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Influenza A viruses (IAVs) from the H5N1 2.3.4.4b clade are circulating in dairy farms in the USA.; ruminants were presumed not to be hosts for IAVs. Previously, IAV-positive mammalian species were hunters and scavengers, possibly getting infected while feeding on infected birds. It is now recognized that H5N1 viruses that circulate in US dairy cattle transmit through a mammary gland route, in contrast to transmission by aerosols via the respiratory tract. The sialome in the cow mammary and respiratory tract is so far solely defined using plant lectins. Here, we used recombinant HA proteins representing current circulating and classical H5 viruses to determine the distribution of IAV receptors in the respiratory and mammary tract tissues of cows. We complemented our study by mapping the glycan distribution of the upper and lower respiratory tracts of horses and pigs. Most of the sialome of the cow respiratory tract is lined with sialic acid modifications, such as N-glycolyl and O-acetyl, which are not bound by IAV. Interestingly, the H5 protein representing the cow isolates is bound significantly in the mammary gland, whereas classical H5 proteins failed to do so. Furthermore, whereas the 9-O-acetyl modification is prominent in all tissues tested, the 5-N-glycolyl modification is not, resulting in the display of receptors for avian IAV hemagglutinins. This could explain the high levels of virus found in these tissues and milk, adding supporting data to this virus transmission route.IMPORTANCEH5N1 influenza viruses, which usually affect birds, have been found on dairy farms in the USA. Surprisingly, these viruses are spreading among dairy cows, and there is a possibility that they do not spread through the air but through their milk glands. To understand this better, we studied how the virus attaches to tissues in the cow's respiratory tract and mammary glands using specific viral proteins. We found that the cow-associated virus binds strongly to the mammary glands, unlike older versions infecting birds. This might explain why the virus is found in cow's milk, suggesting a new way the virus could be spreading., Competing Interests: The authors declare no conflict of interest.

How avian influenza virus will continue to spread and circulate among wildlife is unclear.

Since April 2024, sporadic infections with highly pathogenic avian influenza (HPAI) A(H5) viruses have been detected among dairy farm workers in the United States. To date, infections have mostly been detected through worker monitoring, and have been mild despite the possibility of more severe illness. During June-August 2024, CDC collaborated with the Michigan Department of Health and Human Services and the Colorado Department of Public Health and Environment to implement cross-sectional serologic surveys to ascertain the prevalence of recent infection with HPAI A(H5) virus among dairy workers. In both states, a convenience sample of persons who work in dairies was interviewed, and blood specimens were collected. Among 115 persons, eight (7%; 95% CI = 3.6%-13.1%) had serologic evidence of recent infection with A(H5) virus; all reported milking cows or cleaning the milking parlor. Among persons with serologic evidence of infection, four recalled being ill around the time cows were ill; symptoms began before or within a few days of A(H5) virus detections among cows. This finding supports the need to identify and implement strategies to prevent transmission among dairy cattle to reduce worker exposures and for education and outreach to dairy workers concerning prevention, symptoms, and where to seek medical care if the workers develop symptoms. Timely identification of infected herds can support rapid initiation of monitoring, testing, and treatment for human illness, including mild illness, among exposed dairy workers., Competing Interests: All authors have completed and submitted the International Committee of Medical Journal Editors form for disclosure of potential conflicts of interest. No potential conflicts of interest were disclosed.

Currently A(H5Nx) avian influenza viruses are globally widespread and continue to evolve. Since their emergence in 2020 novel highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b reassortant viruses have become predominant in the world and caused multiple infections in mammals. It was shown that some of A(H5N1) viruses mostly isolated from mammals contain an E627K mutation in the PB2 protein which can lead to adaptation of influenza viruses to mammalian cells. In 2023 in Russia we have isolated two highly pathogenic avian influenza A(H5N1) clade 2.3.4.4b viruses from birds one of which contained an E627K mutation in the PB2 protein. This virus had increased virulence in mice. Limited airborne transmission of the virus with the PB2-E627K mutation was observed between ferrets, in which infectious virus was detected in the nasal washings of the three of the twelve recipient ferrets, and clinical symptoms of the disease were observed in one case. Both viruses showed dominant binding to avian-type sialoside receptors, which was most likely the reason for the limited transmissibility. Thus, this study indicates a possible limited increase in the pandemic potential of A(H5N1) 2.3.4.4b viruses and highlights the importance of continuous avian influenza surveillance for pandemic preparedness and response., (© 2024. The Author(s).)

Highly pathogenic avian influenza (HPAI) poses a substantial threat to several raptors. Between 2021 and 2023, HPAI viruses (HPAIVs) of the Goose/Guangdong lineage H5 clade 2.3.4.4b became widespread in wild birds in Norway, and H5N1 and H5N5 viruses were detected in 31 white-tailed eagles ( Haliaeetus albicilla , WTEs). Post-mortem examinations of four WTEs revealed no macroscopic pathological findings. Microscopic examinations showed the presence of myocardial and splenic necroses and a few lesions in the brain. In situ hybridization revealed the presence of the virus in several organs, suggesting a multisystemic infection. The detection of HPAIV H5N5 in a WTE in February 2022 marked the first recorded occurrence of this subtype in Norway. Since then, the virus has persisted, sporadically being detected in WTEs and other wild bird species. Phylogenetic analyses reveal that at least two distinct incursions of HPAIV H5N1 Eurasian (EA) genotype C affected WTEs, likely introduced by migratory birds from Eurasia and seabirds entering from Western and Central Europe. Some WTE isolates from 2021 to 2022 clustered with those from Canada and Ireland, aligning with the transatlantic spread of H5N1. Others were related to the 2021 mass mortality of great skuas in the UK or outbreaks in seabird populations, including gannets, gulls and terns, during 2022 in the North Sea region. This suggests that the WTEs were likely preying on the affected birds. Our study highlights that WTEs can act as sentinels for some HPAIV strains, but the absence of several known circulating genotypes in WTEs suggests varying pathogenic effects on this species.

Background: H9N2 avian influenza viruses have been circulating in Bangladesh since 2006, affecting multiple avian species and resulting in economic losses. The recent emergence of tribasic strains, along with co-infections, has increased the risk to poultry health. Therefore, the study aimed to compare the immune responses of Sonali (crossbred) and commercial broiler chickens infected with tribasic H9N2 low pathogenic avian influenza (LPAI) virus., Methods: Following H9N2 infection, proinflammatory (IL-6, IL-8, IL-1β and TNF-α) and antiviral (IFN-β and IFN-γ) cytokine expressions were observed in the trachea, lungs, intestine, and lymphoid tissues in Sonali and broiler chickens from 1 day post infection (dpi) to 10 dpi by qPCR., Results: Sonali chickens exhibited significantly higher proinflammatory and antiviral cytokine expressions in the trachea at 3-7 days post infection (dpi), while broiler chickens showed lower immune responses. Broiler chickens displayed prolonged IL-6, IL-8, and IL-1β expression in lungs at 3-10 dpi compared to Sonali chickens. In the intestine, broiler chickens showed higher IL-6 and IL-8 expression that peaks at 1-3 dpi, while in Sonali chickens only IL-1β elevated at 10 dpi. In response to the H9N2 viruses, broiler chickens exhibited a stronger early IFN-β responses and a delayed IFN-γ responses in their lymphoid organs compared to Sonali chickens., Conclusion: This suggests distinct immune profiles between the chicken types in response to the H9N2 infection. The information sheds light on the function of innate immunity in the pathophysiology of currently circulating tribasic H9N2 virus and could assist in effective controlling of avian influenza virus spread in poultry and designing vaccines., (© 2024. The Author(s).)

The guinea fowl (Numida meleagris) holds significant agricultural importance in Ghana, particularly in the Northern, Upper East and Upper West Regions. Despite their economic and cultural significance, guinea fowls face a potential threat from avian influenza, a global concern for its adverse impact on poultry populations. This study assessed the seroprevalence of the virus in mature guinea fowls in the Upper East Region. A cross-sectional survey was conducted in three districts within the Upper East Region from April to June 2023. Blood samples were collected from 397 guinea fowls that are over 4 weeks old, and seroprevalence was determined using ID Screen Influenza A Antibody Competition Enzyme-linked immunosorbent assay (ELISA). The study analysed demographic factors such as sex, age and source of birds, employing statistical methods to establish associations. Among the sampled guinea fowls, 24.7% tested positive for avian influenza antibodies, whereas 75.3% were seronegative. Age did not show statistically significant associations with seroprevalence, but intriguing patterns were observed. Adult guinea fowls exhibited higher seroprevalence (23.7%) compared to growers (1.0%). The source of birds showed no significant association, but birds from slaughter points demonstrated higher seroprevalence (11.6%) compared to households (5.0%) and live bird markets (8.1%). In conclusion, the study underscores the importance of monitoring avian influenza in guinea fowls to implement effective control measures. The presence of antibodies suggests guinea fowls may contribute to virus transmission in the Upper East Region. The study recommends ongoing nationwide surveillance to assess the true prevalence of avian influenza in guinea fowls across Ghana., (© 2024 The Author(s). Veterinary Medicine and Science published by John Wiley & Sons Ltd.)

Vaccine adjuvants, serving as non-specific immune enhancers, play a pivotal role in the immunoprevention and control of animal diseases. This study utilized prokaryotic expression systems to express and purify chicken-derived cIFITM1, cIFITM3, and cViperin, which were then formulated as adjuvants with H9N2 avian influenza virus antigens to create inactivated vaccines. These vaccines were administered to SPF chickens to investigate their immunopotentiating functions. Additionally, the proteins were assessed for their ability to act as standalone immune enhancers. The results demonstrated that cIFITM1, cIFITM3, and cViperin significantly elevated serum hemagglutination inhibition (HI) antibody titers. Notably, when used individually, these proteins markedly enhanced the antiviral capabilities of challenged chickens, leading to alleviated clinical symptoms, reduced tracheal virus replication, diminished virus shedding, and lessened histopathological damage, with cIFITM1 exhibiting the most pronounced effect. Furthermore, the protective efficacy of two H9N2 recombinant virus inactivated vaccines supplemented with cIFITM1 adjuvant was validated, achieving a 100 % vaccine protection efficiency. In conclusion, cIFITM1, cIFITM3, and cViperin as adjuvants for influenza vaccines effectively inhibit virus replication and shedding, highlighting their significant potential in influenza prevention and control., Competing Interests: Declaration of Competing Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The H9N2 inactivated avian influenza vaccine cannot induce cellular and mucosal immune responses, while the attenuated Salmonella vector as an intracellular bacterium can induce dominant cellular and mucosal immune responses. However, it provides low protection against the virus when delivering viral antigens and needs further optimization. Chicken C-C motif chemokine ligand 5 (chCCL5) is an important CC chemokine associated with immune cell chemotaxis, migration, and viral infection. This study connected the sequence of chCCL5 (CCL5) with the hemagglutinin sequence of the H9N2 avian influenza virus (yH9HA), utilizing the attenuated Salmonella typhimurium vector containing the delayed lysis system MazE/F regulated by arabinose as a carrier. A vaccine strain of recombinant attenuated Salmonella typhimurium and H9N2 avian influenza virus HA, rSC0130 (pS0017-yH9HA-CCL5), was successfully constructed. The experimental results indicate that yH9HA-CCL5 can be expressed in 293 T cells; compared to the strain without CCL5, rSC0130 (pS0017-yH9HA-CCL5) can induce significantly increased cellular immune responses and provide better protective effects in H9N2 virus challenge experiments. The above results indicate that chCCL5 can significantly enhance the protective effect of Salmonella delivering H9N2 avian influenza virus HA protein vaccine against H9N2 avian influenza virus infection, providing valuable theoretical support for further improving the protective efficiency of recombinant attenuated Salmonella vectors for delivering viral antigens., 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 © 2024 Elsevier B.V. All rights reserved.)

The H9N2 subtype of the avian influenza virus (AIV) poses a significant threat to the poultry industry and human health. Recombinant vaccines are the preferred method of controlling H9N2 AIV, and Marek's disease virus (MDV) is the ideal vector for recombinant vaccines. During this study, we constructed two recombinant MDV type 1 strains that carry the hemagglutinin (HA) gene of AIV to provide dual protection against both AIV and MDV. To assess the effects of different MDV insertion sites on the protective efficacy of H9N2 AIV, the HA gene of H9N2 AIV was inserted in UL41 and US2 of the MDV type 1 vector backbone to obtain recombinant viruses rMDV-UL41/HA and rMDV-US2/HA, respectively. An indirect immunofluorescence assay showed sustained expression of HA protein in both recombinant viruses. Additionally, the insertion of the HA gene in UL41 and US2 did not affect MDV replication in cell cultures. After immunization of specific pathogen-free chickens, although both the rMDV-UL41/HA and rMDV-US2/HA groups exhibited similar levels of hemagglutination inhibition antibody titers, only the rMDV-UL41/HA group provided complete protection against the H9N2 AIV challenge, and also offered complete protection against challenge with MDV. These results demonstrated that rMDV-UL41/HA could be used as a promising bivalent vaccine strain against both H9N2 avian influenza and Marek's disease in chickens., Competing Interests: Declaration of Competing Interest The authors of this study have no conflicts of interest to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Avian influenza viruses (AIV) pose a continuous challenge to global health and economy. While countermeasures exist to control outbreaks in poultry, the persistent circulation of AIV in wild aquatic and shorebirds presents a significant challenge to effective disease prevention efforts. PB1-F2 is a non-structural protein expressed from a second open reading frame (+1) of the polymerase basic 1 (PB1) segment. The sequence and length of the PB1-F2 protein can vary depending on the host of origin. While avian isolates typically carry full-length PB1-F2, isolates from mammals, often express truncated forms. The selective advantage of the full-length PB1-F2 in avian isolates is not fully understood. Most research on the role of PB1-F2 in influenza virus replication has been conducted in mammalian systems, where PB1-F2 interfered with the host immune response and induced apoptosis. Here, we used Low Pathogenicity (LP) AIV H7N7 expressing full-length PB1-F2 as well as a knockout mutant. We found that the full-length PB1-F2 of LPAIV prolonged survival of infected cells by limiting apoptotic cell death. Furthermore, PB1-F2 knockout LPAIV significantly decreased MHC-I expression on fibroblasts, delayed tissue healing and increased phagocytic uptake of infected cells, whereas LPAIV expressing PB1-F2 has limited effects. These findings indicate that full-length PB1-F2 enables AIV to cause prolonged infections without severely harming the avian host. Our observations may explain maintenance of AIV in the natural bird reservoir in absence of severe clinical signs., 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 © 2024. Published by Elsevier B.V.)

Avian influenza viruses (AIV) are capable of infecting a considerable proportion of the world's population each year, leading to severe epidemics with high rates of morbidity and mortality. The methods now used to diagnose influenza virus A include the Western blot test (WB), hemagglutination inhibition (HI), and enzyme-linked immunosorbent assays (ELISAs). But because of their labor-intensiveness, lengthy procedures, need for costly equipment, and inexperienced staff, these approaches are considered inappropriate. The present review elucidates the recent advancements in the field of avian influenza detection through the utilization of nanomaterials-based immunosensors between 2014 and 2024. The classification of detection techniques has been taken into account to provide a comprehensive overview of the literature. The review encompasses a detailed illustration of the commonly employed detection mechanisms in immunosensors, namely, colorimetry, fluorescence assay, surface plasmon resonance (SPR), surface-enhanced Raman spectroscopy (SERS), electrochemical detection, quartz crystal microbalance (QCM) piezoelectric, and field-effect transistor (FET). Furthermore, the challenges and future prospects for the immunosensors have been deliberated upon. The present review aims to enhance the understanding of immunosensors-based sensing platforms for virus detection and to stimulate the development of novel immunosensors by providing novel ideas and inspirations. Therefore, the aim of this paper is to provide an updated information about biosensors, as a recent detection technique of influenza with its details regarding the various types of biosensors, which can be used for this review., 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 © 2024 Elsevier B.V. All rights reserved.)

Avian influenza poses a significant global health threat, with the potential for widespread pandemics and devastating consequences. Hemagglutinin (HA), a critical surface glycoprotein of influenza viruses, plays a pivotal role in viral entry and serves as a primary target for subunit vaccine development. In this study, we successfully cloned, expressed, and purified hemagglutinin from the circulating strain of H5N1 influenza virus using a robust molecular biology approach. The cloning process involved insertion of the synthetic HA gene into the pET21b vector, confirmed through double digestion and sequencing. SDS-PAGE analysis confirmed the presence of the expected 60 kDa protein band post-induction. Following expression, the protein was subjected to purification via Ni-NTA affinity chromatography, yielding pure protein fractions. Native PAGE analysis confirmed the protein's oligomeric forms, essential for optimal antigenicity. Western blot analysis further validated protein identity using anti-His and anti-HA antibodies. MALDI-TOF analysis confirmed the protein's sequence integrity, while hemagglutination assay demonstrated its biological activity in binding to N-acetyl neuraminic acid. These findings underscore the potential of recombinant hemagglutinin as a valuable antigen for diagnosis and biochemical assays as well as for vaccine development against avian influenza. In conclusion, this study represents a critical guide for bacterial production of H5N1 HA, which can be a cost-effective and simpler strategy compared to mammalian protein expression. Further research into optimizing vaccine candidates and production methods will be essential in combating the ongoing threat of avian influenza pandemics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Since its first isolation from migratory birds in Egypt in 2016, highly pathogenic avian influenza (HPAI) H5N8 has caused several outbreaks among domestic poultry in various areas of the country affecting poultry health and production systems. However, the genetic and biological properties of the H5N8 HPAI viruses have not been fully elucidated yet. In this study, we aimed to monitor the evolution of circulating H5N8 viruses and identify the pathogenicity and mammalian adaptation in vitro and in vivo. Three H5N8 HPAI viruses were used in this study and were isolated in 2021-2022 from poultry and wild birds during our routine surveillance. RNA extracts were subjected to full genome sequencing. Genetic, phylogenetic, and antigenic analyses were performed to assess viral characteristics and similarities to previously isolated viruses. Phylogenetic analysis showed that the hemagglutinin genes of the three isolates belonged to clade 2.3.4.4b and grouped with the 2019 viruses from G3 with high similarity to Russian and European lineages. Multiple basic amino acids were observed at cleavage sites in the hemagglutinin proteins of the H5N8 isolates, indicating high pathogenicity. In addition, several mutations associated with increased virulence and polymerase activity in mammals were observed. Growth kinetics assays showed that the H5N8 isolate is capable of replicating efficiently in mammalian cells lines. In vivo studies were conducted in SPF chickens (White Leghorn), mice, and hamsters to compare the virological characteristics of the 2022 H5N8 isolates with previous H5N8 viruses isolated in 2016 from the first introduction. The H5N8 viruses caused lethal infection in all tested chickens and transmitted by direct contact. However, we showed that the 2016 H5N8 virus causes a higher mortality in chickens compared to 2022 H5N8 virus. Moreover, the 2022 virus can replicate efficiently in hamsters and mice without preadaptation causing systemic infection. These findings underscore the need for continued surveillance of H5 viruses to identify circulating strains, determine the commercial vaccine's effectiveness, and identify zoonotic potential., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Moatasim et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

A/goose/Guangdong/1/96-like (GsGd) highly pathogenic avian influenza (HPAI) H5 viruses cause severe outbreaks in poultry when introduced. Since emergence in 1996, control measures in most countries have suppressed local GsGd transmission following introductions, making persistent transmission in domestic birds rare. However, geographical expansion of clade 2.3.4.4 sublineages has raised concern about establishment of endemic circulation, while mechanistic drivers leading to endemicity remain unknown. We reconstructed the evolutionary history of GsGd sublineage, clade 2.3.4.4c, in Taiwan using a time-heterogeneous rate phylogeographic model. During Taiwan's initial epidemic wave (January 2015 - August 2016), we inferred that localised outbreaks had multiple origins from rapid spread between counties/cities nationwide. Subsequently, outbreaks predominantly originated from a single county, Yunlin, where persistent transmission harbours the trunk viruses of the sublineage. Endemic hotspots determined by phylogeographic reconstruction largely predicted the locations of re-emerging outbreaks in Yunlin. The transition to endemicity involved a shift to chicken-dominant circulation, following the initial bidirectional spread between chicken and domestic waterfowl. Our results suggest that following their emergence in Taiwan, source-sink dynamics from a single county have maintained GsGd endemicity up until 2023, pointing to where control efforts should be targeted to eliminate the disease., (© 2024. The Author(s).)

Avian influenza viruses (AIVs) are a major economic burden to the poultry industry and pose serious zoonotic risks, with human infections being reported every year. To date, the vaccination of birds remains the most important method for the prevention and control of AIV outbreaks. Most national vaccination strategies against AIV infection use whole virus-inactivated vaccines, which predominantly trigger a systemic antibody-mediated immune response. There are currently no studies that have examined the antibody repertoire of birds that were infected with and/or vaccinated against AIV. To this end, we evaluate the changes in the H9N2-specific IgM and IgY repertoires in chickens subjected to vaccination(s) and/or infectious challenge. We show that a large proportion of the IgM and IgY clones were shared across multiple individuals, and these public clonal responses are dependent on both the immunisation status of the birds and the specific tissue that was examined. Furthermore, the analysis revealed specific clonal expansions that are restricted to particular H9N2 immunisation regimes. These results indicate that both the nature and number of immunisations are important drivers of the antibody responses and repertoire profiles in chickens following H9N2 antigenic stimulation. We discuss how the repertoire biology of avian B-cell responses may affect the success of AIV vaccination in chickens, in particular the implications of public versus private clonal selection., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Dascalu, Sealy, Sadeyen, Flammer, Fiddaman, Preston, Dixon, Bonsall, Smith and Iqbal.)

The resurgence of H5N1 avian influenza highlights the urgent need for robust surveillance systems to detect zoonotic risks before they evolve into human-to-human transmission. The One Health approach-integrating human, animal, and environmental health-offers a comprehensive framework for addressing these challenges. H5N1, a highly pathogenic avian influenza virus, has caused significant mortality in avian populations and poses a serious threat to human health. While human cases are rare, alarming outbreaks in mammals, such as elephant seals with up to 96% mortality, emphasize the importance of early detection in animal populations. Traditional surveillance methods, which often focus solely on human cases, are reactive and may be too late to prevent widespread transmission. One Health enhances early warning systems by monitoring wildlife, livestock, and environmental samples, identifying viral spillover events at their source, and enabling timely interventions to contain the virus before it reaches human populations., (© 2024. The Author(s).)

Deep learning presents a promising approach to complex biological classifications, contingent upon the availability of well-curated datasets. This study addresses the challenge of analyzing three-dimensional protein structures by introducing a novel pipeline that utilizes open-source tools to convert protein structures into a format amenable to computational analysis. Applying a two-dimensional convolutional neural network (CNN) to a dataset of 12,143 avian influenza virus genomes from 64 countries, encompassing 119 hemagglutinin (HA) and neuraminidase (NA) types, we achieved significant classification accuracy. The pathogenicity was determined based on the presence of H5 or H7 subtypes, and our models, ranging from zero to six mid-layers, indicated that a four-layer model most effectively identified highly pathogenic strains, with accuracies over 0.9. To enhance our approach, we incorporated Principal Component Analysis (PCA) for dimensionality reduction and one-class SVM for abnormality detection, improving model robustness through bootstrapping. Furthermore, the K-nearest neighbor (K-NN) algorithm was fine-tuned via hyperparameter optimization to corroborate the findings. The PCA identified distinct clustering for pathogenic HA, yielding an AUC of up to 0.85. The optimized K-NN model demonstrated an impressive accuracy between 0.96 and 0.97. These combined methodologies underscore our deep learning framework's capacity for rapid and precise identification of pathogenic avian influenza strains, thus providing a critical tool for managing global avian influenza threats.

Background: This study aimed to investigate epidemiologic characteristics of major human infection with avian influenza and explore the factors underlying the spatial distributions, particularly H5N6 and H9N2, as H9N2 could directly infect mankind and contribute partial or even whole internal genes to generate novel human-lethal reassortants such as H5N6. They pose potential threats to public health and agriculture., Methods: This study collected cases of H5N1, H5N6, H9N2, and H7N9 in China, along with data on ecoclimatic, environmental, social and demographic factors at the provincial level. Boosted regression tree (BRT) models, a popular approach to ecological studies, has been commonly used for risk mapping of infectious diseases, therefore, it was used to investigate the association between these variables and the occurrence of human cases for each subtype, as well as to map the probabilities of human infections., Results: A total of 1,123 H5N1, H5N6, H9N2, and H7N9 human cases have been collected in China from 2011 to 2024. Factors including density of pig and density of human population emerged as common significant predictors for H5N1 (relative contributions: 5.3, 5.8%), H5N6 (10.8, 6.4%), H9N2 (11.2, 7.3%), and H7N9 (9.4, 8.0%) infection. Overall, each virus has its own ecological and social drivers. The predicted distribution probabilities for H5N1, H5N6, H9N2, and H7N9 presence are highest in Guangxi, Sichuan, Guangdong, and Jiangsu, respectively, with values of 0.86, 0.96, 0.93 and 0.99., Conclusion: This study highlighted the important role of social and demographic factors in the infection of different avian influenza, and suggested that monitoring and control of predicted high-risk areas should be prioritized., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Qu, Chen, Chen, Cao, Wu, Zhou, Qi, Miao, Yan and Yang.)

The incidence of human infection by zoonotic avian influenza viruses, especially H5N1 and H7N9 viruses, has increased. Current zoonotic H7N9 avian influenza viruses (identified since 2013) emerged during reassortment of viruses belonging to different subtypes. Despite analyses of their genetic background, we do not know why current H7N9 viruses are zoonotic. Therefore, there is a need to identify the factor(s) responsible for the extended host tropism that enables these viruses to infect humans as well as birds. To identify H7N9-specific amino acids that confer zoonotic properties on H7N9 viruses, we performed multiple alignment of the hemagglutinin (HA) amino acid sequences of A/Shanghai/1/2013 (H7N9) and A/duck/Zhejiang/12/2011(H7N3) (a putative, non- or less zoonotic HA donor to the zoonotic H7N9 virus). We also analyze the function of an H7N9 HA-specific amino acid with respect to HA acid stability, and evaluated the effect of acid stability on viral infectivity and virulence in a mouse model. HA2-116D, preserved in current zoonotic H7N9 viruses, was crucial for loss of HA acid stability. The acid-labile HA protein in H7 viruses played an important role in infection of human airway epithelial cells; HA2-116D contributed to infection and replication of H7 viruses. Finally, HA2-116D served as a H7 virulence factor in mice. These results suggest that acid-labile HA harboring HA2-116D confers zoonotic characteristics on H7N9 virus and that future novel zoonotic avian viruses could emerge from non-zoonotic H7 viruses via acquisition of mutations that remove HA acid stability., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Daidoji et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Long-lasting chemiluminescence (CL) emissions are necessary for improving the detection accuracy and expanding the application scope. Here, we have synthesized three oil-in-water (O/W) multicolor protein capsules (LCBA, F/LCBA, and RB/F/LCBA) using a simple ultrasound method and have engineered specific target-triggered catalytic hairpin assembly on their surface and chemiluminescence resonance energy transfer inside. Consequently, three multicolor capsules exhibit excellent structural stability, generate blue-, green-, and red-colored emissions when reacting with H 2 O 2 , have long-lasting CL emission over 1 h, and successfully achieve the accurate multiple visualization detection of avian influenza virus subtype targets. Without the need for complex instruments and analysis procedures, the CL imaging assays can be carried out and recorded with a common smartphone. The detection limits for visualizing H1N1, H7N9, and H5N1 are 5.5, 7.6, and 9.0 pM, respectively. There is a linear range between 20.0 and 625 pM and excellent selectivity against interfering DNA. Furthermore, visualization detection has been successfully applied for the detection of H1N1, H7N9, and H5N1 in healthy human serum samples. With these merits, this facile, ultrasensitive, and multiple visualization sensor has potential applications in point-of-care testing and early diagnosis.

Between 2013 and 2018, the novel A/Anhui/1/2013 (AH/13)-lineage H7N9 virus caused at least five waves of outbreaks in humans, totaling 1,567 confirmed human cases in China. Surveillance data indicated a disproportionate distribution of poultry infected with this AH/13-lineage virus, and laboratory experiments demonstrated that this virus can efficiently spread among chickens but not among Pekin ducks. The underlying mechanism of this selective transmission remains unclear. In this study, we demonstrated the absence of Neu5Gc expression in chickens across all respiratory and gastrointestinal tissues. However, Neu5Gc expression varied among different duck species and even within the tissues of the same species. The AH/13-lineage viruses exclusively bind to acetylneuraminic acid (Neu5Ac), in contrast to wild waterbird H7 viruses that bind both Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The level of Neu5Gc expression influences H7 virus replication and facilitates adaptive mutations in these viruses. In summary, our findings highlight the critical role of Neu5Gc in affecting the host range and interspecies transmission dynamics of H7 viruses among avian species.IMPORTANCEMigratory waterfowl, gulls, and shorebirds are natural reservoirs for influenza A viruses (IAVs) that can occasionally spill over to domestic poultry, and ultimately humans. This study showed wild-type H7 IAVs from waterbirds initially bind to glycan receptors terminated with N-acetylneuraminic acid (Neu5Ac) or N-glycolylneuraminic acid (Neu5Gc). However, after enzootic transmission in chickens, the viruses exclusively bind to Neu5Ac. The absence of Neu5Gc expression in gallinaceous poultry, particularly chickens, exerts selective pressure, shaping IAV populations, and promoting the acquisition of adaptive amino acid substitutions in the hemagglutinin protein. This results in the loss of Neu5Gc binding and an increase in virus transmissibility in gallinaceous poultry, particularly chickens. Consequently, the transmission capability of these poultry-adapted H7 IAVs in wild water birds decreases. Timely intervention, such as stamping out, may help reduce virus adaptation to domestic chicken populations and lower the risk of enzootic outbreaks, including those caused by IAVs exhibiting high pathogenicity., Competing Interests: The authors declare no conflict of interest.

Cases of H5 highly pathogenic avian influenzas (HPAI) are on the rise. Although mammalian spillover events are rare, H5N1 viruses have an estimated mortality rate in humans of 60%. No human cases of H5 infection have been reported in Malaysian Borneo, but HPAI has circulated in poultry and migratory avian species transiting through the region. Recent deforestation in coastal habitats in Malaysian Borneo may increase the proximity between humans and migratory birds. We hypothesise that higher rates of human-animal contact, caused by this habitat destruction, will increase the likelihood of potential zoonotic spillover events. In 2015, an environmentally stratified cross-sectional survey was conducted collecting geolocated questionnaire data in 10,100 individuals. A serological survey of these individuals reveals evidence of H5 neutralisation that persisted following depletion of seasonal H1/H3 HA binding antibodies from the plasma. The presence of these antibodies suggests that some individuals living near migratory sites may have been exposed to H5 HA. There is a spatial and environmental overlap between individuals displaying high H5 HA binding and the distribution of migratory birds. We have developed a novel surveillance approach including both spatial and serological data to detect potential spillover events, highlighting the urgent need to study cross-species pathogen transmission in migratory zones., (© 2024. The Author(s).)

Competing Interests: The authors have declared that no competing interests exist

The ongoing outbreak of the Goose/Guangdong/1/1996 (Gs/Gd) H5 lineage of highly pathogenic avian influenza (HPAI) viruses has caused higher mortality than all other HPAI outbreaks taken together. It is unique in its spillover and adaptation to wild waterfowl, which has facilitated its spread worldwide to many other species. However, avian influenza virus (AIV) surveillance, which historically aims to protect the poultry sector, is inadequate to document the spread and impact of Gs/Gd H5 virus in wild birds and other wildlife in most countries. A positive exception is Canada's AIV surveillance in wild birds, applied in a recent study (J. A. Giacinti, A. V. Signore, M. E. B. Jones, L. Bourque, et al., mBio 15:e03203-23, 2024, https://doi.org/10.1128/mbio.03203-23), which aims to protect wildlife, domestic animals, and human health according to the One Health approach. It is recommended to follow this approach in other countries to fill knowledge gaps in the epidemiology of Gs/Gd H5 virus in wild birds and other wildlife and to help control and, above all, prevent future HPAI outbreaks., Competing Interests: The author declares no conflict of interest.

The influenza neuraminidase (NA) is a potential target for the development of a next-generation influenza vaccine, but its antigenicity is not well understood. Here, we isolate an anti-N6 human monoclonal antibody, named 18_14D, from an H5N6 avian influenza virus (AIV) infected patient. The antibody weakly inhibits enzymatic activity but confers protection in female mice, mainly via ADCC function. The cryo-EM structure shows that 18_14D binds to a unique epitope on the lateral surface of the N6 tetramer, preventing the formation of tightly closed NA tetramers. These findings contribute to the molecular understanding of protective immune responses to NA of AIVs in humans and open an avenue for the rational design of NA-based vaccines., (© 2024. The Author(s).)

The lack of consolidated information regarding the response of wild bird species to infection with avian influenza virus (AIV) is a challenge to both conservation managers and researchers alike, with related sectors also impacted, such as public health and commercial poultry. Using two independent searches, we reviewed published literature for studies describing wild bird species experimentally infected with avian influenza to assess host species' relative susceptibility to AIVs. Additionally, we summarize broad-scale parameters for elements such as shedding duration and minimum infectious dose that can be used in transmission modelling efforts. Our synthesis shows that waterfowl (i.e. Anatidae) compose the vast majority of published AIV pathobiology studies, whereas gulls and passerines are less represented in research despite evidence that they also are susceptible and contribute to highly pathogenic avian influenza disease dynamics. This study represents the first comprehensive effort to compile available literature regarding the pathobiology of AIVs in all wild birds in over a decade. This database can now serve as a tool to all researchers, providing generalized estimates of pathobiology parameters for a variety of wild avian families and an opportunity to critically examine and assess what is known and identify where further insight is needed.

The current highly pathogenic avian influenza H5N1 panzootic is having substantial impacts on wild birds and marine mammals. Following major and widespread outbreaks in South America, an incursion to Antarctica occurred late in the austral summer of 2023/2024 and was confined to the region of the Antarctic Peninsula. To infer potential underlying processes, we compiled H5N1 surveillance data from Antarctica and sub-Antarctic Islands prior to the first confirmed cases., (© 2024 The Author(s). Influenza and Other Respiratory Viruses published by John Wiley & Sons Ltd.)

BackgroundOutbreaks of highly pathogenic avian influenza (HPAI) on poultry farms and in wild birds worldwide persists despite intensified control measures. It causes unprecedented mortality in bird populations and is increasingly affecting mammalian species. Better understanding of HPAI introduction pathways into farms are needed for targeted disease prevention and control. The relevance of airborne transmission has been suggested but research involving air sampling is limited and unequivocal evidence on transmission routes is lacking.AimWe aimed to investigate whether HPAI virus from wild birds can enter poultry houses through air inlets by characterising host materials through eukaryote DNA sequencing.MethodsWe collected particulate matter samples in and around three HPAI-affected poultry farms which were cleared and decontaminated before sampling. Indoor measurements (n = 61) were taken directly in the airflow entering through air inlets, while outdoor air samples (n = 60) were collected around the poultry house. Positive controls were obtained from a bird rehabilitation shelter. We performed metabarcoding on environmental DNA by deep sequencing 18S rRNA gene amplicons.ResultsWe detected waterbird DNA in air inside all three, and outside of two, poultry farms. Sequences annotated at species level included swans and tufted ducks. Waterbird DNA was present in all indoor and outdoor air samples from the bird shelter.ConclusionAirborne matter derived from contaminated wild birds can potentially introduce HPAI virus to poultry houses through air inlets. The eDNA metabarcoding could assess breaches in biosecurity for HPAI virus and other pathogens potentially transmitted through air via detection of their hosts.

In this study the pathogenicity, infectivity, and transmissibility of H5N1 highly pathogenic avian influenza (HPAI) clade 2.2.1.2 and H5N8 HPAI clade 2.3.4.4b viruses were evaluated in commercial broilers on days 24 and 31. The mortality rate was 100 % in both challenge viruses and in contact birds either on day 24 or day 31 which confirmed the highly pathogenicity of both clades (2.2.1.2/ 2.3.4.4b) in commercial broilers. Both clades (H5N8 clade 2.3.4.4b/ H5N1 clade 2.2.1.2 viruses) were efficiently replicate within and transmitted between commercial broilers. The H5N8-infected birds shed high titer of viruses from oropharynx and cloaca, which associated with the field spread of AIV-H5N8 in commercial broilers. Mean lesion score in both challenged clades showed similar scores, which confirmed the pathogenicity of both clades in commercial broilers' organs (mainly spleen, cerebellum, thymus, Bursa, Lung) which confirm the neurogenic affinity of the virus. In the central nervous system, non-suppurative encephalitis consisting in multifocal areas of necrosis in cerebral hemispheres, intense spongiosis, neuronal chromatolysis and gliosis were commonly observed. In cerebrum, chromatolysis of Purkinje neurons was a common finding. In the lung, interstitial pneumonia consisting of moderate to severe increase of the cellularity (macrophages and lymphoid cells) in air capillaries and focal areas of necrosis associated with intense viral replication was commonly observed. In lymphoid tissues, including spleen, thymus, and bursa of Fabricius, multifocal areas of necrosis/apoptosis of variable intensity in mononuclear cells were present. Particularly, diffuse necrotic areas were present in the spleen. In the liver, we detected focal areas of necrosis with mild distention of hepatic sinusoids. To conclude the AIV either H5N1 or H5N8 have neurological affinity with immune suppression effect based on necrosis and apoptosis of lymphoid tissues., 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 © 2024 Elsevier Ltd. All rights reserved.)

Highly pathogenic avian influenza A(H5N1) virus was detected in dead seals on Tyuleniy Island in eastern Russia, in the Sea of Okhotsk. Viruses isolated from dead northern fur seals belong to clade 2.3.4.4b and are closely related to viruses detected predominantly in the Russian Far East and Japan in 2022-2023.

On March 25, 2024 an outbreak of highly pathogenic avian influenza (HPAI) A H5N1 was identified in dairy cows across multiple farms in the United States. Zoonotic cases originating in individuals with close contact to infected herds and poultry flocks have been subsequently identified. Spillover events such as this raise the specter of recent pandemics including COVID-19 and Mpox and may lead clinical laboratories to assess their capacity for diagnosis of HPAI H5N1. In this review, we detail the origins of the H5N1 clade 2.3.4.4b outbreak as well as the existing capacity to identify HPAI H5N1 as influenza A virus by commercially available assays. Furthermore, we highlight the absence of commercially available influenza A H5 subtyping assays and limitations associated with the current 510(k)-cleared assay. This outbreak also serves as an early opportunity to assess the new and unknown regulatory challenges faced by laboratory-developed tests in light of the FDA's final rule on in vitro diagnostic devices. National agencies along with public health and clinical laboratories all serve an essential role in the response to HPAI H5N1. To most effectively utilize each group's strength requires open communication and willingness to embrace novel approaches., 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Deadly outbreaks among poultry, wild birds, and carnivorous mammals by the highly pathogenic H5N1 virus of the clade 2.3.4.4b have been reported in South America. The increasing virus incidence in various mammal species poses a severe zoonotic and pandemic threat. In Uruguay, the clade 2.3.4.4b viruses were first detected in February 2023, affecting wild birds and backyard poultry. Three months after the first reported case in Uruguay, the disease affected a population of 23 coatis (Nasua) in an ecological park. Most animals became infected, likely directly or indirectly from wild birds in the park, and experienced sudden death. Five animals from the colony survived, and four of them developed antibodies. The genomes of the H5N1 strains infecting coatis belonged to the B3.2 genotype of the clade 2.3.4.4b. Genomes from coatis were closely associated with those infecting backyard poultry, but transmission likely occurred through wild birds. Notable, two genomes have a 627K substitution in the RNA polymerase PB2 subunit, a hallmark amino acid linked to mammalian adaptation. Our findings support the ability of the avian influenza virus of the 2.3.4.4b clade to infect and transmit among terrestrial mammals with high pathogenicity and undergo rapid adaptive changes. It also highlights the coatis' ability to develop immunity and naturally clear the infection., 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 © 2024 Elsevier Ltd. All rights reserved.)

This study analyzes the effect of avian influenza outbreaks on retail price premiums in the US poultry market. We estimate hedonic price models for eggs, chickens, and turkeys, controlling for quality characteristics, unobserved time, and regional factors. To measure the impact of avian influenza outbreaks we use 2 proxies. The first proxy is a measure of the number of new bird infections at the production level. The second proxy measures online search queries related to the outbreak. The results show that, on average, prices increase across product categories, that is, egg, broiler, and turkey markets, during avian influenza outbreaks. Furthermore, we observe price convergence and reduced dispersion within product categories, which is consistent with the economic theory of asymmetric substitutability between conventional and premium products. Our analysis finds that the HPAI outbreak caused a reduction of the price gap between conventional and premium products., Competing Interests: DISCLOSURES 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian influenza remains a global public health concern for its well-known point mutation and genomic segment reassortment, through which plenty of serum serotypes are generated to escape existing immune protection in animal and human populations. Some occasional cases of human infection of avian influenza viruses (AIVs) since 2020 posed a potential pandemic risk through human-to-human transmission. Both east-west and north-south migratory birds fly through and linger in the Hebei Province of China as a stopover habitat, providing an opportunity for imported AIVs to infect the local poultry and for viral gene reassortment to generate novel stains. In this study, we collected more than 6000 environmental samples (mostly feces) in Hebei Province from 2021 to 2023. Samples were screened using real-time RT-PCR, and virus isolation was performed using the chick embryo culture method. We identified 10 AIV isolates, including a novel reassortant H3N3 isolate. Sequencing analysis revealed these AIVs are highly homologous to those isolated in the Yellow River Basin. Our findings supported that AIVs keep evolving to generate new isolates, necessitating a continuous risk assessment of local avian influenza in wild waterfowl in Hebei, China., Competing Interests: Declaration of Competing Interest No potential conflict of interest was reported by the author(s)., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In July 2022, an outbreak of highly pathogenic avian influenza A(H5N1) virus clade 2.3.4.4b occurred among migratory birds at Qinghai Lake in China. The virus circulated in June, and reassortants emerged after its introduction into the area. Surveillance in 2023 showed that the virus did not establish a stable presence in wild waterfowl.

Nigeria recorded one of the earliest outbreaks of the Highly Pathogenic Avian Influenza (HPAI) virus H5N1 in 2006, which spread to other African countries. In 2023, 18 countries reported outbreaks of H5N1 in poultry, with human cases documented in Egypt, Nigeria, and Djibouti. There is limited information on the molecular epidemiology of HPAI H5N1 in Nigeria. We determined the molecular epidemiology and genetic evolution of the virus from 2006 to 2021. We investigated the trend and geographical distribution across Nigeria. The evolutionary history of 61 full-length genomes was performed from 13 countries worldwide, and compared with sequences obtained from the early outbreaks in Nigeria up to 2021. MEGA 11 was used to determine the phylogenetic relationships of H5N1 strains, which revealed close ancestry between sequences in Nigeria and those from other African countries. Clade classification was performed using the subspecies classification tool for Bacterial and Viral Bioinformatics Research Center (BV-BRC) version 3.35.5. H5N1 Clade 2.2 was observed in 2006, with 2.3.2, 2.3.2.1f clades observed afterwards and 2.3.4.4b in 2021. Our findings underscore the need for genomics surveillance to track antigenic variation and clades switching to monitor the epidemiological of the virus and safeguard human and animal health.Impacts Specific variations in the hemagglutinin (HA) and neuraminidase (NA) genes of Avian influenza virus are consistent in different geographical regions. H5N1 Clade 2.2 was reported in 2006, with 2.3.2, 2.3.2.1f afterwards and 2.3.4.4b in 2021. Nigeria is an epicentre for avian influenza with three major migratory routes for wild birds transversing the country. It is plausible that the Avian influenza in Northern Nigeria may be linked to wild bird sanctuaries in the region., (© 2024. The Author(s).)

Scaup, including both Lesser and Greater (Aythya affinis and Aythya marila, respectively), are a grouping of populous and widespread North American diving ducks. Few influenza type A viruses (IAV) have been reported from these species despite a high prevalence of antibodies to IAV being reported. Existing virologic and serologic data indicate that IAV infection routinely occurs in scaup, yet it is unknown which IAV subtypes are linked to these infections. In this study, we aimed to gain a more complete picture of IAV natural history in Lesser and Greater Scaup from two coastal flyways in North America in 2015-18 (302 samples from California in the Pacific Flyway and 471 samples from Maryland in the Atlantic Flyway). Low prevalence of active IAV infection was detected by real-time reverse-transcription PCR in Lesser Scaup sampled in Maryland and California (2.8% and 8.1%, respectively). A single IAV (H1N1) was isolated in embryonated chicken eggs from a bird sampled in California. Similarly low levels were observed in Greater Scaup in California (3.3%). Antibodies to the nucleoprotein as detected with a commercial blocking ELISA were observed in all species and flyway combinations. Antibody seroprevalence estimates were higher in adult Lesser Scaup than in juveniles at both the ≤0.5 (P<0.001, z=-3.582) and ≤0.7 serum-sample-to-negative-control absorbance thresholds (P=0.003, z=-2.996). Neutralizing antibodies to H1-H12, H14, and H15 were detected using a microtiter virus neutralization assay, with the highest prevalence of antibodies against H1 (38%), H6 (36%), and H11 (35%). The high prevalence of antibodies to IAV and evidence of previous exposure to numerous subtypes are consistent with a high level of population immunity and a low prevalence of infection. These results must be interpreted in the context of season (winter sampling), as results may vary with the annual influx of naïve juvenile birds., (© Wildlife Disease Association 2024.)

Purpose of Review: This review aims to discuss the current state of human infections with Avian Influenza A (H5) and (H9) viruses, to support awareness of the global epidemiology among clinicians and public health professionals interested in emerging respiratory infections., Recent Findings: Among increasing numbers of detections in avian species of Avian Influenza A(H5N1) clade 2.3.4.4b globally, reported human cases of severe infection have been rare.Enhanced surveillance of persons exposed to avian species infected with Influenza A (H5N1) clade 2.3.4.4b in different countries has identified small numbers of asymptomatic individuals with Avian Influenza A (H5N1) detected by PCR from the upper respiratory tract; some of these instances have been considered to represent contamination rather than infection.There have also been recent sporadic human cases of Avian Influenza A(H9N2) internationally, including in China and Cambodia., Summary: Human infections with Avian Influenza A(H5) and (H9) viruses remain of interest as an emerging infection both to clinicians and public health professionals. While maintaining effective surveillance is essential, one health strategies to control infection in avian species will be key to mitigating these risks., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Infections of dairy cattle with clade 2.3.4.4b H5N1 highly pathogenic avian influenza virus (HPAIV) were reported in March 2024 in the U.S. and viable virus was detected at high levels in raw milk from infected cows. This study aimed to determine the potential quantities of infectious HPAIV in raw milk in affected states where herds were confirmed positive by USDA for HPAIV (and therefore were not representative of the entire population), and to confirm that the commonly used continuous flow pasteurization using the FDA approved 72 °C (161°F) for 15 s conditions for high-temperature short time (HTST) processing, will inactivate the virus. Double-blinded raw milk samples from bulk storage tanks from farms (n = 275) were collected in four affected states. Samples were screened for influenza A using quantitative real-time RT-PCR (qrRT-PCR) of which 158 (57.5%) were positive and were subsequently quantified in embryonating chicken eggs. Thirty-nine qrRT-PCR positive samples (24.8%) were positive for infectious virus with a median titer of 3.5 log 10 50% egg infectious doses (EID 50 ) per mL. To closely simulate commercial milk pasteurization processing systems, a pilot-scale continuous flow pasteurizer was used to evaluate HPAIV inactivation in artificially contaminated raw milk using the most common legal conditions in the US: 72 °C (161°F) for 15 s. Among all replicates at two flow rates (n = 5 at 0.5 L/min; n = 4 at 1 L/min), no viable virus was detected. A mean reduction of ≥5.8 ± 0.2 log 10 EID 50 /mL occurred during the heating phase where the milk is brought to 72.5 °C before the holding tube. Estimates from heat-transfer analysis support that standard U.S. continuous flow HTST pasteurization parameters will inactivate >12 log 10 EID 50 /mL of HPAIV, which is ∼9 log 10 EID 50 /mL greater than the median quantity of infectious virus detected in raw milk from bulk storage tank samples. These findings demonstrate that the US milk supply is safe when pasteurized., 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., (Published by Elsevier Inc.)

Influenza A viruses (IAV) pose substantial burden on human and animal health. Avian, swine and human IAV bind sialic acid on host glycans as receptor, whereas some bat IAV require MHC class II complexes for cell entry. It is unknown how this difference evolved and whether dual receptor specificity is possible. Here we show that human H2N2 IAV and related avian H2N2 possess dual receptor specificity in cell lines and primary human airway cultures. Using sialylation-deficient cells, we reveal that entry via MHC class II is independent of sialic acid. We find that MHC class II from humans, pigs, ducks, swans and chickens but not bats can mediate H2 IAV entry and that this is conserved in Eurasian avian H2. Our results demonstrate that IAV can possess dual receptor specificity for sialic acid and MHC class II, and suggest a role for MHC class II-dependent entry in zoonotic IAV infections., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Avian chlamydiosis is a serious avian infection that carries a significant zoonotic danger to the poultry industry. The respiratory co-infections caused by the low pathogenic avian influenza virus H9N2 (LPAIV H9N2) also cause significant financial losses in the poultry industry. The purpose of this study was to examine the pathogenicity of Chlamydophila psittaci, and LPAIV H9N2 individually and in combination in broiler chickens, as well as to determine whether or not aqueous neem (Azadirachta indica) leaf extract is effective against infections caused by these pathogens. Therefore, 120 broiler cobb chicks were equally divided into 4 groups (30 birds each) with triplicates with 10 birds. Broilers in group 1 (G1) were infected with only C. psittaci, broilers in group 2 (G2) were infected with only LPAIV H9N2, broilers in group 3 (G3) were infected with C. psittaci and LPAIV H9N2, and broilers in group 4 (G4) remained not challenged and non-treated with any therapeutic or preventive treatment (negative control). At 21 d postinfection (dpi), birds in G1, G2, and G3 were divided into 3 subgroups of 10 birds each: subgroup (A) remained infected and untreated (positive control), subgroup (B) infected and received oxytetracycline for 5 consecutive d, and subgroup (C) infected and received 8% aqueous neem leaf extract for 5 consecutive d. The multiplication of C. psittaci in birds in G1, in various tissues was evaluated using Giemsa staining and the data showed that multiplication was much higher in the lung, spleen, and liver from 6 h to 21 dpi, but low in the heart from 8 to 21 dpi. During simultaneous co-infection in G3, the birds developed significant clinical symptoms and postmortem lesions (PM). Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect viral shedding from oropharyngeal and cloacal swabs between 2 dpi and 8 dpi, with cycle threshold (CT) values ranging from 22 to 24. In contrast, bacterial shedding began 6 h after infection and continued until 21 dpi, with CT values ranging from 23 to 26. Administration of an aqueous neem leaf extract at an 8% concentration (Group C) resulted in a numerical rise in average body weight across all treatment groups in the third and fourth week, as well as a reduction in LPAIV H9N2 and C. psittaci replication in the respiratory and gut of treated birds compared to those treated with oxytetracycline (Group B). Overall, respiratory co-infections pose a considerable risk to the poultry business, which is a big threat. To control C. psittaci and LPAIV H9N2 in broiler chickens, oral supplementation of 8% aqueous neem leaf extract is recommended. This treatment improves the birds' performance, as evidenced by an increase in their average body weight. In addition, the application of 8% aqueous neem leaf extract lowers C. psittaci replication within tissues and diminishes LPAIV H9N2 shedding., Competing Interests: DISCLOSURES The authors declare no conflicts of interest., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Highly pathogenic avian influenza (HPAI) poses a significant threat to animal and public health, with outbreaks occurring globally. HPAI poses significant challenges due to its high mortality rate and public health concerns, with outbreaks spreading globally since the emergence of the H5N1 virus in 2003. In Japan, HPAI outbreaks have been particularly prevalent during autumn and winter seasons, with the 2022-2023 winter experiencing the most severe outbreak to date. However, limited research has directly examined the association between HPAI outbreaks and weather conditions in Japan. Here we show that specific weather conditions are associated with an increased risk of HPAI outbreaks on poultry farms in Japan. By analyzing databases of HPAI cases and meteorological data from 2020-2023, we found that higher average air temperatures two to three weeks prior, lower average wind speeds four weeks prior, and longer sunlight hours two and four weeks prior to outbreaks were significantly associated with increased risk of HPAI outbreaks in Japan. These results suggest that weather may influence environmental survival and transmission of the virus, as well as patterns of wild bird movement that could seed new outbreaks. These findings enhance our understanding of the factors influencing HPAI transmission dynamics and highlight the importance of integrating weather forecasts into disease surveillance and prevention strategies.

After viral infection, the virus relies on the host cell's complex metabolic and biosynthetic machinery for replication. However, the impact of avian influenza virus (AIV) on metabolites and gene expression in poultry cells remains unclear. To investigate this, we infected chicken embryo fibroblasts DF1 cells with H9N2 AIV at an MOI of 3. Our aim was to explore how H9N2 AIV alters DF1 cells metabolic pathways to facilitate its replication. We employed metabolomics and transcriptomics techniques to analyze changes in metabolite content and gene expression. Metabolomics analysis revealed a significant increase in glutathione-related metabolites, including reduced glutathione (GSH), oxidized glutathione (GSSG) and total glutathione (T-GSH) upon H9N2 AIV infection in DF1 cells. Elisa results confirmed elevated levels of GSH, GSSG, and T-GSH consistent with metabolomics findings, noting a pronounced increase in GSSG compared to GSH. Transcriptomics showed significant alterations in genes involved in glutathione synthesis and metabolism post-H9N2 infection. However, adding the glutathione synthesis inhibitor BSO exogenously significantly promoted H9N2 replication in DF1 cells. This was accompanied by increased mRNA levels of pro-inflammatory cytokines (IL-1β, IFN-γ) and decreased mRNA levels of anti-inflammatory cytokines (TGF-β, IL-13). BSO also reduced catalase (CAT) gene expression and inhibited its activity, leading to higher reactive oxygen species (ROS) and malondialdehyde (MDA) level in DF1 cells. qPCR results indicated decreased mRNA levels of Nrf2, NQO1, and HO-1 with BSO, ultimately increasing oxidative stress in DF1 cells. Therefore, the above results indicated that H9N2 AIV infection in DF1 cells activated the glutathione metabolic pathway to enhance the cell's self-defense mechanism against H9N2 replication. However, when GSH synthesis is inhibited within the cells, it leads to an elevated oxidative stress level, thereby promoting H9N2 replication within the cells through Nrf2/HO-1 pathway. This study provides a theoretical basis for future rational utilization of the glutathione metabolic pathway to prevent viral replication., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In search of novel therapeutic options to treat influenza virus (IV) infections, we previously identified a series of inhibitors that act by disrupting the interactions between the PA and PB1 subunits of the viral RNA polymerase. These compounds showed broad-spectrum antiviral activity against human influenza A and B viruses and a high barrier to the induction of drug resistance in vitro. In this short communication, we investigated the effects of combinations of the PA-PB1 interaction inhibitor 54 with oseltamivir carboxylate (OSC), zanamivir (ZA), favipiravir (FPV), and baloxavir marboxil (BXM) on the inhibition of influenza A and B virus replication in vitro. We observed a synergistic effect of the 54/OSC and 54/ZA combinations and an antagonistic effect when 54 was combined with either FPV or BXM. Moreover, we demonstrated the efficacy of 54 against highly pathogenic avian influenza viruses (HPAIVs) both in cell culture and in the embryonated chicken eggs model. Finally, we observed that 54 enhances OSC protective effect against HPAIV replication in the embryonated eggs model. Our findings represent an advance in the development of alternative therapeutic strategies against both human and avian IV infections., 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 © 2024. Published by Elsevier B.V.)

Competing Interests: We declare no competing interests.

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV., Competing Interests: DISCLOSURES 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 © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475 LRGVRVSK 482 and 528 TITYSSPMMW 537 . Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

The highly pathogenic avian influenza (HPAI) H5N1 virus clade 2.3.4.4b has caused the death of millions of domestic birds and thousands of wild birds in the USA since January 2022 (refs. 1-4 ). Throughout this outbreak, spillovers to mammals have been frequently documented 5-12 . Here we report spillover of the HPAI H5N1 virus to dairy cattle across several states in the USA. The affected cows displayed clinical signs encompassing decreased feed intake, altered faecal consistency, respiratory distress and decreased milk production with abnormal milk. Infectious virus and viral RNA were consistently detected in milk from affected cows. Viral distribution in tissues via immunohistochemistry and in situ hybridization revealed a distinct tropism of the virus for the epithelial cells lining the alveoli of the mammary gland in cows. Whole viral genome sequences recovered from dairy cows, birds, domestic cats and a raccoon from affected farms indicated multidirectional interspecies transmissions. Epidemiological and genomic data revealed efficient cow-to-cow transmission after apparently healthy cows from an affected farm were transported to a premise in a different state. These results demonstrate the transmission of the HPAI H5N1 clade 2.3.4.4b virus at a non-traditional interface, underscoring the ability of the virus to cross species barriers., (© 2024. The Author(s).)