Search Results (130)

Background/Objectives: Outbreaks of highly pathogenic avian influenza virus (AIV) result in significant financial losses and the death or depopulation of millions of domestic birds. Early and rapid detection and surveillance are needed to slow the spread of AIV and prevent its spillover to humans. The volatile metabolome (i.e., the pattern of volatile metabolites emitted by a living subject) represents one such source of health information that can be monitored for disease diagnosis. Indeed, dogs have been successfully trained to recognize patterns of “body odors” associated with many diseases. Because little is known regarding the mechanisms involved in the alteration of the volatile metabolome in response to health perturbation, questions still arise regarding the specificity, or lack thereof, of these alterations. Methods: To address this concern, we experimentally infected twenty mallard ducks with one of two different strains of low-pathogenic AIV (ten ducks per strain) and collected cloacal swabs at various time points before and after infection. Results: Headspace analyses revealed that four volatiles were significantly altered following infection, with distinct profiles associated with each viral strain. The volatiles that differed between strains among post-infection sampling periods included ethylbenzyl ether (p = 0.00006), 2-phenoxyethanol (p = 0.00017), 2-hydroxybenzaldehyde (p = 0.00022), and 6-methyl-5-hepten-2-one (p = 0.00034). Conclusions: These findings underscore that AIV-induced changes to the volatile metabolome are strain-specific, emphasizing the need for disease-specific profiling in diagnostic development. Full article

Highly pathogenic avian influenza (HPAI) H5N1, particularly clade 2.3.4.4b, has demonstrated an unprecedented capacity for cross-species transmission, with recent reports confirming its presence in dairy cattle in the United States of America (USA) in 2024. This unexpected spillover challenges traditional understanding of the virus’s host range and raises serious public health and veterinary concerns. Infected cattle presented with clinical signs such as decreased milk production, thickened or discolored milk, respiratory issues, and lethargy. Pathological findings revealed inflammation of the mammary glands and the detection of a virus in nasal secretions and raw milk, suggesting a potential for both intra- and interspecies transmission. While the current risk of human-to-human transmission remains low, the detection of H5N1 in a human exposed to infected cattle highlights the need for heightened surveillance and protective measures. Moreover, the presence of infectious viruses in the food chain, particularly in unpasteurized milk, introduces a new dimension of zoonotic risk. This review synthesizes emerging evidence on the epidemiology, pathology, diagnostic findings, and zoonotic implications of HPAI H5N1 infection in cattle. It also highlights the importance of genomic surveillance, intersectoral collaboration, and One Health approaches in managing this evolving threat. As the virus continues to circulate and adapt across diverse hosts, including wild birds, domestic poultry, and now mammals, the potential for reassortment and emergence of novel strains remains a significant concern. Immediate actions to strengthen biosecurity, monitor viral evolution, and protect both animal and human populations are critical to mitigate the global risk posed by this expanding panzootic. Full article

Avian influenza is an economically significant disease affecting poultry worldwide and is caused by influenza A viruses that can range from low to highly pathogenic strains. These viruses primarily target the respiratory, digestive, and nervous systems of birds, leading to severe outbreaks that threaten poultry production and pose zoonotic risks. The ectodomain of the avian influenza virus (AIV) matrix protein 2 (M2e), known for its high conservation across influenza strains, has emerged as a promising candidate for developing a universal influenza vaccine in a mouse model. However, the efficacy of such expression against poultry AIVs remains limited. The objective of this study was to evaluate the immunogenicity of nodavirus-like particles displaying the M2e proteins. In this study, three synthetic heterologous M2e genes originated from AIV strains H5N1, H9N2 and H5N2 were fused with the nodavirus capsid protein (NVC) of the giant freshwater prawn Macrobrachium rosenbergii (NVC-3xAvM2e) prior to immunogenicity characterisations in chickens. The expression vector pTRcHis-TARNA2 carrying the NVC-3xAvM2e gene cassette was introduced into E. coli TOP-10 cells. The recombinant proteins were purified, inoculated into one-week-old specific pathogen-free chickens subcutaneously and analysed. The recombinant protein NVC-3xAvM2e formed virus-like particles (VLPs) of approximately 25 nm in diameter when observed under a transmission electron microscope. Dynamic light scattering (DLS) analysis revealed that the VLPs have a polydispersity index (PDI) of 0.198. A direct ELISA upon animal experiments showed that M2e-specific antibodies were significantly increased in vaccinated chickens after the booster, with H5N1 M2e peptides having the highest mean absorbance value when compared with those of H9N2 and H5N2. A challenge study using low pathogenic AIV (LPAI) strain A/chicken/Malaysia/UPM994/2018 (H9N2) at 10^6.5 EID₅₀ showed significant viral load in the lung and cloaca, but not in the oropharyngeal of vaccinated animals when compared with the unvaccinated control group. Collectively, this study suggests that nodavirus-like particles displaying three heterologous M2e have the potential to provide protection against LPAI H9N2 in chickens, though the vaccine’s efficacy and cross-protection across different haemagglutinin (HA) subtypes should be further evaluated. Full article

The avian influenza virus, particularly the highly pathogenic H5N1 subtype, represents a significant public health threat due to its interspecies transmission potential and growing resistance to current antiviral therapies. To address this, the identification of novel and effective neuraminidase (NA) inhibitors is critical. In this study, an integrated in silico strategy was employed, beginning with the generation of an energy-optimized pharmacophore model (e-pharmacophore, ADDN) based on the reference inhibitor Zanamivir. A virtual screening of 47,781 natural compounds from the PubChem database was performed, followed by molecular docking validated through an enrichment assay. Promising hits were further evaluated via ADMET predictions, density functional theory (DFT) calculations to assess chemical reactivity, and molecular dynamics (MD) simulations to examine the stability of the ligand–protein complexes. Three lead compounds (C1: CID 102209473, C2: CID 85692821, and C3: CID 45379525) demonstrated strong binding affinity toward NA. Their ADMET profiles predicted favorable bioavailability and low toxicity. The DFT analyses indicated suitable chemical reactivity, particularly for C2 and C3. The MD simulations confirmed the structural stability of all three ligand–NA complexes, supported by robust and complementary intermolecular interactions. In contrast, Zanamivir exhibited limited hydrophobic interactions, compromising its binding stability within the active site. These findings offer a rational foundation for further experimental validation and the development of next-generation NA inhibitors derived from natural sources. Full article

Infectious bronchitis virus (IBV) and low-pathogenic avian influenza virus (LPAIV) H9N2 are commonly identified in poultry, individually or in association with other pathogens. This study monitored 183 broiler farms affected by respiratory diseases across seven regions of Morocco from January 2021 to December 2023. Among these farms, 87.98% were vaccinated against IBV, while 57.92% were against AI H9N2. Abnormally high mortality rates were observed in 44.26% of the farms, with 24.69% of cases attributed to IBV, 50.62% to LPAI H9N2, and 13.58% due to coinfection with both IBV and H9N2. RT-PCR analysis of tissue samples and cloacal and tracheal swabs collected from 183 broiler farms revealed that 33.33% were positive for IBV and 34.97% for H9N2. Coinfection by IBV and H9N2 was detected in 12.57% of cases, peaking at 17% in 2022. Co-infected flocks exhibited severe clinical signs and lesions, such as reduced food consumption, diarrhea, and renal issues. The predominant lesions were in the respiratory tract, affecting 91.26% of infected broilers. Additionally, among the 183 flocks, 50 farms that tested positive for IBV infection were randomly selected from the seven regions of Morocco for further investigation of other respiratory pathogens, including Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), and infectious laryngotracheitis (ILT), using real-time RT-PCR. Detection rates for these pathogens were 26% for MG, 30% for MS, 4% for ILTv (vaccine strain), and 18% for ILTw (wild strain). Detection rates for single, dual, triple, and quadruple infections were 34%, 42%, 18%, and 4%, respectively. The most common dual and triple coinfections were IBV + H9N2 (14%) and IBV + MG + MS (10%). Phylogenetic analysis of the S gene identified two main IBV genotypes, namely, 793B and D181, with the latter being a strain circulating for the first time in Moroccan poultry. This underscores the urgent need to establish surveillance systems to track pathogen circulation and implement strategies to control virus spread, ensuring the protection of animals and public health. Full article

Introduction: Highly pathogenic avian influenza (HPAI) H5N1, a zoonotic virus primarily affecting birds, has shown increasing cross-species transmission, including to domestic animals such as cats. Recent reports of cat infections, often associated with contact with infected birds or the consumption of raw milk from H5N1-positive cattle, raise concerns about their role in viral adaptation and zoonotic transmission. Objective: To assess the global prevalence and characteristics of H5N1 infections in cats (Felis catus) through a systematic review and meta-analysis. Methods: Following PRISMA guidelines, we conducted a systematic search across PubMed, Scopus, and Web of Science up to 1 March 2025. Observational studies reporting the prevalence or seroprevalence of H5N1 in cats (Felis catus) were included. Data extraction and quality assessment were performed independently by four reviewers. Meta-analyses were conducted using a random-effects model, and heterogeneity was assessed via I² statistics. Results: Twenty-one studies met the inclusion criteria, of which eight were included in the meta-analysis (n = 3586 cats). The pooled global prevalence of Felis catus infections due to H5N1 influenza was 0.7% (95%CI: 0.3–1.1%), with high heterogeneity (I² = 86.5%). The prevalence varied by the diagnostic method, region, cat type, and time. Domestic cats and those in Africa had higher infection rates (20.0% and 32.0%, respectively). Case reports (n = 35) revealed a high mortality (74%), predominantly from clade 2.3.4.4b, with neurological and respiratory manifestations. Conclusions: Although the overall prevalence is low, H5N1 infection in cats is increasing, particularly in clade 2.3.4.4b. Their close contact with humans and other animals highlights the need for enhanced surveillance, diagnostics, and One Health strategies to mitigate zoonotic risks. Full article

A characteristic feature of influenza A viruses is their high capacity for reassortment, significantly increasing their genetic diversity. This can lead to the formation of influenza A virus variants with unique phenotypic characteristics, particularly those with pandemic potential. Representatives of the H4N9 subtype are low-pathogenic influenza A (LPAI) viruses. Despite their low pandemic potential, these viruses may represent an important reservoir of genes for genetic exchange with other IAVs. Here, we analyzed the reassortment events of H4N9 viruses using all publicly available sequences. Several computational approaches, including phylogenetic reconstructions and reassortment detection algorithms (PDDM and PDCP), were used to identify phylogenetic incongruences. Numerous reassortment events were detected in H4N9 viruses, especially in the NS segment. This suggests extensive genetic exchange with other avian and mammalian IAVs. In addition, a comparison of phylogenetic and geographic patterns suggests that H4N9 viruses have undergone multiple trans-regional transmissions. These results suggest that LPAI viruses make a significant contribution to the overall influenza gene pool, increasing the likelihood of the emergence of new IAV variants with unpredictable phenotypic characteristics. However, our results suggest that the current understanding of the real distribution and genetic diversity is fragmented. Therefore, better monitoring and surveillance of H4N9 viruses should improve influenza pandemic preparedness. Full article

Background: Avian influenza (AI), caused by orthomyxoviruses, is a globally significant disease affecting avian and non-avian species. It manifests in two variants, according to the two biovariants of the virus differentiated as highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) strains, both of which compromise animal welfare, reduce productivity, and cause substantial economic loss. The zoonotic potential of HPAI strains, particularly the currently dominant clade 2.3.4.4b, raises concerns about public health and epidemic risks. This review assesses the results of current vaccine trials targeting HPAI clade 2.3.4.4b, emphasizing these studies because most outbreak strains in domestic poultry currently belong to this dominant clade. Methods: Multiple scientific databases comprised reports of research trials on vaccine efficacy against HPAI clade 2.3.4.4b. The Boolean term “Clade 2.3.4.4b AND vaccine” was entered into the following databases: PubMed, PubAg, Scopus, Cochrane Library, and ScienceDirect. Results: The resulting papers were analyzed. Studies revealed that antigenic similarity between vaccine and field strains enhances protective efficacy (PE), reduces viral shedding, and improves hemagglutination inhibition titers. While multivalent vaccines showed potential, results were inconsistent and varied depending on strain compatibility. Single-dose vaccines may provide sufficient PE for poultry, though ducks and geese often require multiple doses, and long-term PE is yet unknown. It was discovered that vector vaccines can provide appropriate PE against clade 2.3.4.4.b. Conclusions: Further analysis is needed as their effects may be short-lived, and subsequent doses may be required. Limited research exists on the long-term efficacy of these vaccines and their effectiveness in many avian species. Addressing these gaps is crucial for optimizing vaccination strategies. A re-evaluation of vaccination strategies is recommended but essential to implement adequate biosecurity measures on in poultry farms. This review synthesizes current evidence and may assist veterinarians and authorities in deciding whether to apply or license vaccines to reduce economic losses caused by AI. Full article

The novel H10N3 avian influenza virus (AIV) has infected four individuals since 2021 and caused severe respiratory damage, posing a significant threat to public health. However, its pathogenic mechanisms remain poorly understood. Our findings revealed that H10N3 infection induces severe lung damage and causes death in mice, even at low doses. The elevated levels of multiple pro-inflammatory factors in the bronchoalveolar lavage fluid were significantly increased during infection, displaying hallmarks of a cytokine storm. Transcriptome sequencing further revealed systematic activation of inflammation-related pathways, predicting that viral infection induces multiple forms of programmed cell death, including apoptosis, pyroptosis, and necroptosis. Protein-level validation showed that the activation of key cell death markers, including Caspase-3, GSDMD, and MLKL, significantly increased as the infection progressed, with their dynamic changes correlating strongly with the expression pattern of viral proteins. This study elucidates the central role of the synergistic effect between the cytokine storm and multiple cell death pathways in H10N3 pathogenesis. These findings not only advance our understanding of the pathogenic mechanisms of AIVs but also provide a critical theoretical basis for the development of targeted therapeutic strategies. Full article

Highly Pathogenic Avian Influenza H5N1 (HPAI H5N1) was first detected in chickens in Scottland in 1959 and has since circulated globally, causing regular outbreaks among different animal species, as well as incidental infections in humans. In this scoping review, the epidemiology and impact of HPAI H5N1 among migratory birds, poultry, and cattle in the United States were analyzed, with a particular focus on outbreaks since January 2022. Following PRISMA guidelines, a total of 27 articles were identified for this review. Publicly available data and reports from the USDA and CDC were also evaluated and summarized. The identified articles primarily included epidemiological studies of detections in wild birds, mammals, and case reports on H5N1 and transmission dynamics among cattle, with a notable absence of poultry-focused reports. Wild birds, especially migratory species, have played an important role in virus dissemination. Studies among mammals, including seals, bears, and domestic cats, along with the emerging outbreak among cattle, highlight the virus’s ability to adapt to diverse hosts, with the possibility of mammal-to-mammal transmission. Despite the low number of human infections, the zoonotic risk of the disease and the possibility of a human outbreak remain significant. The complexity and risks associated with the virus, in comparison with the limited current scientific studies in the United States, demand further investigations to mitigate its impact on animals, ecosystems, and human health. Full article

Background/Objective: Highly pathogenic (HP) H5Nx and low-pathogenicity (LP) H9N2 avian influenza viruses (AIVs) pose global threats to the poultry industry and public health, highlighting the critical need for a dual-protective vaccine. Methods: In this study, we generated a model PR8-derived recombinant H5N2 vaccine strain with hemagglutinin (HA) and neuraminidase (NA) genes from clade 2.3.2.1c H5N1 and Y439-like H9N2 viruses, respectively. To enhance the immunogenicity of the recombinant H5N2 vaccine strain, N-glycans of the HA2 subunit, NA, and M2e were modified. Additionally, we replaced M2e with avian M2e to enhance the antigenic homogeneity of AIVs for better protection. We also replaced PR8 PB2 with 01310 PB2, which is the PB2 gene derived from an LP H9N2 avian influenza virus, to eliminate pathogenicity in mammals. The productivity of the model vaccine strain (rvH5N2-aM2e-vPB2) in embryonated chicken eggs (ECEs), its potential risk of mammalian infection, and the immunogenicity associated with different inactivation methods (formaldehyde (F/A) vs. binary ethyleneimine (BEI)) were evaluated. Results: The rvH5N2-aM2e-vPB2 strain demonstrated high productivity in ECEs and exhibited complete inhibition of replication in mammalian cells. Furthermore, compared with using F/A inactivation, inactivation using BEI significantly enhanced the immune response, particularly against NA. This enhancement resulted in increased virus neutralization titers, supporting its efficacy for dual protection against H5Nx and H9N2 avian influenza viruses. Furthermore, we demonstrated that M2e-specific immune responses, difficult to induce with inactivated vaccines, can be effectively elicited with live vaccines, suggesting a strategy to enhance M2e immunogenicity in whole influenza virus vaccines. Conclusions: Finally, the successful development of the model rH5N2 vaccine strain is described; this strain provides dual protection, has potential applicability in regions where avian influenza is endemic, and can be used to promote the development of versatile H5N2 recombinant vaccines for effective avian influenza control. Full article

The development of effective and innovative vaccination strategies is urgently needed to better control the spread and transmission of the low-pathogenic avian influenza H9N2 subtype (LPAI-H9N2) in poultry. In addition, the enhancement of innate immunity by some of these innovative inactivated vaccines has not yet been investigated. Here, an experiment was conducted in commercial broiler chickens to compare the immune response to two different inactivated H9N2 vaccines. For this, Group 1 (G1) broilers were vaccinated with vaccine 1 [Nobilis^® H9N2-P (pathogen-associated molecular patterns—PAMP) technology], broilers in G2 were vaccinated with vaccine 2 [an inactivated whole H9N2 virus (IWV) autogenous oil emulsion vaccine], while birds in G3 were not vaccinated. The study lasted 34 days. Innate immune parameters (phagocytic activity, nitric oxide, and lysozyme), cytokine signaling (IL-1β, IL-6, IL-8), humoral immunity using the hemagglutination inhibition (HI) test, and the gene expressions of IFN-γ and TLR-21 were assessed. The results showed a significant increase in innate immunity and modulatory cytokines at 24–48 h after the vaccination of G1 broilers, with a continuous increase until the end of the experiment. In addition, a significant increase in geometric mean HI titers was observed in G1 at 11 days post-vaccination (dpv), and a significant (p ˂ 0.05) upregulation of IFN-γ and TLR-21 was observed in the same group, G1, at 31 dpv compared to G2 and G3. Nobilis^® H9N2-P may induce faster and stronger innate and active humoral immunity compared to another IWV, which may contribute to the protection of broilers against early H9N2 infections. However, challenge protection studies for several IWV vaccines, including PAMP-H9N2 against LPAI-H9N2, should be further evaluated in both specific pathogen-free (SPF) and commercial broilers. Full article

The introduction of HPAI H5N1 clade 2.3.4.4b viruses to North America in late 2021 resulted in avian influenza outbreaks in poultry, mortality events in many wild bird species, and spillovers into many mammalian species. Reassortment events with North American low-pathogenic virus were identified as early as February 2022 and over 100 genotypes have been characterized. Such diversity increases the complexity and time required for monitoring virus evolution. Here, we performed ordination and clustering analyses on sequence data from H5N1 viruses identified in North America between January 2020 and December 2023 to visualize the genotypic diversity of viruses in poultry and wildlife populations. Our results reveal that ordination- and cluster-based approaches can complement traditional phylogenetic analyses specifically for the preliminary assignment of H5N1 viruses to genotypic groups or to identify novel genotypes. Our study expands current knowledge on the genotypic diversity of H5N1 viruses in North America and describes a rapid approach for early virus genotype assignment. Full article

Genetically diverse avian influenza viruses (AIVs) are maintained in wild aquatic birds with increasingly frequent spillover into mammals, yet these represent a small proportion of the overall detections. The isolation of AIVs in marine mammals, including seals, has been reported sporadically over the last 45 years. Prior to 2016, all reports of AIVs detected in seals were of low-pathogenicity AIVs. In spite of this, the majority of reported AIV outbreaks caused fatal respiratory diseases, with harbor seals particularly susceptible to infection. The H5 clade 2.3.4.4b highly pathogenic AIV (HPAIV) was detected in seals for the first time in 2016. Recently, many cases of mass seal die-offs have occurred because of 2.3.4.4b HPAIV and are attributed to spillover from wild bird species. The potential for seal-to-seal transmission has been considered after the mass mortality of southern elephant seals off the coast of Argentina. Close contact between seals and wild birds, the rapid evolution of H5N1 AIVs, and the possibility of efficient mammal-to-mammal transmission are increasing concerns due to the potential for the establishment of a marine mammal reservoir and public health risks associated with the pandemic potential of the virus. This manuscript details the detection of AIVs in the seal population, comparing interesting features of various subtypes with an emphasis on avian-to-mammal-to-mammal transmission. Phylogenetic characterizations of the representative seal isolates were performed to demonstrate the relationships within the different virus isolates. Furthermore, we demonstrate that the reassortment events between different LPAIVs occurred before and after the viruses reached the seal population. The reassortment of viral segments plays an important role in the evolution of influenza viruses. Taken together, these data report on the 45 year history between seals and AIVs. Full article

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms. Full article