherlands.

Surveillance of avian influenza viruses in South Korea between 2012 and 2014.

BACKGROUND: National surveillance of avian influenza virus (AIV) in South Korea has been annually conducted for the early detection of AIV and responses to the introduction of highly pathogenic avian influenza (HPAI) virus. In this study, we report on a nationwide surveillance study of AIV in domestic poultry and wild birds in South Korea between 2012 and 2014. METHODS: During the surveillance programs between 2012 and 2014, 141,560 samples were collected. Of these, 102,199 were from poultry farms, 8215 were from LBMs, and 31,146 were from wild bird habitats. The virus isolation was performed by inoculation of embryonated chicken eggs and AIV isolates were detected using hemagglutination assay. For subtying of AIV, the hemagglutinin and neuraminidase genes were confirmed by sequencing. Phylogenetic analysis of the H5 subtypes was performed using 28 H5 AIV isolates. RESULTS: Between 2012 and 2014, a total of 819 AIV were isolated from 141,560 samples. Virus isolation rates for AIV were 0.6, 0.4, 0.1, and 2.7% in wild birds (n = 202), domestic ducks (n = 387), minor poultry (n = 11), and the live bird market (LBM) (n = 219), respectively. In wild birds, various subtypes were found including H1-H7 and H9-H13. The major subtypes were H5 (n = 48, 23.9%: N3 (n = 4) and N8 (n = 44)), H4 (n = 39, 19.4%), and H1 (n = 29, 14.4%). In domestic poultry, mainly ducks, the H5N8 (n = 275, 59.3%), H3 (n = 30, 17.2%), and H6 (n = 53, 11.4%) subtypes were predominantly found. The most frequently detected subtypes in LBM, primarily Korean native chicken, were H9 (n = 169, 77.2%). H3 (n = 10, 4%) and H6 (n = 30, 13.7%) were also isolated in LBM. Overall, the prevalence of AIV was found to be higher between winter and spring and in western parts of South Korea. The unusual high prevalence of the H5 subtype of AIV was due to the large scale outbreak of H5N8 HPAI in wild birds and domestic poultry in 2014. CONCLUSIONS: Enhanced surveillance and application of effective control measures in wild birds and domestic poultry, including LBM, should be implemented to control AI and eradicate HPAI.

Experimental infection of SPF and Korean native chickens with highly pathogenic avian influenza virus (H5N8).

In 2014, an H5N8 outbreak of highly pathogenic avian influenza (HPAI) occurred in South Korea. The H5N8 strain produced mild to moderate clinical signs and mortality rates in commercial chicken farms, especially Korean native chicken farms. To understand the differences between their pathogenicity in SPF chicken and Korean native chicken., we evaluated the mean bird lethal doses (BLD50) of the Korean representative H5N8 virus (A/broiler duck/Korea/Buan2/2014) The BLD50values of the H5N8 virus were 10(5.3)EID50 and 10(6.7)EID50 in SPF and Korean native chickens, respectively. In addition, the mean death time was much longer, and the viral titers in tissues of H5N8-infected chickens were significantly lower, in the Korean group than in the SPF group. These features of the H5N8 virus likely account for its mild-to-moderate pathogenicity in commercial chicken farms, especially Korean native chicken flocks, despite the fact that it is a highly pathogenic virus according to the OIE criteria. To improve current understanding and management of HPAI, pathogenic characterization of novel emerging viruses should be performed by natural route in major poultry species in each country.

Pathologic Changes in Wild Birds Infected with Highly Pathogenic Avian Influenza A(H5N8) Viruses, South Korea, 2014.

In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus.

Novel reassortant influenza A(H5N8) viruses among inoculated domestic and wild ducks, South Korea, 2014.

An outbreak of highly pathogenic avian influenza, caused by a novel reassortant influenza A (H5N8) virus, occurred among poultry and wild birds in South Korea in 2014. The aim of this study was to evaluate the pathogenesis in and mode of transmission of this virus among domestic and wild ducks. Three of the viruses had similar pathogenicity among infected domestic ducks: the H5N8 viruses were moderately pathogenic (0%-20% mortality rate); in wild mallard ducks, the H5N8 and H5N1 viruses did not cause severe illness or death; viral replication and shedding were greater in H5N8-infected mallards than in H5N1-infected mallards. Identification of H5N8 viruses in birds exposed to infected domestic ducks and mallards indicated that the viruses could spread by contact. We propose active surveillance to support prevention of the spread of this virus among wild birds and poultry, especially domestic ducks.

Supplementation of influenza split vaccines with conserved M2 ectodomains overcomes strain specificity and provides long-term cross protection.

Current influenza vaccines do not provide good protection against antigenically different influenza A viruses. As an approach to overcome strain specificity of protection, this study demonstrates significantly improved long-term cross protection by supplementing split vaccines with a conserved molecular target, a repeat of the influenza M2 ectodomain (M2e) expressed on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Intramuscular immunization with H1N1 split vaccine (A/California/07/2009) supplemented with M2e5x VLPs induced M2e-specific humoral and cellular immune responses, and shaped the host responses to the vaccine in the direction of T-helper type 1 responses inducing dominant IgG2a isotype antibodies as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge, M2e5x VLP-supplemented vaccination lowered lung viral loads and induced long-term cross protection against H3N2 or H5N1 subtype influenza viruses over 12 months. M2e antibodies, CD4 T cells, and CD8 T cells were found to contribute to improving heterosubtypic cross protection. In addition, improved cross protection by supplemented vaccination with M2e5x VLPs was mediated via Fc receptors. The results support evidence that supplementation with M2e5x VLPs is a promising approach for overcoming the limitation of strain-specific protection by current influenza vaccination.

Genetic evolution of H5 highly pathogenic avian influenza virus in domestic poultry in Vietnam between 2011 and 2013.

In spite of highly pathogenic avian influenza H5N1 vaccination campaigns for domestic poultry, H5N1 viruses continue to circulate in Vietnam. To estimate the prevalence of avian influenza virus in Vietnam, surveillance was conducted between November 2011 and February 2013. Genetic analysis of 312 highly pathogenic avian influenza H5 viruses isolated from poultry in Vietnam was conducted and possible genetic relationships with strains from neighboring countries were investigated. As previously reported, phylogenetic analysis of the avian influenza virus revealed two H5N1 HPAI clades that were circulating in Vietnam. Clade 1.1, related to Cambodian strains, was predominant in the southern provinces, while clade 2.3.2.1 viruses were predominant in the northern and central provinces. Sequence analysis revealed evidence of active genetic evolution. In the gene constellation of clade 2.3.2.1, genotypes A, B, and B(II) existed during the 2011/2012 winter season. In June 2012, new genotype C emerged by reassortment between genotype A and genotype B(II), and this genotype was predominant in 2013 in the northern and central provinces. Interestingly, enzootic Vietnamese clade 2.3.2.1C H5 virus subsequently reassorted with N2, which originated from wild birds, to generate H5N2 highly pathogenic avian influenza, which was isolated from duck in the northeast region. This investigation indicated that H5N1 outbreaks persist in Vietnam and cause genetic reassortment with circulating viruses. It is necessary to strengthen active influenza surveillance to eradicate highly pathogenic avian influenza viruses and sever the link between highly pathogenic avian influenza and other circulating influenza viruses.

Genetic and pathogenic characteristics of H1 avian and swine influenza A viruses.

This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation. The titres of domestic duck viruses in chickens were much higher than those of wild-bird viruses. Both domestic duck and wild-bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titres in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.

Virus-like particles containing multiple M2 extracellular domains confer improved cross-protection against various subtypes of influenza virus.

The extracellular domain of M2 (M2e), a small ion channel membrane protein, is well conserved among different human influenza A virus strains. To improve the protective efficacy of M2e vaccines, we genetically engineered a tandem repeat of M2e epitope sequences (M2e5x) of human, swine, and avian origin influenza A viruses, which was expressed in a membrane-anchored form and incorporated in virus-like particles (VLPs). The M2e5x protein with the transmembrane domain of hemagglutinin (HA) was effectively incorporated into VLPs at a several 100-fold higher level than that on influenza virions. Intramuscular immunization with M2e5x VLP vaccines was highly effective in inducing M2e-specific antibodies reactive to different influenza viruses, mucosal and systemic immune responses, and cross-protection regardless of influenza virus subtypes in the absence of adjuvant. Importantly, immune sera were found to be sufficient for conferring protection in naive mice, which was long-lived and cross-protective. Thus, molecular designing and presenting M2e immunogens on VLPs provide a promising platform for developing universal influenza vaccines without using adjuvants.

Molecular epidemiology of Newcastle disease viruses in Vietnam.

Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in Southeast Asia. In the present study, 12 field isolates of NDV were recovered from dead village chickens in Vietnam between 2007 and 2012, and were characterized. All the field isolates were classified as velogenic. Based on the sequence analysis of the F variable region, two distinct genetic groups (Vietnam genetic groups G1 and G2) were recognized. Phylogenetic analysis revealed that all the 12 field isolates fell into the class II genotype VII cluster. Ten of the field isolates, classified as Vietnam genetic group G1, were closely related to VIIh viruses that had been isolated from Indonesia, Malaysia, and Cambodia since the mid-2000s, while the other two field isolates, of Vietnam genetic group G2, clustered with VIId viruses, which were predominantly circulating in China and Far East Asia. Our results indicate that genotype VII viruses, especially VIIh viruses, are predominantly responsible for the recent epizootic of the disease in Vietnam.

Research Note: Comparative evaluation of pathogenicity in SPF chicken between different subgroups of H5N6 high pathogenicity avian influenza viruses.

Since 2014, periodic outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4 H5 HPAI virus (HPAIV) have resulted in huge economic losses in the Korean poultry industry. During the winter season of 2016-2017, clade 2.3.4.4e H5N6 HPAIVs classified into 5 subgroups (C1-5) were introduced into South Korea. Interestingly, it was revealed that the subgroup C2 and C4 viruses were predominantly distributed throughout the country, whereas detection of the subgroup C3 viruses was confined in a specific local region. In the present study, we conducted comparative evaluation of the pathogenicity of viruses belonging to subgroups C2 and C3 (H15 and HN1 strains) in specific pathogen-free (SPF) chickens, and further compared them with previously determined pathogenicity of subgroup C4 (ES2 strain) virus. The HN1 strain showed lower viral replication in tissues, less transmissibility, and higher mean chicken lethal dose than the H15 and ES2 strains in SPF chickens. Considering that the HN1 strain has a different NS gene segment from the H15 and ES2 strains, the reassortment of the NS gene segment likely affects their infectivity and transmissibility in chickens. These findings emphasize the importance of monitoring the genetic characteristics and pathogenic features of HPAIVs to effectively control their outbreaks in the field.

Development and evaluation of a multiplex real-time RT-PCR assay for simultaneous detection of H5, H7, and H9 subtype avian influenza viruses.

H5, H7 and H9 are the major subtypes of avian influenza virus (AIV) that cause economic losses in the poultry industry and sporadic zoonotic infection. Early detection of AIV is essential for preventing disease spread. Therefore, molecular diagnosis and subtyping of AIV via real-time RT-PCR (rRT-PCR) is preferred over other classical diagnostic methods, such as egg inoculation, RT-PCR and HI test, due to its high sensitivity, specificity and convenience. The singleplex rRT-PCRs for the Matrix, H5 and H7 gene used for the national surveillance program in Korea have been developed in 2017; however, these methods were not designed for multiplexing, and does not reflect the sequences of currently circulating strains completely. In this study, the multiplex H5/7/9 rRT-PCR assay was developed with sets of primers and probe updated or newly designed to simultaneously detect the H5, H7 and H9 genes. Multiplex H5/7/9 rRT-PCR showed 100% specificity without cross-reactivity with other subtypes of AIVs and avian disease-causing viruses or bacteria, and the limit of detection was 1-10 EID50/0.1 ml (50% egg infectious dose). Artificial mixed infections with the three different subtypes could be detected accurately with high analytical sensitivity even under highly biased relative molecular ratios by balancing the reactivities of each subtype by modifying the concentration of the primers and probes. The multiplex H5/7/9 rRT-PCR assay developed in this study could be a useful tool for large-scale surveillance programs for viral detection as well as subtyping due to its high specificity, sensitivity and robustness in discriminating viruses in mixed infections, and this approach would greatly decrease the time, cost, effort and chance of cross-contamination compared to the conventional method of testing three subtypes by different singleplex rRT-PCR methods in parallel or in series.

Surveillance and Genetic Analysis of Low-Pathogenicity Avian Influenza Viruses Isolated from Feces of Wild Birds in Mongolia, 2021 to 2023.

The introduction of novel highly pathogenic (HPAI) viruses into Korea has been attributed to recombination events occurring at breeding sites in the Northern Hemisphere. This has increased interest in monitoring and genetically analyzing avian influenza viruses (AIVs) in northern regions, such as Mongolia, which share migratory bird flyways with Korea. AIVs in Mongolia were monitored by analyzing 10,149 fecal samples freshly collected from wild birds from April to October in 2021 to 2023. The prevalence of AIVs in wild birds was 1.01%, with a total of 77 AIVs isolated during these 3 years. These 77 AIVs included hemagglutinin (HA) subtypes H1, H2, H3, H4, H6, H10 and H13 and neuraminidase (NA) subtypes N1, N2, N3, N6, N7 and N8. The most frequently detected subtype combinations were H3N8 (39.0%) and H4N6 (19.5%), although HPAI viruses were not detected. Genetic analysis indicated that theses AIVs isolated from Mongolian samples were closely related to AIVs in wild birds in Korea, including those of Eurasian lineage. These findings indicate the necessity of continuous AIV surveillance and monitoring, as HPAI viruses introduced into Korea may derive from strains in Mongolia.

Genetic and antigenic characteristics of H4 subtype avian influenza viruses in Korea and their pathogenicity in quails, domestic ducks and mice.

In Korea, a nationwide surveillance programme was implemented in 2003 to identify highly pathogenic avian influenza viruses (AIVs). AIVs belonging to one of the most common haemagglutinin subtypes, H4, were isolated from two domestic ducks and 52 wild birds between 2004 and 2010. These H4 AIVs could be further classified into three neuraminidase subtypes: H4N6 (94.4%), H4N2 (3.7%) and H4N3 (1.9%). Phylogenetic analysis revealed that the H4 AIVs had a variety of genetic constellations, with at least nine different genotypes represented. The pathogenicity of these H4 viruses was assessed in quails, domestic ducks and mice. None of the H4 AIVs induced clinical signs in quails or domestic ducks. Viral shedding in quails was relatively high, and virus was recovered up to 5-7 days post-inoculation (p.i.) in oropharyngeal swabs, but the viruses replicated poorly in domestic ducks. Quails may act as an intermediate host in which AIVs are amplified and transmitted to other species. In mice, all of the AIVs were recovered efficiently at relatively high titres from the lungs up to 7 days p.i., demonstrating the potential for AIVs to infect mice directly without prior adaptation. None of the AIVs induced clinical signs nor was any lethal to infected mice. However, there was significant loss of body weight in mice infected with viruses of duck origin. It is suggested that the active surveillance of influenza viruses needs to be enhanced in domestic poultry as well as in wild birds, and that it should include assessment of pathogenicity in animal models.

Molecular epidemiological investigation of velogenic Newcastle disease viruses from village chickens in Cambodia.

Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.