H5N1 Avian Influenza Virus Research Articles

Enhanced Immunogenicity of Chicken H9N2 Influenza Inactivated Vaccine Through a Novel Dual-Targeting Fusion Protein Strategy

Background/Objectives: Targeted delivery of antigens to dendritic cells (DCs) is an effective strategy for enhancing vaccine efficacy. Methods: In this study, dual-targeting fusion proteins (GRFT-VHH54 and GRFT-VHH74) were constructed by fusing Griffithsin (GRFT), an algae-derived lectin with enveloped virus-binding properties, to DC-specific binding nanobodies (VHH54 and VHH74). Vaccines were formulated by combining the inactivated H9N2 avian influenza virus with these fusion proteins, and the potential of the fusion proteins to enhance vaccine-induced immunity in chickens was systematically evaluated. For parallel comparison, control groups included H9N2 avian influenza vaccines containing the inactivated virus alone, the inactivated virus with the immune enhancer CVCVA5, and a commercial H9N2 avian influenza inactivated vaccine. Results: At 4 weeks post-immunization, chickens vaccinated with the inactivated H9N2 virus combined with the GRFT-VHH74 fusion protein (1/2 H9+GRFT-VHH74) exhibited significantly enhanced humoral, mucosal, and cellular immune responses compared to those vaccinated with the inactivated H9N2 virus alone or the commercial H9N2 vaccine (p < 0.05). Additionally, chickens in the 1/2 H9+GRFT-VHH74 group exhibited enhanced resistance to the heterologous H9N2 subtype avian influenza virus, achieving a 90% protection rate, which was higher than that of the other groups. Conclusions: These results indicate that the GRFT-VHH74 fusion protein has significant potential for advancing the development of inactivated vaccines against the H9N2 subtype avian influenza. Furthermore, it provides valuable insights for enhancing the immunogenicity and efficacy of inactivated vaccines targeting other avian influenza subtypes.

Analyzing Molecular Traits of H9N2 Avian Influenza Virus Isolated from a Same Poultry Farm in West Java Province, Indonesia, in 2017 and 2023

Background Indonesia is one of the countries that is endemic to avian influenza virus subtype H9N2. This study aims to compare the molecular characteristics of avian influenza virus (AIV) subtype H9N2 from West Java. Methods Specific pathogen-free (SPF) embryonated chicken eggs were used to inoculate samples. RNA extraction and RT–qPCR confirmed the presence of H9 and N2 genes in the samples. RT–PCR was employed to amplify the H9N2-positive sample. Nucleotide sequences were obtained through Sanger sequencing and analyzed using MEGA 7. Homology comparison and phylogenetic tree analysis, utilizing the neighbor-joining tree method, assessed the recent isolate’s similarity to reference isolates from GenBank. Molecular docking analysis was performed on the HA1 protein of the recent isolate and the A/Layer/Indonesia/WestJava-04/2017 isolate, comparing their interactions with the sialic acids Neu5Ac2-3Gal and Neu5Ac2-6Gal. Results RT–qPCR confirmed the isolate samples as AIV subtype H9N2. The recent virus exhibited 11 amino acid residue differences compared to the A/Layer/Indonesia/WestJava-04/2017 isolate. Phylogenetically, the recent virus remains within the h9.4.2.5 subclade. Notably, at antigenic site II, the recent isolate featured an amino acid N at position 183, unlike A/Layer/Indonesia/WestJava-04/2017. Molecular docking analysis revealed a preference of HA1 from the 2017 virus for Neu5Ac2-3Gal, while the 2023 virus displayed a tendency to predominantly bind with Neu5Ac2-6Gal. Conclusion In summary, the recent isolate displayed multiple mutations and a strong affinity for Neu5Ac2-6Gal, commonly found in mammals.

A human-infecting H10N5 avian influenza virus: Clinical features, virus reassortment, receptor-binding affinity, and possible transmission routes.

A human-infecting H10N5 avian influenza virus: Clinical features, virus reassortment, receptor-binding affinity, and possible transmission routes.

In-laboratory inactivation of H5N1 in raw whole milk through milk acidification: Results from a pilot study.

In-laboratory inactivation of H5N1 in raw whole milk through milk acidification: Results from a pilot study.

Phosphorylation of PA at serine 225 enhances viral fitness of the highly pathogenic H5N1 avian influenza virus in mice.

Phosphorylation of PA at serine 225 enhances viral fitness of the highly pathogenic H5N1 avian influenza virus in mice.

Molecular patterns of matrix protein 1 (M1): A strong predictor of adaptive evolution in H9N2 avian influenza viruses

The H9N2 subtype of avian influenza virus (AIV) emerges as a significant member of the influenza A virus family. However, the varying degrees of epidemiological dominance among different lineages or clades of H9N2 AIVs have not been fully clarified. The matrix protein M1, a key structural component of the virion, plays a crucial role in maintaining the viral structure and lifecycle. To elucidate the intrinsic relationship between the genetic patterns of M1 and the adaptive dynamics of H9N2 AIVs, this study focused on the five major evolutionary patterns of M1 and conducted in vitro and in vivo investigations from the perspectives of vRNP release after viral uncoating, polymerase activity, mRNA and vRNA levels, the nuclear export of vRNPs, plasma membrane-binding capacity, proliferation capacity, growth competitiveness, and transmission potential. The results revealed a strong correlation between the epidemiological dominance of H9N2 AIVs and the specific patterns of M1, with M1P5 standing out as particularly significant. This finding highlights the pivotal influence of the M1 gene patterns on the replication and transmission dynamics of H9N2 AIVs, thereby offering valuable insights into the mechanisms driving differences in adaptive evolution and shifts in epidemiological dominance within the H9N2 AIV population.

Surveillance of H7N9 avian influenza virus in farmers’ markets in Beijing in 2019–2023

Avian influenza viruses (AIVs) present an ongoing threat of human infections. Continuous surveillance is important for detecting new infections and verifying prevention and control measures. Swabs of the external environment and throat swabs of employees were collected from six farmers’ markets in Beijing to detect influenza A virus. Positive samples were sequenced, and their genetic characteristics analyzed. In total, 3251 environmental samples were collected from 2019 to 2023, 11 of which were positive for influenza A virus (positivity rate of 0.34%), including nine for H9N2 and two for H7N9. In a genetic analysis, all H7N9 samples showed low pathogenicity, and no mutations at highly pathogenic sites were detected. All 1135 throat swab samples from staff were negative for influenza A virus. At present, the detection rate of AIVs in farmers’ markets is very low, and no adaptive mutations allowing cross-host transmission were found, indicating a low risk of AIV infection among the people of Beijing.

Highly Pathogenic Avian Influenza Virus H5N1 in Double-crested Cormorants (Nannopterum auritum) of the Chesapeake Bay, USA.

Double-crested Cormorants (Nannopterum auritum) have historically exhibited low levels of infection and antibodies to avian influenza virus (AIV). The recent global expansion of clade 2.3.4.4b A/goose/Guangdong/1/1996 highly pathogenic (HP) avian influenza virus H5N1 (HPAI H5N1) has resulted in large-scale mortalities across diverse waterbird taxa including cormorants. We sampled 32 and 29 Double-crested Cormorants breeding in the Chesapeake Bay, US, during the summers of 2023 and 2024, respectively, to assess HPAI H5N1 infection and AIV antibodies. Although no mortality was observed in the area, one bird sampled in 2023 was infected with HPAI H5N1. Additionally, 21/31 individuals in 2023 and 10/25 individuals in 2024 for which sera were collected had AIV antibodies. Based on additional testing using hemagglutination inhibition, virus neutralization, and an enzyme-linked lectin assay, 94 and 100% (2023 and 2024, respectively) of the seropositive birds tested positive for antibodies to both H5 and N1, suggesting previous infection with HPAI H5N1. These results are consistent with survival and limited clinical effects related to HPAI H5N1 infections. Furthermore, these results suggest that population immunity to HPAI H5N1 within the Chesapeake Bay might reduce future infections and potential population impacts should HP H5N1 remain on the landscape, though immunity may be waning across time. Because results are based on a single population, additional testing for both infection and antibodies as well as continued monitoring could enhance understanding of antibody persistence.

Influenza a virus subtype H9N2 infection induces respiratory microbiota dysbiosis in chickens via type-I interferon-mediated mechanisms.

Avian influenza virus (AIV) poses significant threats to poultry and human health. This study investigates the impact of H9N2 AIV infection on the respiratory microbiota of chickens using 16S rRNA gene sequencing. Total 48 one-day-old specific pathogen-free chickens were assigned to six groups: a control and five post-infection groups (days 1, 3, 5, 7, and 9). After a 15-day microbiota stabilization period, the infected chickens received a viral inoculum (107 TCID50/ml) via ocular, intra-nasal, and intra-tracheal routes. Tracheal and broncho-alveolar lavage samples were analyzed. Significant reductions in microbiota diversity were observed on days 5, 7, and 9 post-infection, compared to d0 controls. Permutational Multivariate Analysis of Variance confirmed significant beta diversity differences (P = 0.001) between infected and uninfected groups. The microbial shifts from d5 to d9 were marked by increased Proteobacteria, decreased Actinobacteria and Firmicutes, and a rise in Dickeya. Elevated type-I interferon (IFN-β) and viperin gene expression at d5 coincided with reduced microbiota diversity, highlighting the respiratory microbiota's role in modulating host responses to AIV H9N2 infection and suggesting potential biomarkers for respiratory dysbiosis.

Hemagglutinin with a polybasic cleavage site confers high virulence on H7N9 avian influenza viruses.

Hemagglutinin with a polybasic cleavage site confers high virulence on H7N9 avian influenza viruses.

Receptor binding, structure, and tissue tropism of cattle-infecting H5N1 avian influenza virus hemagglutinin.

Receptor binding, structure, and tissue tropism of cattle-infecting H5N1 avian influenza virus hemagglutinin.

Spray vaccination with a safe and bivalent H9N2 recombinant chimeric NDV vector vaccine elicits complete protection against NDV and H9N2 AIV challenge

Newcastle disease virus (NDV) and H9N2 avian influenza virus (AIV) represent significant pathogenic risks to the poultry industry, leading to considerable economic losses. Vaccination is a widely used preventive measure against these pathogens, yet the lack of a live bivalent vaccine targeting NDV and H9N2 AIV imposes a heavy vaccination burden. Previously, we constructed a genotype-matched chimeric NDV vector, LX-OAI4S, in which the genotype I NDV backbone was replaced with the ectodomain of haemagglutinin-neuraminidase (HN) and modified using the attenuated F gene from the genotype VII vaccine strain A-VII. Based on the LX-OAI4S vector, we successfully generated three H9N2 recombinant viruses: LX-OAI4S-NPU-HA, LX-OAI4S-MU-HA, and LX-OAI4S-HNU-HA. These recombinants incorporated the H9N2 HA gene, flanked by untranslated regions (UTRs) from the NP, M, or HN gene of the NDV LX strain, inserted between the P and M genes of LX-OAI4S. The vaccine candidate LX-OAI4S-NPU-HA induced a more robust immune response in chickens against H9N2 AIV and NDV than the other two recombinants. This response effectively protects against virus shedding and lethal virus challenge. Furthermore, spray vaccination with LX-OAI4S-NPU-HA showed protective efficacy against H9N2 AIV and NDV. This study offers a promising strategy for comprehensive protection in regions threatened by H9N2 AIV and NDV.

Avian influenza mRNA vaccine encoding hemagglutinin provides complete protection against divergent H5N1 viruses in specific-pathogen-free chickens

BackgroundThe rapid mutation of avian influenza virus (AIV) poses a significant threat to both the poultry industry and public health. Herein, we have successfully developed an mRNA-LNPs candidate vaccine for H5 subtype highly pathogenic avian influenza and evaluated its immunogenicity and protective efficacy.ResultsIn experiments on BALB/c mice, the vaccine candidate elicited strong humoral and a certain cellular immune responses and protected mice from the heterologous AIV challenge. Antibody and splenocyte passive transfer assays in mice suggested that antibodies played a crucial role in providing protection. Experiments involving SPF chickens have revealed that two doses of the 5 µg vaccine candidate in this study provided 100% complete protection against homologous strains, but only 50% complete protection against heterologous strains. Even immunization with two doses of the 15 µg vaccine candidate resulted in 90% complete protection against heterologous strains. To enhance the immune efficacy of the candidate vaccine, we designed 6 sequences with different secondary structures and screened out the candidate sequence with the highest expression (SY2-HA mRNA). Experiments on SPF chickens showed that two doses of 5 µg SY2-HA mRNA-LNP vaccine provided 100% complete protection against homologous and heterologous H5N1 AIV strains. Immunization tests with the SY2-HA mRNA-LNP vaccine were repeated in the SPF chicken model, inducing antibody production levels that are consistent with previous tests and providing 100% complete protection against both homologous and heterologous strains of the virus, indicating that the vaccine has a stable immune efficacy.ConclusionsThe vaccine developed in this study provides complete protection against divergent H5N1 AIV strains in chickens, offering a promising approach for the future development of mRNA vaccines against multivalent avian influenza subtypes.Graphical

Molecular characterization and genetic analysis of highly pathogenic H5N1 clade 2.3.4.4b in seagulls from Dukan Lake, Iraq.

Avian influenza virus (AIV) remains a significant global threat, with periodic reemergence in Iraq. This study marks the first molecular characterization of the highly pathogenic avian influenza (HPAI) H5N1 clade 2.3.4.4b in seagulls. The H5N1 AIV was identified during outbreaks in 2024 at Dukan Lake in Sulaimani province. Phylogenetic analysis of the HA gene revealed that the Dukan Lake strain belongs to subclade 2.3.4.4b, clustering closely with Kazakhstan HPAI strains (A/mute swan/Mangystau/1-S24R-2/2024(H5N1) and (A/Cygnus cygnus/Karakol lake/01/2024(H5N1)), with DNA identities of 99.38% and 98.82%, respectively. Genetic analysis showed a polybasic amino acid cleavage site motif (PLREKRRKRGLF) in the HA gene. Additionally, receptor-binding domain (RBD) analysis indicated a preference for the avian α-2, 3 Sialic acid (SA) receptor over the mammalian α-2, 6 SA receptor. The NA gene analysis revealed amino acid residues D199, I223, S247, and H275, which are susceptible to antiviral drugs. The molecular analysis of the H5N1 Dukan Lake seagull strain provides insights into how the virus spreads among different species and countries, which is crucial for global health security and the development of effective control measures.

The Generation of a H9N2 Avian Influenza Virus with HA and C3d-P29 Protein Fusions and Vaccine Development Applications.

Maternal-derived antibody (MDA) interferes with immune responses, leading to the failure of H9N2 avian influenza vaccinations in poultry. So far, none of the commercially available H9N2 avian influenza vaccines used in poultry have been able to overcome MDA interference. To develop a vaccine that can overcome MDA interference, one or multiple copies of the minimum-binding domain (P29) from the complement protein C3d were inserted in between the signal peptide and the head domain of the hemagglutinin (HA) protein on a H9N2 avian influenza virus (A/Chicken/Shanghai/H514/2017, named H514). The HA proteins containing P29 stimulated stronger type I interferences than wild-type HA proteins in vitro. The modified viruses with the HA proteins containing one copy of P29 (rH514-P29.1) and two copies of P29.2 (rH514-P29.2) were successfully rescued using reverse genetics. The inactivated vaccines developed with rH514-P29.1 or rH514-P29.2 induced higher and faster humoral immune responses than the vaccine developed with rH514 in specific pathogen-free (SPF) chickens. To evaluate the vaccines' efficacy in the presence of MDA and to ensure a uniform level of MDA, passively transferred antibody (PTA) was used as a model to mimic MDA in 1-day-old SPF chickens. Our results showed that the rH514-P29.2 inactivated vaccine induced significantly higher HI titers than the rH514 inactivated vaccine in the presence of PTA. More importantly, it reduced viral shedding after being challenged with H514 in the presence of PTA. Our results suggest that vaccine antigens fused with two copies of P29 can decrease the interference of MDA on immunity in chickens. Overall, our results provide a new strategy for overcoming MDA interference.

Isoleucine at position 137 of haemagglutinin acts as a mammalian adaptation marker of H9N2 avian influenza virus

ABSTRACT The H9N2 subtype of avian influenza virus (AIV) is widely distributed among poultry and wild birds and is also a threat to humans. During AIV active surveillance in Liaoning province from 2015 to 2016, we identified 10 H9N2 strains exhibiting different lethality to chick embryos. Two representative strains, A/chicken/China/LN07/2016 (CKLN/07) and A/chicken/China/LN17/2016 (CKLN/17), with similar genomic background but different chick embryo lethality, were chosen to evaluate the molecular basis for this difference. A series of reassortants between CKLN/07 and CKLN/17 were generated and their chick embryo lethality was assessed. We found that the isoleucine (I) residue at position 137 (H3 numbering) in the haemagglutinin (HA) was responsible for the chick embryo lethality of the H9N2 virus. Further studies revealed that the threonine (T) to I mutation at HA position 137 enhanced viral replication in vitro and in vivo. Moreover, the HA-T137I substitution in H9N2 avian influenza virus increased the guinea pig transmission efficiency. We also found that the HA-T137I substitution was critical for α2,6-linked sialic acid binding preference and HA activation and stability of H9N2 virus. Our findings demonstrated that HA-137I is a key molecular marker for mammalian adaptation of H9N2 AIV.

Infection Tracing and Virus Genomic Analysis of Two Cases of Human Infection with Avian Influenza A(H5N6) - Fujian Province, China, April-May 2024.

Global human cases of zoonotic influenza A(H5N6) have increased significantly in recent years, primarily due to widespread circulation of clade 2.3.4.4b virus since 2020. Concurrent with this trend, sporadic human infections with clade 2.3.4.4h H5N6 avian influenza virus continue to occur. The high mortality rate associated with H5N6 virus infections has emerged as a critical public health concern. Through comprehensive field epidemiological investigations and laboratory analyses, we identified the infection sources for these cases and conclusively ruled out human-to-human transmission. Genetic analyses revealed that while the virus maintains its avian host tropism, it has acquired mutations that may enhance human receptor binding affinity, viral replication capacity, pathogenicity, and neuraminidase inhibitor resistance. The ongoing viral mutations increase the potential for H5 subtype avian influenza viruses to overcome species barriers and cause human epidemics. Enhanced surveillance strategies incorporating advanced technologies, such as metagenomic sequencing, are essential for early risk detection and management. Special attention should be directed toward cancer patients and immunocompromised individuals, who demonstrate increased susceptibility to avian influenza virus infections and require targeted prevention and control measures.

Proteomic Analysis of Differentially Expressed Proteins in A549 Cells Infected with H9N2 Avian Influenza Virus.

Influenza A viruses (IAVs) are highly contagious pathogens that cause zoonotic disease with limited availability of antiviral therapies, presenting ongoing challenges to both public health and the livestock industry. Unveiling host proteins that are crucial to the IAV life cycle can help clarify mechanisms of viral replication and identify potential targets for developing alternative host-directed therapies. Using a four-dimensional (4D), label-free methodology coupled with bioinformatics analysis, we analyzed the expression patterns of cellular proteins that changed following H9N2 virus infection. Compared to the control group, the H9N2 infected group displayed 732 differentially expressed proteins (DEPs), with 298 proteins showing upregulation and 434 proteins showing downregulation. Gene Ontology (GO) functional analysis showed that DEPs were catalog in 11 biological processes, three cellular components, and eight molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that DEPs were involved in processes including cytokine signaling pathways induced by virus infection and protein digestion and absorption. Proteins including TP53, DDX58, and STAT3 were among the top hub proteins in the protein-protein interaction (PPI) analysis, suggesting that these signaling cascades could be essential for the propagation of IAVs. Furthermore, the host protein SNAPIN was chosen to ascertain the accuracy of expression changes identified through a proteomic analysis. The results indicated that SNAPIN was downregulated following infection with IAVs both in vitro and in vivo, which is consistent with the proteomics results, suggesting that SNAPIN may serve as a key regulatory factor in the viral life cycle of IAVs. Our research delineates an extensive interaction map of IAV infection within the A549 cells, facilitating the discovery of pivotal proteins that contribute to the virus's propagation, potentially offering target candidates to screen for antiviral therapeutics.

HA198 mutations in H9N2 avian influenza: molecular dynamics insights into receptor binding.

The H9N2 avian influenza virus is widely disseminated in poultry and poses a zoonotic threat, despite vaccination efforts. Mutations at residue 198 of hemagglutinin (HA) are critical for antigenic variation and receptor-binding specificity, but the underlying molecular mechanisms remain unclear. This study explores the molecular mechanisms by which mutations at the HA 198 site affect the antigenicity, receptor specificity, and binding affinity of the H9N2 virus. Using the sequence of the A/Chicken/Jiangsu/WJ57/2012 strain, we constructed recombinant H9N2 viruses, including rWJ57, rWJ57/HA198A, and rWJ57/HA198T, using reverse genetics. These variants were analyzed through hemagglutination inhibition (HI) assays, receptor-destroying enzyme (RDE) assays, enzyme-linked immunosorbent assays (ELISA) and solid-phase receptor binding assays. Additionally, molecular dynamics (MD) simulations were performed to further dissect the atomic-level interactions between HA and sialic acids (SA). The results demonstrated that HA mutations significantly altered the receptor-binding properties of the virus. Specifically, rWJ57 (HA198V) exhibited 4-fold and 16-fold higher overall receptor-binding avidity compared to rWJ57/HA198A and rWJ57/HA198T, respectively. Furthermore, HA198V/T mutations significantly enhanced viral binding to human-type α2,6 SA receptors (p < 0.001), whereas the HA198A mutation exhibited a marked preference for avian-type α2,3 SA receptors (p < 0.001). Additionally, these mutations altered interactions with non-specific antibodies but not specific antibodies, with high-avidity receptor binding mutations exhibiting reduced non-specific antibody binding, suggesting a potential novel mechanism for immune evasion. MD simulations revealed HA198V/T formed stable complexes with the α2,6 SA, mediated by specific residues and water bridges, whereas HA198A formed stable complexes with the α2,3 SA. Interestingly, residue 198 interacted with the α2,6 SA via water bridges but had with showed minimal direct interaction with α2,3 SA. This study provides new insights into the molecular basis of receptor specificity, binding affinity, and antigenic drift in H9N2 viruses, highlighting the critical role of HA 198 mutations in regulating host adaptation. These findings are of great significance for H9N2 virus surveillance, vaccine development, and zoonotic transmission risk assessment.

Based on the MaxEnt model the analysis of influencing factors and simulation of potential risk areas of human infection with avian influenza A (H7N9) in China.

Exposure to infected animals and their contaminated environments may be the primary cause of human infection with the H7N9 avian influenza virus. However, the transmission characteristics and specific role of various influencing factors in the spread of the epidemic are not clearly understood. Therefore, it is of great significance for scientific research and practical application to explore the influencing factors related to the epidemic. Based on the data of relevant influencing factors and case sample points, this study used the MaxEnt model to test the correlation between human infection with H7N9 avian influenza and influencing factors in China from 2013 to 2017, and scientifically simulated and evaluated the potential risk areas of human infection with H7N9 avian influenza in China. The simulation results show that the epidemic risk is increasing year by year, and the eastern and southeastern coasts have always been high-risk areas. After verification, the model simulation results are generally consistent with the actual outbreak of the epidemic. Population density was the main influencing factor of the epidemic, and the secondary influencing factors included vegetation coverage, precipitation, altitude, poultry slaughter, production value, and temperature. The study revealed the spatial distribution and diffusion rules of the H7N9 epidemic and clarified the key influencing factors. In the future, more variables need to be included to improve the model and provide more accurate support for prevention and control strategies.