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.

Novel Avian Influenza A(H5N6) Virus in Wild Birds, South Korea, 2023.

We isolated novel reassortant avian influenza A(H5N6) viruses containing genes from clade 2.3.4.4b H5N1 virus and low pathogenicity avian influenza viruses in carcasses of whooper swans and bean geese in South Korea during December 2023. Neuraminidase gene was from a clade 2.3.4.4b H5N6 virus infecting poultry and humans in China.

Seroprevalence of Avian Influenza A(H5N6) Virus Infection, Guangdong Province, China, 2022.

In 2022, we assessed avian influenza A virus subtype H5N6 seroprevalence among the general population in Guangdong Province, China, amid rising numbers of human infections. Among the tested samples, we found 1 to be seropositive, suggesting that the virus poses a low but present risk to the general population.

Emerging Novel Reassortant Influenza A(H5N6) Viruses in Poultry and Humans, China, 2021.

A novel highly pathogenic avian influenza A(H5N6) clade 2.3.4.4b virus was isolated from a poultry market in China that a person with a confirmed case had visited. Most genes of the avian and human H5N6 isolates were closely related. The virus also exhibited distinct antigenicity to the Re-11 vaccine strain.

Epidemiologic, Clinical, and Genetic Characteristics of Human Infections with Influenza A(H5N6) Viruses, China.

The recent rise in the frequency of influenza A(H5N6) infections in China has raised serious concerns about whether the risk for human infection has increased. We surveyed epidemiologic, clinical, and genetic data of human infections with A(H5N6) viruses. Severe disease occurred in 93.8% of cases, and the fatality rate was 55.4%. Median patient age was 51 years. Most H5N6 hemagglutinin (HA) genes in human isolates in 2021 originated from subclade 2.3.4.4b; we estimated the time to most recent common ancestor as June 16, 2020. A total of 13 genotypes with HA genes from multiple subclades in clade 2.3.4.4 were identified in human isolates. Of note, 4 new genotypes detected in 2021 were the major causes of increased H5N6 virus infections. Mammalian-adapted mutations were found in HA and internal genes. Although we found no evidence of human-to-human transmission, continuous evolution of H5N6 viruses may increase the risk for human infections.

Novel Reassortant Avian Influenza A(H5N6) Virus, China, 2021.

Although reports of human infection with influenza A(H5N6) increased in 2021, reports of similar H5N6 virus infection in poultry are few. We detected 10 avian influenza A(H5N6) clade 2.3.4.4b viruses in poultry from 4 provinces in China. The viruses showed strong immune-escape capacity and complex genetic reassortment, suggesting further transmission risk.

Susceptibility of common family Anatidae bird species to clade 2.3.4.4e H5N6 high pathogenicity avian influenza virus: an experimental infection study.

BACKGROUND: There were large outbreaks of high pathogenicity avian influenza (HPAI) caused by clade 2.3.4.4e H5N6 viruses in the winter of 2016-2017 in Japan, which caused large numbers of deaths among several endangered bird species including cranes, raptors, and birds in Family Anatidae. In this study, susceptibility of common Anatidae to a clade 2.3.4.4e H5N6 HPAI virus was assessed to evaluate their potential to be a source of infection for other birds. Eurasian wigeons (Mareca penelope), mallards (Anas platyrhynchos), and Northern pintails (Anas acuta) were intranasally inoculated with 106, 104, or 102 50% egg infectious dose (EID50) of clade 2.3.4.4e A/teal/Tottori/1/2016 (H5N6). RESULTS: All birds survived for 10 days without showing any clinical signs of infection. Most ducks inoculated with ≥ 104 EID50 of virus seroconverted within 10 days post-inoculation (dpi). Virus was mainly shed via the oral route for a maximum of 10 days, followed by cloacal route in late phase of infection. Virus remained in the pancreas of some ducks at 10 dpi. Viremia was observed in some ducks euthanized at 3 dpi, and ≤ 106.3 EID50 of virus was recovered from systemic tissues and swab samples including eyeballs and conjunctival swabs. CONCLUSIONS: These results indicate that the subject duck species have a potential to be a source of infection of clade 2.3.4.4e HPAI virus to the environment and other birds sharing their habitats. Captive ducks should be reared under isolated or separated circumstances during the HPAI epidemic season to prevent infection and further viral dissemination.

Highly Pathogenic Avian Influenza Clade 2.3.4.4 Subtype H5N6 Viruses Isolated from Wild Whooper Swans, Mongolia, 2020.

We identified clade 2.3.4.4 highly pathogenic avian influenza A(H5N6) viruses from whooper swans (Cygnus cygnus) found dead in Mongolia. The identification of these infections in wild birds in this area is of concern because of the potential for virus dissemination during fall migration.

Highly Pathogenic Avian Influenza A(H5N6) Virus Clade 2.3.4.4h in Wild Birds and Live Poultry Markets, Bangladesh.

Migratory birds play a major role in spreading influenza viruses over long distances. We report highly pathogenic avian influenza A(H5N6) viruses in migratory and resident ducks in Bangladesh. The viruses were genetically similar to viruses detected in wild birds in China and Mongolia, suggesting migration-associated dissemination of these zoonotic pathogens.

Emergence of a young case infected with avian influenza A (H5N6) in Anhui Province, East China during the COVID-19 pandemic.

In the context of the coronavirus disease 2019 pandemic, we investigated the epidemiological and clinical characteristics of a young patient infected by avian influenza A (H5N6) virus in Anhui Province, East China, and analyzed genomic features of the pathogen in 2020. Through the cross-sectional investigation of external environment monitoring (December 29-31, 2020), 1909 samples were collected from Fuyang City. It was found that the positive rate of H5N6 was higher than other areas obviously in Tianma poultry market, where the case appeared. In addition, dual coinfections were detected with a 0.057% polymerase chain reaction positive rate the surveillance years. The virus was the clade 2.3.4.4, which was most likely formed by genetic reassortment between H5N6 and H9N2 viruses. This study found that the evolution rates of the hemagglutinin and neuraminidase genes of the virus were higher than those of common seasonal influenza viruses. The virus was still highly pathogenic to poultry and had a preference for avian receptor binding.

N-linked glycosylation at site 158 of the HA protein of H5N6 highly pathogenic avian influenza virus is important for viral biological properties and host immune responses.

Since 2014, clade 2.3.4.4 has become the dominant epidemic branch of the Asian lineage H5 subtype highly pathogenic avian influenza virus (HPAIV) in southern and eastern China, while the H5N6 subtype is the most prevalent. We have shown earlier that lack of glycosylation at position 158 of the hemagglutinin (HA) glycoprotein due to the T160A mutation is a key determinant of the dual receptor binding property of clade 2.3.4.4 H5NX subtypes. Our present study aims to explore other effects of this site among H5N6 viruses. Here we report that N-linked glycosylation at site 158 facilitated the assembly of virus-like particles and enhanced virus replication in A549, MDCK, and chicken embryonic fibroblast (CEF) cells. Consistently, the HA-glycosylated H5N6 virus induced higher levels of inflammatory factors and resulted in stronger pathogenicity in mice than the virus without glycosylation at site 158. However, H5N6 viruses without glycosylation at site 158 were more resistant to heat and bound host cells better than the HA-glycosylated viruses. H5N6 virus without glycosylation at this site triggered the host immune response mechanism to antagonize the viral infection, making viral pathogenicity milder and favoring virus spread. These findings highlight the importance of glycosylation at site 158 of HA for the pathogenicity of the H5N6 viruses.

Genetic characterization of an H5N6 avian influenza virus with multiple origins from a chicken in southern China, October 2019.

BACKGROUND: Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype pose a great threat to the poultry industry and human health. In recent years, H5N6 subtype has rapidly replaced H5N1 as the most predominate HPAIV subtype circulating in domestic poultry in China. In this study, we describe the genetic and phylogenetic characteristics of a prevalent H5N6 strain in Guangdong, China. RESULTS: Nucleotide sequencing identified a H5N6 subtype HPAIV, designated as A/chicken/Dongguan/1101/2019 (DG/19), with a multibasic cleavage site in the hemagglutinin (HA). Phylogenetic analysis revealed DG/19 was a reassortant of H5N1, H5N2, H5N8, and H6N6 subtypes of avian influenza viruses. A number of mammalian adaptive markers such as D36N in the HA were identified. CONCLUSIONS: Our results showed that HPAIV H5N6 strains still emerge in well-managed groups of chicken farms. Considering the increasing prevalence of H5N6 HPAIV, and the fact that H5N6 HPAIVs are well adapted to migratory birds, an enhanced surveillance for the East Asian-Australasian flyway should be undertaken to prevent potential threats to the poultry industry and human health.

Plant-based, adjuvant-free, potent multivalent vaccines for avian influenza virus via Lactococcus surface display.

Influenza epidemics frequently and unpredictably break out all over the world, and seriously affect the breeding industry and human activity. Inactivated and live attenuated viruses have been used as protective vaccines but exhibit high risks for biosafety. Subunit vaccines enjoy high biosafety and specificity but have a few weak points compared to inactivated virus or live attenuated virus vaccines, especially in low immunogenicity. In this study, we developed a new subunit vaccine platform for a potent, adjuvant-free, and multivalent vaccination. The ectodomains of hemagglutinins (HAs) of influenza viruses were expressed in plants as trimers (tHAs) to mimic their native forms. tHAs in plant extracts were directly used without purification for binding to inactivated Lactococcus (iLact) to produce iLact-tHAs, an antigen-carrying bacteria-like particle (BLP). tHAs BLP showed strong immune responses in mice and chickens without adjuvants. Moreover, simultaneous injection of two different antigens by two different formulas, tHAH5N6 + H9N2 BLP or a combination of tHAH5N6 BLP and tHAH9N2 BLP, led to strong immune responses to both antigens. Based on these results, we propose combinations of plant-based antigen production and BLP-based delivery as a highly potent and cost-effective platform for multivalent vaccination for subunit vaccines.

Outbreaks of Highly Pathogenic Avian Influenza (H5N6) Virus Subclade 2.3.4.4h in Swans, Xinjiang, Western China, 2020.

In January 2020, the subclade 2.3.4.4h of highly pathogenic avian influenza (H5N6) virus infected migratory whooper swans and mute swans in Xinjiang, western China. The virus is lethal to chickens and ducks but has low pathogenicity in mice. Antigenically, this subclade is similar to the H5N1 vaccine seed virus Re-11.

Efficacy of Neuraminidase Inhibitors against H5N6 Highly Pathogenic Avian Influenza Virus in a Nonhuman Primate Model.

Attention has been paid to H5N6 highly pathogenic avian influenza virus (HPAIV) because of its heavy burden on the poultry industry and human mortality. Since an influenza A virus carrying N6 neuraminidase (NA) has never spread in humans, the potential for H5N6 HPAIV to cause disease in humans and the efficacy of antiviral drugs against the virus need to be urgently assessed. We used nonhuman primates to elucidate the pathogenesis of H5N6 HPAIV as well as to determine the efficacy of antiviral drugs against the virus. H5N6 HPAIV infection led to high fever in cynomolgus macaques. The lung injury caused by the virus was severe, with diffuse alveolar damage and neutrophil infiltration. In addition, an increase in interferon alpha (IFN-α) showed an inverse correlation with virus titers during the infection process. Oseltamivir was effective for reducing H5N6 HPAIV propagation, and continuous treatment with peramivir reduced virus propagation and the severity of symptoms in the early stage. This study also showed pathologically severe lung injury states in cynomolgus macaques infected with H5N6 HPAIV, even in those that received early antiviral drug treatments, indicating the need for close monitoring and further studies on virus pathogenicity and new antiviral therapies.

Diversity of avian influenza A(H5N6) viruses in wild birds in southern China.

The predominance of H5N6 in ducks and continuous human cases have heightened its potential threat to public health in China. Therefore, the detection of emerging variants of H5N6 avian influenza viruses has become a priority for pandemic preparedness. Questions remain as to its origin and circulation within the wild bird reservoir and interactions at the wild-domestic interface. Samples were collected from migratory birds in Poyang Lake, Jiangxi Province, PR China during the routine bird ring survey in 2014-16. Phylogenetic and coalescent analyses were conducted to uncover the evolutionary relationship among viruses circulating in wild birds. Here, we report the potential origin and phylogenetic diversity of H5N6 viruses isolated from wild birds in Poyang Lake. Sequence analyses indicated that Jiangxi H5N6 viruses most likely evolved from Eurasian-derived H5Nx and H6N6 viruses through multiple reassortment events. Crucially, the diversity of the HA gene implies that these Jiangxi H5N6 viruses have diverged into two primary clades - clade 2.3.4.4 and clade 2.3.2.1 c. Phylogenetic analysis revealed two independent pathways of reassortment during 2014-16 that might have facilitated the generation of emerging variants within wild bird populations as well as inter-species infections. Our findings contribute to our understanding of the genetic diversification of H5N6 viruses in the wild bird population. These results highlight the necessity of large-scale surveillance of wild birds in the Poyang Lake area to address the threat of regional epizootic epidemics and attendant pandemics.

Clinical and Immunological Characteristics of Human Infections With H5N6 Avian Influenza Virus.

BACKGROUND: H5N6 avian influenza virus (AIV) has caused sporadic, recurring outbreaks in China and Southeast Asia since 2013, with 19 human infections and 13 deaths. Seventeen of these infections occurred since December 2015, indicating a recent rise in the frequency of H5N6 cases. METHODS: To assess the relative threat of H5N6 virus to humans, we summarized and compared clinical data from patients infected with H5N6 (n = 19) against data from 2 subtypes of major public health concern, H5N1 (n = 53) and H7N9 (n = 160). To assess immune responses indicative of prognosis, we compared concentrations of serum cytokines/chemokines in patients infected with H5N6, H5N1, H7N9, and 2009 pandemic H1N1 and characterized specific immune responses from 1 surviving and 2 nonsurviving H5N6 patients. RESULTS: H5N6 patients were found to have higher incidences of lymphopenia and elevated alanine aminotransferase and lactate dehydrogenase levels compared with H5N1 and H7N9 patients. Hypercytokinemia was detected at substantially higher frequencies from H5N6 patients compared to those infected with other AIV subtypes. Evaluation of adaptive immunity showed that both humoral and cellular responses could be detected in the H5N6-infected survivor, but cellular responses were absent in the nonsurvivors. In addition, the surviving patient had lower concentrations of both pro- and anti-inflammatory cytokines/chemokines compared to the nonsurvivors. CONCLUSIONS: Our results support that H5N6 virus could potentially be a major public health threat, and suggest it is possible that the earlier acquisition of cellular immunity and lower concentrations of cytokines/chemokines contributed to survival in our patient. Analysis of more patient samples will be needed to draw concrete conclusions.

Antigenic Variation of Avian Influenza A(H5N6) Viruses, Guangdong Province, China, 2014-2018.

Market surveillance showed continuing circulation of avian influenza A(H5N6) virus in live poultry markets in Guangdong Province in 2017, despite compulsory vaccination for avian influenza A(H5Nx) and A(H7N9). We analyzed H5N6 viruses from 2014-2018 from Guangdong Province, revealing antigenic drift and decreased antibody response against the vaccine strain in vaccinated chickens.

Genetic Characterization of Avian Influenza A(H5N6) Virus Clade 2.3.4.4, Russia, 2018.

Timely identification of pandemic influenza threats depends on monitoring for highly pathogenic avian influenza viruses. We isolated highly pathogenic avian influenza A(H5N6) virus clade 2.3.4.4, genotype G1.1, in samples from a bird in southwest Russia. The virus has high homology to human H5N6 influenza strains isolated from southeast China.

Genetic Characterization and Zoonotic Potential of Highly Pathogenic Avian Influenza Virus A(H5N6/H5N5), Germany, 2017-2018.

We genetically characterized highly pathogenic avian influenza virus A(H5N6) clade 2.3.4.4b isolates found in Germany in 2017-2018 and assessed pathogenicity of representative H5N5 and H5N6 viruses in ferrets. These viruses had low pathogenicity; however, continued characterization of related isolates is warranted because of their high potential for reassortment.