H5N1 Subtype Research Articles

Self-assembling nanoparticle vaccine elicits a robust protective immune response against avian influenza H5N6 virus in chickens

The continuous circulation and evolution of the H5N6 subtype highly pathogenic avian influenza virus (HPAIV) challenge the development of the global poultry industry and human public health security. To address the potential threat of the H5N6 virus, a secure and efficacious vaccine is urgent. In our research, a self-assembling nanoparticle vaccine presenting the hemagglutinin of the H5N6 AIV was developed based on the ferritin antigen display platform. The results showed that a single-dose vaccination of this nanoparticle vaccine elicited potent hemagglutination inhibition (HI) antibody responses and neutralizing antibody responses in the chickens. Meanwhile, the fused HA-ferritin nanoparticle vaccine induced significantly higher levels of Th1/Th2 immune responses. After a lethal attack with the H5N6 virus, the fused HA-ferritin nanoparticle vaccine conferred chickens with 100 % (10/10) challenge protection. Importantly, the fused HA-ferritin nanoparticle with only 28 hemagglutination units (HAU) provided chickens with immune protection comparable to commercial inactivated vaccines and protected the chickens from severe lung pathological damage. These results in our study support the superiority of ferritin as an antigen display platform and suggest that self-assembled nanoparticle vaccines based on this platform possess the potential as an avian influenza candidate vaccine.

Rapid and Safe Neutralization Assay for Circulating H5N1 Influenza Virus in Dairy Cows.

Rapid and safe neutralization assays are required for highly pathogenic avian influenza viruses, including a clade 2.3.4.4b H5N1 subtype recently found in cows. Here, we report a neutralization assay using luminescent virus-like particles. This assay has lower biosafety requirements and provides a larger dynamic range than conventional methods. We applied this technique to evaluate the cross-reactivity of neutralizing antibodies induced by clade 2.3.4.4b candidate vaccine viruses (CVVs) with the cow-derived H5N1 virus. Our findings indicate that these CVVs share antigenic characteristics with the cow-derived H5N1 virus, suggesting the potential efficacy of vaccines developed using these CVVs.

Low-inflammatory lipid nanoparticle-based mRNA vaccine elicits protective immunity against H5N1 high-pathogenicity avian influenza virus with reduced adverse reactions.

Messenger RNA vaccines based on lipid nanoparticles (mRNA-LNPs) are promising vaccine modalities. However, mRNA-LNP vaccines frequently cause adverse reactions such as swelling and fever in humans, partly due to the inflammatory nature of LNP. Modification of the ionizable lipids used in LNP is one approach to avoid these adverse reactions. Herein, we report the development of mRNA-LNP vaccines with better protective immunity and reduced adverse reactions using LNP, which contains a disulfide (SS)-cleavable bond and pH-activated lipid-like materials with oleic acid (ssPalmO) as an ionizable lipid (LNPssPalmO). We used mRNA expressing H5N1 subtype high-pathogenicity avian influenza virus-derived hemagglutinin or neuraminidase to generate mRNA-LNP vaccines against H5N1 influenza. Compared with conventional LNP, mRNA-LNPssPalmO induced comparable antigen-specific antibodies and better interferon-gamma (IFN-γ)-producing T-helper type-1 (Th1) responses in mice. Both mRNA-LNPssPalmO and conventional mRNA-LNP conferred strong protection against homologous H5N1 virus challenge. In addition, mRNA-LNPssPalmO showed better cross-protection against heterologous H5N1 virus challenge compared with conventional mRNA-LNPs. Furthermore, we observed that mRNA-LNPssPalmO induced less inflammatory responses (e.g., inflammatory cytokine production and vascular hyperpermeability) and fewer adverse reactions (e.g., weight loss and fever) compared with conventional mRNA-LNP. These results suggest that mRNA-LNPssPalmO would be a safe alternative to conventional vaccines to overcome mRNA-LNP vaccine hesitancy.

Rapid Onset of Innate Response, Cytokine Signaling and Humoral Immunity in Inactivated LPAI-H9N2-Vaccinated Broilers

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.

Epidemiological and genetic characterization of the influenza A (H1N1) virus in Hangzhou City in 2023.

To explore and describe the epidemiological and genetic variation characteristics of the influenza A (H1N1) virus in Hangzhou City. Respiratory throat swab specimens collected from the fever clinic of the 903rd Hospital of the Chinese People's Liberation Army (PLA) between January and March 2023 were collected. The respiratory pathogen antigens were identified using the colloidal gold method, and those testing positive for influenza A virus antigens were confirmed and subtyped by RT-qPCR. Seventeen H1N1 isolates were selected to amplify hemagglutinin (HA) and neuraminidase (NA) gene sequences via RT-PCR, and sequencing was completed following the identification of the amplified products. The sequenced HA and NA sequences were spliced using DNASTAR software (version 5.0), and a phylogenetic tree was constructed using MEGA software (version 11.0) for genetic characterization. A total of 2,376 respiratory samples were tested, with 680 cases testing positive for influenza A. Of these, 129 positive cases of influenza A were randomly selected for typing, resulting in the isolation of 112 H1N1 subtypes and 17 H3N2 subtypes. The HA genes of 17 strains of influenza A (H1N1) were randomly selected for amino acid homology comparisons with two vaccine strains recommended by the WHO for 2023 (A/Wisconsin/67/2022 (H1N1) and A/Victoria/4897/2022 (H1N1)). The HA gene results showed identities of 98.24 to 98.65% and 98.41 to 98.82%, respectively, and the NA gene results were 98.79 to 99.15% and 98.94 to 99.29%, respectively. Fourteen amino acid sites were altered in the HA gene of the 17 strains, with some strains contributing to the Sa and Ca antigenic determinants, respectively. Seventeen strains had mutations in the NA gene at sites 13, 50, 200, 339, 382, and 469. The sequenced strains, vaccine strains, and some 2023 domestic representative strains independently formed a branch 6B.1A.5a.2a. The continuous evolutionary mutations of the H1N1 virus genes in Hangzhou City suggest the possibility of the virus escaping from the immune response. This study provides an experimental basis for evaluating the protective effect of the vaccine and formulating preventive measures against influenza in Hangzhou City.

Specific detection of duck adeno-associated virus using a TaqMan-based real-time PCR assay.

Duck adeno-associated Virus (DAAV) is a novel pathogen that was recently discovered in ducks. To establish a molecular detection assay for DAAV for further epidemiological investigation and pathogenic mechanism. Here, we designed specific primers and probes according to the sequence characteristics of the newly discovered DAAV and then established a TaqMan real-time PCR method (TaqMan-qPCR) for the detection of DAAV. Our data showed that the established TaqMan-qPCR for detecting DAAV had high sensitivity, with the lowest detection limit of 29.1 copies/μL. No cross reaction was found with duck circovirus (DuCV), H9N2 subtype avian influenza virus (AIV), avian Tembusu virus (ATmV). duck hepatitis A virus 1 and 3 (DHAV-1 and DHAV-3), duck adenovirus A (DAdV-A), duck adenovirus 3 (DAdV-3), or duck enteritis virus (DEV). The repeatability was excellent, with the coefficients of variation of repeated intragroup and intergroup tests ranging from 0.12-0.21% and 0.62-1.42%, respectively. Seventy-eight clinical samples collected from diseased or deceased ducklings were tested. The results showed that the DAAV positive rate was 21.79%, and a triple infection (DAAV+MDPV+GPV) was found. These data provide technical support for further molecular epidemiological surveillance and pathogenic mechanism studies of DAAV infection.

The Thermal Stability of Influenza Viruses in Milk.

Highly pathogenic avian influenza viruses (HPAIVs) of the H5N1 subtype (clade 2.3.4.4b) have been detected in raw milk from infected cows. Several studies have examined the time and temperature parameters to ascertain whether influenza viruses in milk can be inactivated completely under commercial pasteurization conditions, yielding conflicting results. This study aimed to investigate whether milk could help protect influenza viruses from heat treatment. After heat treatment at 49 °C for one hour, the titer reduction of the influenza A/WSN/1933 (A/H1) virus in milk was approximately 1.6 log10TCID50/mL, which was significantly lower than that (3 log10TCID50/mL) observed in the Dulbecco's Modified Eagle Medium (DMEM) control media. The influenza D/bovine/CHN/JY3002/2022 (D/Yama2019) virus in milk retained a high residual infectivity (4.68 × 103 log10TCID50/mL) after treatment at 53 °C; however, the virus in DMEM completely lost its infectivity under the same conditions. Moreover, the influenza A/chicken/CHN/Cangzhou03/2023 (A/H5) virus in DMEM could be inactivated completely using any of the three heat treatment methods: 63 °C for 30 min, 72 °C for 15 s, or 80 °C for 15 s. For the virus present in milk, only heat treatment at 80 °C for 15 s completely inactivated it. These results suggest that milk prevents influenza viruses from pasteurization inactivation.

H9 Consensus Hemagglutinin Subunit Vaccine with Adjuvants Induces Robust Mucosal and Systemic Immune Responses in Mice by Intranasal Administration.

The H9N2 subtype avian influenza viruses mainly cause respiratory symptoms, reduce the egg production and fertility of poultry, and result in secondary infections, posing a great threat to the poultry industry and human health. Currently, all H9N2 avian influenza commercial vaccines are inactivated vaccines, which provide protection for immunized animals but cannot inhibit the spread of the virus and make it difficult to distinguish between the infected animals and vaccinated animals. In this study, a trimeric consensus H9 hemagglutinin (HA) subunit vaccine for the H9N2 subtype avian influenza virus based on a baculovirus expression system was first generated, and then the effects of three molecular adjuvants on the H9 HA subunit vaccine, Cholera toxin subunit B (CTB), flagellin, and granulocyte-macrophage colony-stimulating factor (GM-CSF) fused with H9 HA, and one synthetic compound, a polyinosinic-polycytidylic acid (PolyI:C) adjuvant, were evaluated in mice by intranasal administration. The results showed that these four adjuvants enhanced the immunogenicity of the H9 HA subunit vaccine for avian influenza viruses, and that GM-CSF and PolyI:C present better mucosal adjuvant activity for the H9 HA subunit vaccine. These results demonstrate that we have developed a potential universal H9 HA mucosal subunit vaccine with adjuvants in a baculovirus system that would be helpful for the prevention and control of H9N2 subtype avian influenza viruses.

Establishment and application of a duplex fluorescent quantitative PCR assay for H9N2 subtype avian influenza virus and infectious bronchitis virus.

The H9N2 subtype of avian influenza virus (AIV) and infectious bronchitis virus (IBV) are important avian viruses that cause respiratory symptoms in poultry, and can form mixed infections. In this study, primers and probes were designed based on the HA gene of H9N2 and the 5' noncoding region of IBV, respectively, and a fluorescent quantitative RT-PCR assay was established for simultaneous detection of these two pathogens. The reaction system and conditions were optimized. The method only detected AIV subtype H9N2 and IBV and no other viruses, confirming its high specificity. The assay detected 13.5 copies/μL and 1.66 copies/μL of H9N2 and IBV in clinical samples, respectively. The coefficients of variation for intra- and interassay repeatability were < 3%. The established method was used to analyze 254 clinical samples (oropharyngeal and cloacal swabs) from Hubei Province, China; 98.82% were positive for both pathogens. In summary, a duplex fluorescent quantitative RT-PCR method capable of simultaneously detecting AIV subtype H9N2 and IBV was established. It is specific, sensitive, and reproducible, and can be used for diagnosis of a variety of clinical samples. It provides a technological means for the rapid and simultaneous detection of both pathogens, and thus can facilitate clinical diagnosis and epidemiological investigations.

Effect of stabilizers on the detection of swine influenza A virus (swIAV) in spiked oral fluids over time

BackgroundAggregated samples such as oral fluids (OFs) display an animal friendly and time and cost-efficient sample type for swine Influenza A virus (swIAV) monitoring. However, further molecular and biological characterization of swIAV is of particular significance. The reportedly inferior suitability of aggregated samples for subtyping of swIAV presents a major drawback compared to nasal swabs, still considered the most appropriate sample type for this purpose (Garrido-Mantilla et al. BMC Vet Res 15(1):61, 2019). In addition, the viral load in the original sample, storage conditions and characteristics of different swIAV strains might further compromise the eligibility of aggregated samples for molecular detection and subtyping. Therefore, the present study aimed to evaluate the suitability of stabilizing media to minimize the degradation of viral RNA and thus increase the detection and subtyping rate of swIAV by RT-qPCR in spiked OFs under different conditions (virus strain, storage temperature and viral load in the original sample) over a time span of 14 days.ResultsThe use of stabilizing media in spiked OFs resulted in a significant higher probability to detect swIAV RNA compared to OFs without stabilizers (OR = 46.1, p < 0.001). In addition, swIAV degradation over time was significantly reduced in samples suspended with stabilizer (OR = 5.80, p < 0.001), in samples stored at 4 °C (OR = 2.53, p < 0.001) and in samples spiked with the avian derived H1N2 subtype (OR = 2.26, p < 0.01). No significant differences in swIAV RNA detection and degradation of swIAV RNA in spiked OFs over time were observed between the three different stabilizing media.ConclusionAddition of stabilizers and storage of samples under cooled conditions significantly improved detection and subtyping of swIAV in spiked OFs.

The inhibitory effect of Hypericum japonicum on H9N2 avian influenza virus

The H9N2 subtype of avian influenza virus (AIV) causes severe immunosuppression and high mortality in view of its frequent co-infection with other pathogens, resulting in significant economic losses in the poultry industry. Current vaccines provide suboptimal immune protection against H9N2 AIV owing to antigenic variations, highlighting the urgent need for safe and effective antiviral drugs for the prevention and treatment of this virus. This study aimed to investigate the inhibitory effects of Hypericum japonicum extract on H9N2 AIV. Our findings revealed that the extract obtained through resin column separation using 60% ethanol (S06-60%) inhibited H9N2 AIV replication in Madin-Daby canine kidney cells in a dose-dependent manner. The maximum safe concentration of the water-soluble S06-60% extract was determined to be 0.05 mg/mL. Time-course experiments indicated that S06-60% primarily exerted its antiviral effects during the viral pretreatment and adsorption stages. Furthermore, in vivo experiments conducted on specific pathogen-free chickens confirmed the effectiveness of S06-60% in inhibiting H9N2 AIV infection and mitigating associated damage to tracheal and lung tissues. Overall, our study highlights the therapeutic potential of Hypericum japonicum extract S06-60% as a viable antiviral candidate against H9N2 AIV, offering promising implications for its application in poultry health management to reduce the economic impact on the poultry industry.

Computational screening of phytocompounds from C. amboinicus identifies potential inhibitors of influenza A (H3N2) virus by targeting hemagglutinin

The H3N2 subtype of the influenza A virus continues to be a notable public health issue due to its association with seasonal epidemics and severe human morbidity. The constrained effectiveness of current antiviral medications, combined with the inevitable emergence of drug-resistant variants, mandates the exploration of innovative therapeutic approaches. This study focuses on the identification of phytocompounds from Coleus amboinicus with the potential to target hemagglutinin, viral protein involved in viral entry by binding to sialyl glycoconjugates receptors on the surface of host cells. Molecular docking studies were carried out to assess the efficacy of C. amboinicus phytocompounds with hemagglutinin receptor-binding site. The study revealed that among the 84 signature phytocompounds, isosalvianolic acid and salvianolic acid C showed the highest docking scores and favourable intermolecular interactions. Pharmacokinetic analysis and Pan-assay interference compounds (PAINS) filtering confirmed that isosalvianolic acid meets the criteria outlined in Lipinski’s rule of five, exhibits favourable ADMET profiles and passes PAINS filters. Furthermore, the molecular dynamics simulations followed by radius of gyration (Rg), solvent accessible surface area (SASA), and MM-PBSA calculations for binding free energy, verified the stability of the docked complexes. Together, the study identifies isosalvianolic acid as a promising inhibitor of the H3N2 virus by binding to hemagglutinin, indicating its potential as a strategy for therapeutic intervention.

Identification of novel Influenza virus H3N2 nucleoprotein inhibitors using most promising synthetic Epicatechin derivatives

Influenza A virus is a leading cause of acute respiratory tract infections, posing a significant global health threat and resulting in substantial annual financial losses due to seasonal epidemics. Current treatment options are limited and increasingly ineffective due to viral mutations. This study aimed to identify potential drug candidates targeting the nucleoprotein of the H3N2 subtype of Influenza A virus. We focused on epicatechin derivatives and employed a suite of cheminformatics approaches, including ADMET profiling, drug-likeness evaluation, PASS predictions, molecular docking, molecular dynamics simulations, Principal Component Analysis (PCA), dynamic cross-correlation matrix (DCCM) analyses, and free energy landscape assessments. Molecular docking and dynamics simulations revealed strong and stable binding interactions between the derivatives and the target protein, with complexes 01 and 81 exhibiting the highest binding affinities. Additionally, ADMET profiling indicated favorable pharmacokinetic properties for these compounds, supporting their potential as effective antiviral agents. Compound 81 demonstrated exceptional quantum chemical descriptors, including a small HOMO-LUMO energy gap, high electronegativity, and significant softness, suggesting high chemical reactivity and strong electron-accepting capabilities. These properties enhance Compound 81’s potential to interact effectively with the H3N2 nucleoprotein. Experimental validation is strongly recommended to advance these compounds toward the development of novel antiviral therapies to address the global threat of influenza.

Low detection of H5N1 virus in commercial chickens with a low-level of vaccination coverage against H5N1 virus infection in Bangladesh

BackgroundBangladesh has reported > 560 H5N1 outbreaks in poultry and eight human cases since 2007. Commercial chicken farms were mostly affected. Commercial chicken farms across the country use imported vaccines against H5N1 virus; however, these vaccines did not use local circulatory isolates of H5N1virus. Vaccination may have limited effectiveness in chicken because of the mismatch in terms of subtypes and clades. To test this, we conducted a mixed-method study to assess the impact of ongoing vaccination against H5N1 virus on H5N1 viral shedding through freshly dropped feces of chickens raised in commercial farms that exclusively vaccinated or did not vaccinate their chickens.MethodsInitially, we collected vaccination coverage data from all active farms in a subdistrict of each of eight division. In each district, 25 vaccinated and 25 non-vaccinated chicken farms were selected randomly for sample collection. All samples were tested to detect avian influenza viruses using rRT-PCR.ResultsA total of 5092 poultry farms were surveyed; among them 1284 (25%) chicken farms administered vaccine against H5N1 virus. In total 21 of 400 tested farms (5%) had chicken feces samples that tested positive for AIVs; of these three were positive for H5N1 subtype of clade 2.3.2.1. Out of three H5N1 positive farms, 1 (33%) was vaccinated and 2 (67%) were unvaccinated. The chicken farms that administered vaccine against H5N1 was found protective for the detection of H5N1 viral RNA (aOR 0.39, 95% CI: 0.32–0.48). The H5N1 isolates of clade 2.3.2.1 sequenced in this study formed a cluster with the vaccine strain A/duck/Guangdong/S1322/2010 (H5N1) [Re-6].ConclusionsThe overall low vaccination coverage with low detection of H5N1 virus in commercial chickens makes it difficult to assess the effectiveness of the vaccine in reducing H5N1 viral shedding.

Features of influenza virus hemagglutinin genes and their recoding possibilities

The world has already entered the stage of increasing odds for a new pandemic, which prompts to seek out for new flu vaccines, because existing vaccines demonstrate only suboptimal effectiveness. With the Covid-19 pandemic, the possibility of using mRNA vaccines has been opened up, and a prospect of finding hemagglutinin (HA) gene mRNA-based new influenza vaccines seems very attractive. As a rule, the mRNA vaccine is a product of recoding, which ensures the mRNA stability. However, the results of mRNA recoding can be ambiguous. The purpose of this report is to analyze the features of genes and proteins and to consider opportunities and limitations in their recoding. Primary structures of NA proteins and relevant genes were retrieved from Internet publicly available databases. The amino acid composition and frequency of dipeptides, nucleotide and dinucleotide compositions, %GC, translational code and compositions of neighboring di- and tricodones, distribution along primary structure for explicit and synonymous mutations were determined. H1N1 and H3N2 subtypes have both specific and general features (limitations) in their genes, differing not only in the number of protein substitutions, but also in the number and distribution of gene synonymous codons, which do not manifest in the protein primary structure, but appear, apparently, as a hidden factor, which causes the low effectiveness of classical influenza vaccines. The identification of several limitations in gene structure suggests that its any modification (in any gene) must not contradict each of the restrictions established by nature. The frequency of CpG dinucleotides in all studied strains is low, but a potential for optimizing it in H1N1 strains due to the prohibition of the quartet in the gene for arginine-encoding codons is especially limited and can be implemented through synonymous codons of other amino acids (alanine, proline, threonine or serine). Compared to the H1N1 subtype, the H3N2 subtype can be expected to have more possibilities in constructing stable NA gene mRNA.

Single-cell transcriptomic analysis reveals the commonality of immune response upon H1N1 influenza virus infection in mice and humans

Seasonal influenza A virus (IAV), particularly the H1N1 subtype, poses a significant public health threat because of its substantial morbidity and mortality rates worldwide. Understanding the immune response to H1N1 is crucial for developing effective treatments and vaccines. In this study, we deciphered the single-cell transcriptomic landscape of peripheral blood mononuclear cells (PBMCs) from H1N1-infected humans and lung tissue samples from H1N1-infected mice by mining HIN1-related single-cell RNA sequencing data from the GEO database. We observed similar changes in immune cell composition following H1N1 infection, with an increase in macrophages but a decrease in T cells in both species. Moreover, significant transcriptional changes in bystander immune cells upon H1N1 infection were identified, with the upregulation of the chemokine CCL2 in human PBMCs and increased expression of interferon-stimulated genes such as Ifit3, Ifit1 and Isg15 in mouse pulmonary immune cells. Intercellular cross-talk analysis highlighted enhanced interactions among bystander immune cells during H1N1 infection, with neutrophils in humans and macrophages in mice showing the most remarkable increases in interaction intensity. Transcription factor analysis revealed the conserved upregulation of key antiviral regulons, including STAT1 and IRF7, in T cells across both species, highlighting their pivotal roles in antiviral defense. These results suggest that humans and mice exhibit common immune responses to H1N1 infection, underscoring the similarity of vital immune mechanisms across species. The conserved immune mechanisms identified in this study provide potential therapeutic targets for enhancing antiviral immunity. Our research underscores the importance of understanding species-specific and conserved immune responses to H1N1 and offers insights that could inform the development of novel antiviral therapies and improve clinical outcomes for individuals affected by influenza.

A human isolate of bovine H5N1 is transmissible and lethal in animal models.

The outbreak of clade 2.3.4.4b highly pathogenic avian influenza viruses of the H5N1 subtype (HPAI H5N1) in dairy cows in the US has so far resulted in spillover infections of at least thirteen farm workers1-3, who presented with mild respiratory symptoms or conjunctivitis, and one individual with no known animal exposure who was hospitalized but recovered3,4. Here, we characterized A/Texas/37/2024 (huTX37-H5N1), a virus isolated from the eyes of an infected farm worker who developed conjunctivitis5. huTX37-H5N1 replicated efficiently in primary human alveolar epithelial cells, but less efficiently in corneal epithelial cells. Despite causing mild disease in the infected worker, huTX37-H5N1 was lethal in mice and ferrets and spread systemically with high titres in respiratory and non-respiratory organs. Importantly, in four independent experiments in ferrets, huTX37-H5N1 transmitted via respiratory droplets in 17%-33% of transmission pairs and five of six exposed ferrets that became infected died. PB2-631L (encoded by bovine isolates), promoted influenza polymerase activity in human cells, suggesting a role in mammalian adaptation like that of PB2-627K (encoded by huTX37-H5N1). Additionally, bovine HPAI H5N1 viruses were found to be susceptible to polymerase inhibitors both in vitro and in mice. Thus, HPAI H5N1 virus derived from dairy cattle transmits by respiratory droplets in mammals without prior adaptation and causes lethal disease in animal models.

Recombinant avian-derived antiviral proteins cIFITM1, cIFITM3, and cViperin as effective adjuvants in inactivated H9N2 subtype avian influenza vaccines

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.

Genetic features of avian influenza (A/H5N8) clade 2.3.4.4b isolated from quail in Egypt

Several genotypes of the highly pathogenic avian influenza (HPAI) virus H5N8 subtype within clade 2.3.4.4b continue to circulate in different species of domestic birds across Egypt. It is believed that quail contribute to virus replication and adaptation to other gallinaceous poultry species and humans. This study provides genetic characterization of the full genome of HPAI H5N8 isolated from quail in Egypt. The virus was isolated from a commercial quail farm associated with respiratory signs. To characterize the genetic features of the detected virus, gene sequencing via Sanger technology and phylogenetic analysis were performed. The results revealed high nucleotide identity with the HPAI H5N8 virus from Egypt, which has multiple basic amino acid motifs PLREKRRKR/GLF at the hemagglutinin (HA) cleavage site. Phylogenetic analysis of the eight gene segments revealed that the quail isolate is grouped with HPAI H5N8 viruses of clade 2.3.4.4b and closely related to the most recent circulating H5N8 viruses in Egypt. Whole-genome characterization revealed amino acid preferences for avian receptors with few mutations, indicating their affinity for human-like receptors and increased virulence in mammals, such as S123P, S133A, T156A and A263T in the HA gene. In addition, the sequencing results revealed a lack of markers associated with influenza antiviral resistance in the neuraminidase and matrix-2 coding proteins. The results of the present study support the spread of HPAIV H5N8 to species other than chickens in Egypt. Therefore, continuous surveillance of AIV in different bird species in Egypt followed by full genomic characterization is needed for better virus control and prevention.

Field production efficiency investigation of broilers immunized with a turkey herpesvirus vector vaccine expressing hemagglutinin from H9N2 subtype avian influenza virus

Turkey herpesvirus (HVT) vector vaccine expressing hemagglutinin from the H9N2 AIV, namely HVT-H9, were demonstrated to block H9N2 AIV infection and transmission in chickens. In this study, we evaluated the protection efficiency and production performance of broilers in HVT-H9 field trials in the presence or absence of the H9N2 AIV natural infection. HI titers against H9N2 AIV in broilers harboring maternal antibodies were successfully induced by HVT-H9. In the presence of H9N2 AIV natural infection, immunization with HVT-H9 blocked H9N2 AIV infection and reduced the mortality rate. Importantly, HVT-H9 vaccination slightly increased broiler weight and decreased the feed conversion rate in the absence of the H9N2 AIV natural infection but significantly reduced mortality rates and increased production efficiency during the H9N2 AIV natural infection. In summary, HVT-H9 immunization might block H9N2 AIV infection and improve production efficiency in the field, especially in the presence of H9N2 AIV natural infection.