The global expansion of avian influenza viruses (AIVs), particularly clade 2.3.4.4b H5 viruses, continues to pose a significant zoonotic threat. Although Chinese vaccination-plus-culling strategy has reduced H5- and H7-exposure disease activity, continued viral evolution and reassortment highlight the need for sustained surveillance at the human-environment interface. Environmental samples from live poultry markets (LPMs), slaughterhouses and poultry farms/backyards, together with respiratory specimens from hospitalized patients with pneumonia of unknown etiology (PUE) were collected in three Chinese cities from December 2021 to December 2023. Specimens were tested by real-time reverse transcription polymerase chain reaction (rRT-PCR) for influenza A virus (IAV), H5, H7, H9 and H10 subtypes. Positive rates among cities and sampling sites were compared using chi-square tests. Monthly positive rates among environmental sample categories were compared using the Kruskal-Wallis test, and paired monthly rates between 2022 and 2023 using the two-sided Wilcoxon signed-rank test. H5, H9 and H10 viruses underwent HA gene sequencing and phylogenetic analysis. Among 8,335 environmental samples, the overall IAV positive rate was 32.2

The H3 subtype avian influenza virus (AIV) has been widely spread in birds and is known as a natural source of mammalian influenza viruses. Based on data from public databases and our surveillance data, we analyzed the ecology, evolution, and spread of H3 AIVs. Sublineages of H3 AIVs have been detected worldwide, infecting various birds, at least 90 species in wild birds and poultry. Important areas for large-scale and local dissemination of H3 AIVs were identified, such as Alaska, Central Asia, and Chinese provinces. The H3 viruses have elevated the HA gene substitution rate after introduction from wild birds to domestic poultry, and even faster in domestic chickens. Our results implied an evolutionary mechanism of H3 AIV cross-species transmission, that viruses from wild birds to domestic poultry have accelerated substitution rate by shorter generation time and host selection. Novel chicken H3 viruses, especially H3N8 G25 viruses that have spilled over to humans, require high attention.

Subtype H10 viruses are known to infect humans in Africa, Oceania, and Asia. In 2021, 2022, and recently in April 2024, a novel H10N3 subtype avian influenza virus was found cause human infection with severe pneumonia. Herein, we comprehensively studied the phylogenetic evolution and biological characteristics of the newly emerged influenza A(H10N3) virus. We found that the human isolated H10N3 virus was generated in early 2019 in domestic poultry. The viruses bound to salic acid α2, 3 receptors, indicating their insufficient ability to infect humans. Although a low pathogenic avian influenza virus, the human isolated H10N3 virus exhibited robust pathogenicity in both BALB/c and C57BL/6 mice, with MLD50 1000 times higher than a homologous environmental isolate. The human isolated H10N3 also showed respiratory droplet transmissibility in ferrets. Considering the continuous circulation in avian populations and repeated transmission to humans, strengthened surveillance of H10 subtype viruses in poultry should be put into effect.

H4 avian influenza viruses (AIVs) have been widely detected in live poultry markets in China. However, the potential public health impact of H4 AIVs remains largely uncertain. Here, we fully analyzed the distribution and phylogenetic relationship of H4 AIVs in China. We obtained 31 isolates of H4 viruses in China during 2009–2022 through surveillance in poultry-associated environments, such as live poultry markets and poultry farms. Genomic sequence analysis together with publicly available data revealed that frequent reassortment and introduction of H4 AIV from wild birds to poultry may have occurred. We identified 62 genotypes among 127 whole genome sequences of H4 viruses in China, indicating that H4 AIVs had great genetic diversity in China. We also investigated molecular markers and found that drug resistance mutations frequently occurred in the M2 protein and a few mutations related to receptor binding and the host signature in H4 AIVs. Our study demonstrates the cross-species transmission potential of H4 AIVs in China and provides some reference significance for its risk assessment.

BackgroundSince the first human infection with H9N2 virus was reported in 1998, the number of cases of H9N2 infection has exceeded one hundred by 2021. However, there is no systematic description of the biological characteristics of H9N2 viruses isolated from humans.MethodsTherefore, this study analyzed the pathogenicity in mice of all available H9N2 viruses isolated from human cases in China from 2013 to 2021.ResultsAlthough most of the H9N2 viruses analyzed showed low or no pathogenicity in mice, the leucine to glutamine substitution at residue 226 (L226Q) in the hemagglutinin (HA) protein rapidly emerged during the adaptation of H9N2 viruses, and was responsible for severe infections and even fatalities. HA amino acid 226Q conferred a remarkable competitive advantage on H9N2 viruses in mice relative to viruses containing 226L, increasing their virulence, infectivity, and replication.ConclusionThus, our study demonstrates that the adaptive substitution HA L226Q rapidly acquired by H9N2 viruses during the course of infection in mice contributed to their high pathogenicity.

Influenza A(H3N8) viruses first emerged in humans in 2022, but their public health risk has not been evaluated. Here, we systematically investigated the biological features of avian and human isolated H3N8 viruses. The human‐origin H3N8 viruses exhibited dual receptor binding profiles but avian‐origin H3N8 viruses bound to avian type (sialic acid α2, 3) receptors only. All H3N8 viruses were sensitive to the antiviral drug oseltamivir. Although H3N8 viruses showed lower virulence than the 2009 pandemic H1N1 (09pdmH1N1) viruses, they induced comparable infectivity in mice. More importantly, the human population is naïve to H3N8 virus infection and current seasonal vaccination is not protective. Therefore, the threat of influenza A(H3N8) viruses should not be underestimated. Any variations should be monitored closely and their effect should be studied in time for the pandemic potential preparedness purpose.

The continuous evolution of avian influenza viruses (AIVs) of subtype H3 in China and the emergence of human infection with AIV subtype H3N8 highlight their threat to public health. Through surveillance in poultry-associated environments during 2009–2022, we isolated and sequenced 188 H3 AIVs across China. Performing large-scale sequence analysis with publicly available data, we identified 4 sublineages of H3 AIVs established in domestic ducks in China via multiple introductions from wild birds from Eurasia. Using full-genome analysis, we identified 126 distinct genotypes, of which the H3N2 G23 genotype predominated recently. H3N8 G25 viruses, which spilled over from birds to humans, might have been generated by reassortment between H3N2 G23, wild bird H3N8, and poultry H9N2 before February 2021. Mammal-adapted and drug-resistance substitutions occasionally occurred in H3 AIVs. Ongoing surveillance for H3 AIVs and risk assessment are imperative for potential pandemic preparedness.

IntroductionThe 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.MethodsIn 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.ResultsOur 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.DiscussionTherefore, 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.

Objective: Influenza B virus is a significant respiratory pathogen responsible for seasonal influenza. In recent years the B/Yamagata lineage has demonstrated a rapid increase, predominantly featuring the neuraminidase (NA)N342K mutation. This study determined the impact of the NAN342K mutation on the pathogenicity of influenza B virus and elucidate the underlying mechanisms. Methods: Gene fragments with specific mutations were generated using site-directed mutagenesis PCR, resulting in recombinant viruses (rAH127 and rAH127/NAN342K). C57BL/6 mice were infected to evaluate the impact of amino acid mutations on virus pathogenicity. Body weight, survival rate, virus replication, and lung pathology were compared among the groups. NA enzyme activity was assessed to determine the mechanisms underlying the effects of amino acid mutations on the pathogenicity of influenza B virus. Results: The NAN342K mutant virus exhibited significantly increased NA enzyme activity (3.19-fold) and viral replication capacity in MDCK cells (6.76-fold) compared to wild-type virus. These changes led to enhanced pathogenicity in mice, characterized by severe weight loss, increased mortality, and heightened lung tissue inflammation. Conclusions: The NAN342K mutation likely enhances virus replication and pathogenicity by increasing NA enzyme activity. These findings contribute to understanding the molecular mechanisms underlying influenza B virus pathogenicity and have implications for targeted therapeutic strategies.

H6 avian influenza virus (AIV) is one of the most prevalent AIV subtypes in birds globally. To investigate the current situation and characteristics of H6 AIVs circulating in China, we analysed the epidemiology, genetic evolution and pathogenic features of this subtype. During 2000-2021, H6 subtype AIVs spread widely through Southern China and presented high host diversity. On analysing 171 H6 viruses isolated during 2009-2021, dynamic reassortments were observed among H6 and other co-circulating AIV subtypes, and these generated a total of 16 different genotypes. A few H6N6 strains possessed L226 and S228 mutations of hemagglutinin (H3 numbering), which may enhance the affinity of H6 viruses to human receptors. H6N6 viruses also exhibited divergent pathogenicity and growth profiles in vivo and in vitro. Some of the H6N6 viruses could infect mice without mammalian adaptation, and even caused death in this species. Therefore, our study demonstrated that the H6 AIVs posed a potential threat to human health and highlighted the urgent need for continued surveillance and evaluation of the H6 influenza viruses circulating in the field.

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.