Avian influenza viruses (AIVs) continue to circulate in live poultry markets (LPMs), posing potential risks for zoonotic exposure. This study investigated the environmental circulation of AIVs and their potential epidemiological associations with human infection in Henan Province, China.

Influenza antivirals (IAs) have been detected in aquatic environments inhabited by dabbling ducks, the natural reservoir of influenza A virus (IAV), raising concerns about the development of antiviral resistance. Because novel human IAV strains often contain genetic material of avian origin, this may contribute to resistance in viruses with pandemic potential. This study aimed to assess the environmental risk posed by four IAs-oseltamivir carboxylate (OC), zanamivir (ZA), peramivir (PE), and amantadine (AM)-based on their potential for environmental release, environmental stability, and induction of antiviral resistance. The assessment combined data from new experiments on (1) environmental release and (2) environmental stability of PE, AM, OC, and ZA, with results from previously published in vivo experiments in a mallard model examining (3) resistance development to OC, PE, and ZA in IAV. The risk of environmental release was assessed as high for OC, AM, and PE, and very high for ZA. Environmental stability ranged from very high to low, in the order PE > AM > OC > ZA. The potential to induce resistance in IAV was similar for PE and OC, and lower for ZA. Overall, the environmental risk ranking was PE > OC > ZA, with PE and OC posing the highest risks. Prudent use of IAs requires balancing the risk of resistance development against clinical benefit. In cases of complicated influenza or in high-risk patient groups, the clinical benefits are substantial and justify IA use. However, in uncomplicated influenza among otherwise healthy individuals, the clinical benefit is limited, and the risk of resistance development should be carefully considered. Among the evaluated antivirals, ZA showed the lowest environmental risk and should be preferred when feasible.

In recent decades, multiple novel reassortant avian influenza viruses (AIVs) carrying internal genes derived from H9N2 viruses have emerged in poultry in China and have repeatedly caused human infections, highlighting the pivotal role of the H9N2 internal genes cassette in facilitating cross-species transmission. In this study, five H6N1 and one H6N2 AIVs were isolated from chickens and Houdan chickens exhibiting respiratory symptoms. Genetic analyses revealed that the heamagglutinin (HA) genes of these H6 AIVs belonged to the ST339-like lineage and originated from H6 AIVs circulating in domestic geese in China, whereas the neuraminidase (NA) genes were derived from H9N2 or H5N1 AIVs, respectively. Notably, all six internal genes of these H6 isolates were derived from the G57 genotype H9N2 virus. In vitro studies demonstrated that these H6 AIVs replicated effectively in both avian and mammalian cells. The H6N2 isolate displayed dual receptor binding affinity for both avian-like and human-like receptors, whereas the five H6N1 viruses showed no detectable receptor binding affinity under the assay conditions in vitro. Reverse-genetics reassortant assay further revealed that the H6N1 HA itself possesses intrinsic dual receptor binding affinity. Furthermore, these H6 isolates exhibited high virulence in mice, and induced obvious respiratory symptoms in chickens, with efficient transmission among birds. Collectively, our findings demonstrate that H6 AIVs carrying the G57 H9N2 internal gene constellation are actively circulating in chicken populations and pose a substantial threat to poultry health, underscoring their potential risk to public health and the need for continued surveillance at the poultry-human interface.

Highly pathogenic avian influenza (HPAI) H5N1 viruses clade 2.3.2.1e has re-emerged in the Indochinese Peninsula, raising concerns about regional spread and zoonotic risk. We characterized eight H5N1 viruses isolated from 32 environmental and poultry-derived samples collected in Lao People's Democratic Republic (Lao PDR) live bird markets during 2023-2024. Whole-genome and phylogenetic analyses showed that all isolates belonged to clade 2.3.2.1e and were distinct from the previously dominant Lao clade 2.3.2.1c, indicating lineage transition in poultry-associated viruses. The Lao isolates were distributed across regional sublineages rather than forming a single local cluster, suggesting repeated introductions and cross-border genetic connectivity. Segment-level nucleotide identities ranged from 97.2 to 99.9%, revealing a complex mosaic genotype constellation. All isolates retained the avian-associated glutamic acid residue at position 627 of polymerase basic protein 2 (PB2-627E) and the aspartic acid residue at position 701 of polymerase basic protein 2 (PB2-701D). Hemagglutinin (HA) analysis showed a conserved polybasic cleavage motif, PQRERRRKRGLF, and conserved HA1-123S, HA1-154N, HA1-263T, and HA2-497L, whereas isolate-level variation was observed at HA1-94, HA1-188, and HA1-223. Structural modeling and Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) analysis of a representative isolate further highlighted receptor binding site (RBS) associated HA features. Importantly, RBS-associated HA variation was detected despite the absence of lysine residue at position 627 of polymerase basic protein 2 (PB2-627 K), suggesting that early molecular changes relevant to mammalian or human adaptation may arise before acquisition of canonical mammalian-adaptive polymerase mutations. These findings support sustained cross-border genomic surveillance and receptor-binding-focused characterization of H5N1 viruses circulating in Lao live bird markets.

Avian influenza virus (AIV) is an increasing driver of the emergence and re-emergence of outbreaks in both animal and human populations worldwide. Its circulation is influenced by migratory birds, human activities, and climate change. Europe and Africa encompass several major migratory flyways that are undergoing substantial environmental and ecological changes. In this systematic review, conducted in accordance with PRISMA guidelines, we analysed published studies reporting AIV detection in migratory birds across Europe and Africa. A total of 72 publications (2005-2025) were included, comprising 53 from Europe and 19 from Africa. Overall, 10,960 migratory birds representing 166 species, 32 families, and 17 orders were reported across the three principal flyways connecting Europe and Africa: the Atlantic, Mediterranean, and Bosphorus flyways. Publication trends aligned with major HxNy outbreak over the past decade. Both low- and high-pathogenic avian influenza viruses (LPAIV and HPAIV) were detected, with a predominance of LPAIV (86% vs. 14% HPAIV). LPAIV were reported consistently throughout the study period, whereas HPAIV detections were largely confined to periods following the 2005 H5N1 outbreak and the outbreak waves after 2020. H5N1 accounted for 11% of AIV detections and was reported in 17 European countries and 4 African countries with a dominance of the clade 2.3.4.4b since 2020. Overall, this review underscores the contribution of diverse avian orders to AIV circulation - particularly in the emergence and re-emergence of H5N1 - and highlights the central role of the Mediterranean flyway in the interconnection of the 3 flyways in AIV surveillance between Europe and Africa.

The indiscriminate use of antimicrobials in food-producing animals is a major contributor to antimicrobial resistance (AMR), a global One Health challenge that threatens the interconnected health of humans, animals, and ecosystems. Hence, this study conducted to assess the association between farm-level biosecurity practices and antimicrobial use in commercial poultry farms in Bangladesh. A cross-sectional study was conducted on 331 poultry farms (broiler: 113, layer: 103, Sonali: 115) across six districts from September to December 2021. A directed acyclic graph informed variable selection and generalized linear models (GLM) and least absolute shrinkage and selection operator (LASSO) regression were used to identify factors associated with antimicrobial use. Overall, 27.8% of farms reported antibiotic use on the day of the interview, and 49.2% had used antibiotics in the preceding 14 days. Antimicrobial use was significantly higher in rural farms and those with dirt floors or floor-based rearing systems. Higher odds of antimicrobial use were also linked to farmer education level, proximity to recent avian influenza outbreaks, unrestricted access of wild animal, and nearby crow roost. Conversely, farms implementing stronger biosecurity including isolating sick birds, and farms rearing layer birds and maintaining improved infrastructure reported significantly lower antibiotic use. These findings indicate that farm infrastructure, wildlife interfaces and hygiene practices are important correlates of antimicrobial use. Strengthening farm-level biosecurity, regulating antimicrobial access, and expanding veterinary extension services, especially in rural areas are critical to reduce antibiotic dependency and mitigating AMR risks. Establishing a national AMR surveillance framework that links animal, environmental, and human health sectors is essential to monitor resistance patterns and guide targeted policy interventions.

Highly pathogenic (HP) H5N1 avian influenza viruses are zoonotic pathogens and are endemic in wild birds and poultry around the world. Pro-inflammatory cytokines are supposed to contribute to the severe clinical symptoms in infected animals and humans. In this study, we determined the infectivity and cytokine induction in primary human lung cells infected with HP H5N1 avian influenza viruses and seasonal H3N2 influenza viruses. MDCK cells were used to measure viral titers in the supernatants of infected human lung cells by log10TCID50/ml. Quantitative real-time PCR was used to detect the induction of cytokine genes using mRNA from the infected primary human lung cells. HP H5N1 avian influenza viruses induced higher viral titers than seasonal H3N2 influenza viruses in supernatants of primary human lung cells. In addition, HP H5N1avian influenza viruses induced the genes of pro-inflammatory cytokines such as IL-23, IL-1α, IL-1β, IL-6, and IL-8 at significantly higher levels than seasonal H3N2 influenza viruses in primary human lung cells. Our data suggest that the high viral replication and the high induction of pro-inflammatory cytokines in the human lung cells infected with HP H5N1 avian influenza viruses may enhance pathogenicity in the infected humans as compared to seasonal H3N2 influenza viruses.

Highly pathogenic avian influenza (HPAI) has circulated in West Africa since Nigeria's first human case in 2006, making the region a key entry point and dispersal hub for multiple virus lineages in sub-Saharan Africa. The 2.3.4.4b virus clade driving the global spread of avian influenza is now infecting mammals more often, a troubling sign that raises the risk of a future pandemic. At the same time, West Africa's ability to control HPAI is held back by fragmented surveillance systems. Animal, human and environmental health services operate in isolation, rely on incompatible data formats and are managed by separate institutions, which undermines early detection and rapid response. This paper describes the existing surveillance silos in West Africa and outlines the consequences of failing to integrate them, including delayed detection of future human or animal health threats. It highlights emerging examples of One Health surveillance efforts in Nigeria, Senegal, Guinea, Benin and Ghana. From an operational standpoint, five conditions are essential for early warning and timely response: interoperable health information systems; shared governance across sectors; sustainable financing; strong communication with poultry-keeping communities; and effective cross-border collaboration. Establishing fully integrated, system-wide national surveillance platforms is indispensable for effective preparedness. Without such integration, West African countries will continue to detect and respond to emerging health threats too late. Given the region's recurrent zoonotic spillover and expanding ecological vulnerabilities, embedding data integration at the core of preparedness strategies is essential for mitigating the risk of future pandemics.

Wastewater sequencing is an increasingly valuable tool in tracking the spread of infectious disease agents across space and time in areas of dense human settlement. Among pathogens that can be readily detected by this approach is influenza A, which follows predictable patterns of prevalence through the winter months in North America. Here, we leverage routine surveillance of a municipal wastewater treatment plant in Northern California to describe an atypical, off-season spike in influenza A concentrations that rivals that of the winter respiratory virus season. Drawing upon metagenomic data generated through hybrid-capture sequencing, we assemble and subsequently characterize fragments of divergent influenza genomes that appear to derive predominantly from the avian H16 clade. These strains exhibit close evolutionary relationships to influenza isolated from migratory shorebirds, hinting at potential host species and mechanisms of geographic spread. Analysis of read abundances suggests that these avian strains dominate the pool of influenza circulating during the summer months, when typical human-infecting strains are essentially absent. Together, our results expand the value of wastewater sequencing to encompass sensitive tracking of outbreaks within animals in interface regions where human settlement abuts wildlands, increasing overall pandemic preparedness.IMPORTANCEResearchers now commonly search municipal wastewater for viral genetic material, which can indicate trends in the diversity and abundance of strains circulating in communities. Here, we show that under certain conditions, municipal wastewater can also capture the signatures of viruses circulating among animal populations, such as birds. Specifically, we draw on a targeted form of nucleic acid sequencing to discover a strain of influenza that is fairly genetically distinct from known relatives and may be circulating among shorebirds in Northern California. These findings both broaden the possible use cases of wastewater sequencing and provide new insights into avian viruses, some of which can jump host species to make other animals or people sick.

Avian influenza viruses circulate naturally among migratory wild birds but can occasionally infect other species, including poultry and a broad range of mammals and humans. Although highly pathogenic avian influenza viruses (AIVs) are known to infect humans, low-pathogenic AIVs, particularly H7N9 and H9N2 subtypes, have also caused human infections and pose an ongoing zoonotic risk. In this study, we used single-cell transcriptomics to examine the cellular responses of human (A549) and chicken (DF-1) cells 24 h after infection with the zoonotic H9N2 virus. Our findings revealed distinct transcriptional responses between the two cell lines. Notably, dipeptidyl peptidase-4 (DPP4) and carboxypeptidase Q (CPQ) genes were expressed at significantly higher levels in infected DF-1 cells than in A549 cells, suggesting a potential role in viral replication. Overexpression of CPQ and DPP4 in A549 cells resulted in significantly greater viral replication compared to mock-transfected wild-type cells infected with the avian influenza H9N2 virus. Furthermore, Gene Set Enrichment Analysis of differentially expressed genes revealed downregulation of lipid metabolism pathways in cell clusters highly expressing H9N2 virus genes in both A549 and DF-1 cells, indicating a potential link between host lipid metabolism and H9N2 virus replication. These results provide insights into the transcriptional responses of A549 and DF-1 cells to H9N2 infection and identify candidate host factors and pathways associated with viral replication. These findings provide a foundation for future studies using physiologically relevant models to investigate the cellular determinants of H9N2 infection.

Highly pathogenic avian influenza (HPAI) A(H5) viruses can be transmitted from infected birds to various mammalian species, including humans. Avian influenza viruses (AIVs), members of the Orthomyxoviridae family, possess segmented RNA genomes prone to reassortment, favoring the emergence of novel genetic traits that may alter transmissibility, pathogenicity, and antigenicity. Although no sustained human-to-human transmission has been reported, the potential adaptation of these viruses poses a significant pandemic threat. This study aimed to evaluate the non-clinical safety, toxicity, and humoral immune responses induced by an adjuvanted H5 influenza vaccine in rats and rabbits, to support future clinical safety trials in humans. Male and female Wistar rats and New Zealand rabbits were observed for 14, 28, and 90 days after receiving two intramuscular doses of the H5N8 vaccine (15 μg HA/dose) formulated with the IB160 oil-in-water emulsion adjuvant. No systemic comorbidities, central nervous system alterations, or relevant clinical signs were observed. Hematological parameters remained within normal ranges, with total and differential leukocyte counts showing only minor fluctuations (<1% of total leukocytes). Mild biochemical variations in urea and hepatic transaminase levels were not correlated with histopathological alterations. The vaccine elicited a robust humoral response soon after immunization, with all groups reaching protective HAI-antibody titers. Although antibody levels declined over time, particularly in males, they remained significantly above baseline, indicating durable immunological memory. Furthermore, the vaccine induced a specific cellular immune response, confirmed by IL-2 and TNF production by antigen-specific T lymphocytes in splenic cell cultures after the booster dose. In conclusion, the H5N8 vaccine with the IB160 adjuvant was well tolerated locally and systemically, without compromising vital organ function. The safety and immunogenicity findings are consistent with expectations for adjuvanted influenza vaccines, demonstrating strong and durable humoral and cellular immune responses.