Genetic diversity of H5N1 and H5N2 high pathogenicity avian influenza viruses isolated from poultry in Japan during the winter of 2022-2023.

High pathogenicity avian influenza viruses (HPAIVs) of the H5N1 and H5N2 subtypes were responsible for 84 HPAI outbreaks on poultry premises in Japan during October 2022-April 2023. The number of outbreaks during the winter of 2022-2023 is the largest ever reported in Japan. In this study, we performed phylogenetic analyses using the full genetic sequences of HPAIVs isolated in Japan during 2022-2023 and those obtained from a public database to identify their genetic origin. Based on the hemagglutinin genes, these HPAIVs were classified into the G2 group of clade 2.3.4.4b, whose ancestors were H5 HPAIVs that circulated in Europe in late 2020, and were then further divided into three subgroups (G2b, G2d, and G2c). Approximately one-third of these viruses were classified into the G2b and G2d groups, which also included H5N1 HPAIVs detected in Japan during 2021-2022. In contrast, the remaining two-thirds were classified into the G2c group, which originated from H5N1 HPAIVs isolated in Asian countries and Russia during the winter of 2021-2022. Unlike the G2b and G2d viruses, the G2c viruses were first detected in Japan in the fall of 2022. Importantly, G2c viruses caused the largest number of outbreaks throughout Japan over the longest period during the season. Phylogenetic analyses using eight segment genes revealed that G2b, G2d, and G2c viruses were divided into 2, 4, and 11 genotypes, respectively, because they have various internal genes closely related to those of avian influenza viruses detected in wild birds in recent years in Asia, Russia, and North America, respectively. These results suggest that HPAIVs were disseminated among migratory birds, which may have generated numerous reassortant viruses with various gene constellations, resulting in a considerable number of outbreaks during the winter of 2022-2023.

Are all avian influenza outbreaks in poultry the same? The predicted impact of poultry species and virus subtype.

AIMS: Outbreaks of avian influenza in poultry farms are currently increasing in frequency, with devastating consequences for animal welfare, farmers and supply chains. Some studies have documented the direct spread of the avian influenza virus between farms. Prevention of spread between farms relies on biosecurity surveillance and control measures. However, the evolution of an outbreak on a farm might vary depending on the virus strain and poultry species involved; this would have important implications for surveillance systems, epidemiological investigations and control measures. METHODS AND RESULTS: In this study, we utilized existing parameter estimates from the literature to evaluate the predicted course of an epidemic in a standard poultry flock with 10,000 birds. We used a stochastic SEIR simulation model to simulate outbreaks in different species and with different virus subtypes. The simulations predicted large differences in the duration and severity of outbreaks, depending on the virus subtypes. For both turkeys and chickens, outbreaks with HPAI were of shorter duration than outbreaks with LPAI. In outbreaks involving the infection of chickens with different virus subtypes, the shortest epidemic involved H7N7 and HPAIV H5N1 (median duration of 9 and 17 days, respectively) and the longest involved H5N2 (median duration of 68 days). The most severe outbreaks (number of chickens infected) were predicted for H5N1, H7N1 and H7N3 virus subtypes, and the least severe for H5N2 and H7N7, in which outbreaks for the latter subtype were predicted to develop most slowly. CONCLUSIONS: These simulation results suggest that surveillance of certain subtypes of avian influenza virus, in chicken flocks in particular, needs to be sensitive and timely if infection is to be detected with sufficient time to implement control measures. The variability in the predictions highlights that avian influenza outbreaks are different in severity, speed and duration, so surveillance and disease response need to be nuanced and fit the specific context of poultry species and virus subtypes.

Mucosal Administration of Lactobacillus casei Surface-Displayed HA1 Induces Protective Immune Responses against Avian Influenza A Virus in Mice.

Avian influenza is a serious threat to both public health and the poultry industry worldwide. This respiratory virus can be combated by eliciting robust immune responses at the site of infection through mucosal immunization. Recombinant probiotics, specifically lactic acid bacteria, are safe and effective carriers for mucosal vaccines. In this study, we engineered recombinant fusion protein by fusing the hemagglutinin 1 (HA1) subunit of the A/Aquatic bird/Korea/W81/2005 (H5N2) with the Bacillus subtilis poly γ-glutamic acid synthetase A (pgsA) at the surface of Lactobacillus casei (pgsA-HA1/L. casei). Using subcellular fractionation and flow cytometry we confirmed the surface localization of this fusion protein. Mucosal administration of pgsA-HA1/L. casei in mice resulted in significant levels of HA1-specific serum IgG, mucosal IgA and neutralizing antibodies against the H5N2 virus. Additionally, pgsA-HA1/L. casei-induced systemic and local cell-mediated immune responses specific to HA1, as evidenced by an increased number of IFN-γ and IL-4 secreting cells in the spleens and higher levels of IL-4 in the local lymphocyte supernatants. Finally, mice inoculated with pgsA-HA1/L. casei were protected against a 10LD50 dose of the homologous mouse-adapted H5N2 virus. These results suggest that mucosal immunization with L. casei displaying HA1 on its surface could be a potential strategy for developing a mucosal vaccine against other H5 subtype viruses.

Spillover of an endemic avian Influenza H6N2 chicken lineage to ostriches and reassortment with clade 2.3.4.4b H5N1 high pathogenicity viruses in chickens.

Prior to 2017, chicken production in South Africa had only ever been affected by an endemic strain of H6N2 low pathogenic avian influenza (LPAI), but since 2017, an outbreak of Goose/Guangdong clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) introduced by wild birds, followed by clade 2.3.4.4b H5N1 HPAI (2021-present), affected the country. In the present study, the viruses from seven cases of H6N2 LPAI from commercial poultry between October 2019 and August 2020 were genome-sequenced along with an H5N2 HPAI virus, and phylogenetic analysis was performed. The H5N2 HPAI virus caused localized outbreaks in a small-scale chicken farm and a large commercial layer farm in the KwaZulu-Natal province between late October and early December 2022. The phylogenetic results confirmed the first incidence of the chicken-adapted H6N2 lineage in commercial ostriches in the Western Cape province, with a likely epidemiological origin in chickens from the KwaZulu Natal province. The results also showed that the H5N2 HPAI virus was a novel reassortant of PB2, PB1, PA, NP and NA genome segments derived from a parental H6N2 virus that circulated in region, whereas the HA, M and NS genome segments were derived from sub-genotype SA10 H5N1 HPAI parental virus that had circulated in the local wild bird reservoir since July 2021.

Efficacy of recombinant H5 vaccines delivered in ovo or day of age in commercial broilers against the 2015 U.S. H5N2 clade 2.3.4.4c highly pathogenic avian Influenza virus.

BACKGROUND: Avian influenza is a highly contagious, agriculturally relevant disease that can severely affect the poultry industry and food supply. Eurasian-origin H5Nx highly pathogenic avian influenza viruses (HPAIV) (clade 2.3.4.4) have been circulating globally in wild birds with spill over into commercial poultry operations. The negative impact to commercial poultry renewed interest in the development of vaccines against these viruses to control outbreaks in the U.S. METHODS: The efficacy of three recombinant H5 vaccines delivered in ovo or day of age were evaluated in commercial broilers challenged with the 2015 U.S. H5N2 clade 2.3.4.4c HPAIV. The recombinant vaccines included an alphavirus RNA particle vaccine (RP-H5), an inactivated reverse genetics-derived (RG-H5) and recombinant HVT vaccine (rHVT-AI) expressing H5 hemagglutinin (HA) genes. In the first experiment, in ovo vaccination with RP-H5 or rHVT-AI was tested against HPAI challenge at 3 or 6 weeks of age. In a second experiment, broilers were vaccinated at 1 day of age with a dose of either 107 or 108 RP-H5, or RG-H5 (512 HA units (HAU) per dose). RESULTS: In experiment one, the RP-H5 provided no protection following in ovo application, and shedding titers were similar to sham vaccinated birds. However, when the RP-H5 was delivered in ovo with a boost at 3 weeks, 95% protection was demonstrated at 6 weeks of age. The rHVT-AI vaccine demonstrated 95 and 100% protection at 3 and 6 weeks of age, respectively, of challenged broilers with reduced virus shedding compared to sham vaccinated birds. Finally, when the RP-H5 and rHVT vaccines were co-administered at one day of age, 95% protection was demonstrated with challenge at either 3 or 6 weeks age. In the second experiment, the highest protection (92%) was observed in the 108 RP-H5 vaccinated group. Significant reductions (p < 0.05) in virus shedding were observed in groups of vaccinated birds that were protected from challenge. The RG-H5 provided 62% protection from challenge. In all groups of surviving birds, antibody titers increased following challenge. CONCLUSIONS: Overall, these results demonstrated several strategies that could be considered to protected broiler chickens during a H5 HPAI challenge.

Sequence Analysis of the Malaysian Low Pathogenic Avian Influenza Virus Strain H5N2 from Duck.

The avian influenza viruses (AIV) of the H5 subtype have the ability to mutate from low pathogenic (LPAI) to highly pathogenic (HPAI), which can cause high mortality in poultry. Little is known about the pathogenic switching apart from the mutations at the haemagglutinin cleavage site, which significantly contributes to the virus virulence switching phenomenon. Therefore, this study aimed to compare the molecular markers in the haemagglutinin (HA), neuraminidase (NA), and matrix (M) genes of a locally isolated LPAI AIV strain H5N2 from Malaysia with the reference HPAI strains using bioinformatics approaches, emphasising the pathogenic properties of the viral genes. First, the H5N2 strain A/Duck/Malaysia/8443/2004 was propagated in SPF eggs. The viral presence was verified by haemagglutination assay, RT-PCR, and sequencing. Results showed successful amplifications of HA (1695 bp), NA (1410 bp), and M (1019 bp) genes. The genes were sequenced and the deduced amino acid sequences were analysed computationally using MEGA 11 and NetNGlyc software. Analysis of the HA protein showed the absence of the polybasic cleavage motif, but presence of two amino acid residues that are known to affect pathogenicity. There were also two glycosylation sites (glycosites) compared to the reference HPAI viruses, which had three or more at the HA globular head domain. No NA stalk deletion was detected but the haemadsorbing and active centres of the studied NA protein were relatively similar to the reference HPAI H5N2 isolates of duck but not chicken origins. Six NA glycosites were also identified. Finally, we observed a consistent M1 and M2 amino acid sequences between our LPAI isolate with the other HPAI H5N1 or H5N2 reference proteins. These data demonstrate distinct characteristics of the Malaysian LPAI H5N2, compared to HPAI H5N2 or H5N1 from ducks or chickens, potentially aiding the epidemiological research on genetic dynamics of circulating AIV in poultry.

A Dutch highly pathogenic H5N6 avian influenza virus showed remarkable tropism for extra-respiratory organs and caused severe disease but was not transmissible via air in the ferret model.

Continued circulation of A/H5N1 influenza viruses of the A/goose/Guangdong/1/96 lineage in poultry has resulted in the diversification in multiple genetic and antigenic clades. Since 2009, clade 2.3.4.4 hemagglutinin (HA) containing viruses harboring the internal and neuraminidase (NA) genes of other avian influenza A viruses have been detected. As a result, various HA-NA combinations, such as A/H5N1, A/H5N2, A/H5N3, A/H5N5, A/H5N6, and A/H5N8 have been identified. As of January 2023, 83 humans have been infected with A/H5N6 viruses, thereby posing an apparent risk for public health. Here, as part of a risk assessment, the in vitro and in vivo characterization of A/H5N6 A/black-headed gull/Netherlands/29/2017 is described. This A/H5N6 virus was not transmitted between ferrets via the air but was of unexpectedly high pathogenicity compared to other described A/H5N6 viruses. The virus replicated and caused severe lesions not only in respiratory tissues but also in multiple extra-respiratory tissues, including brain, liver, pancreas, spleen, lymph nodes, and adrenal gland. Sequence analyses demonstrated that the well-known mammalian adaptation substitution D701N was positively selected in almost all ferrets. In the in vitro experiments, no other known viral phenotypic properties associated with mammalian adaptation or increased pathogenicity were identified. The lack of transmission via the air and the absence of mammalian adaptation markers suggest that the public health risk of this virus is low. The high pathogenicity of this virus in ferrets could not be explained by the known mammalian pathogenicity factors and should be further studied. IMPORTANCE Avian influenza A/H5 viruses can cross the species barrier and infect humans. These infections can have a fatal outcome, but fortunately these influenza A/H5 viruses do not spread between humans. However, the extensive circulation and reassortment of A/H5N6 viruses in poultry and wild birds warrant risk assessments of circulating strains. Here an in-depth characterization of the properties of an avian A/H5N6 influenza virus isolated from a black-headed gull in the Netherlands was performed in vitro and in vivo, in ferrets. The virus was not transmissible via the air but caused severe disease and spread to extra-respiratory organs. Apart from the detection in ferrets of a mutation that increased virus replication, no other mammalian adaptation phenotypes were identified. Our results suggest that the risk of this avian A/H5N6 virus for public health is low. The underlying reasons for the high pathogenicity of this virus are unexplained and should be further studied.

The Evolution of Highly Pathogenic Avian Influenza A (H5) in Poultry in Nigeria, 2021-2022.

In 2021, amidst the COVID-19 pandemic and global food insecurity, the Nigerian poultry sector was exposed to the highly pathogenic avian influenza (HPAI) virus and its economic challenges. Between 2021 and 2022, HPAI caused 467 outbreaks reported in 31 of the 37 administrative regions in Nigeria. In this study, we characterized the genomes of 97 influenza A viruses of the subtypes H5N1, H5N2, and H5N8, which were identified in different agro-ecological zones and farms during the 2021-2022 epidemic. The phylogenetic analysis of the HA genes showed a widespread distribution of the H5Nx clade 2.3.4.4b and similarity with the HPAI H5Nx viruses that have been detected in Europe since late 2020. The topology of the phylogenetic trees indicated the occurrence of several independent introductions of the virus into the country, followed by a regional evolution of the virus that was most probably linked to its persistent circulation in West African territories. Additional evidence of the evolutionary potential of the HPAI viruses circulating in this region is the identification in this study of a putative H5N1/H9N2 reassortant virus in a mixed-species commercial poultry farm. Our data confirm Nigeria as a crucial hotspot for HPAI virus introduction from the Eurasian territories and reveal a dynamic pattern of avian influenza virus evolution within the Nigerian poultry population.

Ecogeographic Drivers of the Spatial Spread of Highly Pathogenic Avian Influenza Outbreaks in Europe and the United States, 2016-Early 2022.

H5Nx highly pathogenic avian influenza (HPAI) viruses of clade 2.3.4.4 have caused outbreaks in Europe among wild and domestic birds since 2016 and were introduced to North America via wild migratory birds in December 2021. We examined the spatiotemporal extent of HPAI viruses across continents and characterized ecological and environmental predictors of virus spread between geographic regions by constructing a Bayesian phylodynamic generalized linear model (phylodynamic-GLM). The findings demonstrate localized epidemics of H5Nx throughout Europe in the first several years of the epizootic, followed by a singular branching point where H5N1 viruses were introduced to North America, likely via stopover locations throughout the North Atlantic. Once in the United States (US), H5Nx viruses spread at a greater rate between US-based regions as compared to prior spread in Europe. We established that geographic proximity is a predictor of virus spread between regions, implying that intercontinental transport across the Atlantic Ocean is relatively rare. An increase in mean ambient temperature over time was predictive of reduced H5Nx virus spread, which may reflect the effect of climate change on declines in host species abundance, decreased persistence of the virus in the environment, or changes in migratory patterns due to ecological alterations. Our data provide new knowledge about the spread and directionality of H5Nx virus dispersal in Europe and the US during an actively evolving intercontinental outbreak, including predictors of virus movement between regions, which will contribute to surveillance and mitigation strategies as the outbreak unfolds, and in future instances of uncontained avian spread of HPAI viruses.

Predictive analysis for pathogenicity classification of H5Nx avian influenza strains using machine learning techniques.

Over the past decades, avian influenza (AI) outbreaks have been reported across different parts of the globe, resulting in large-scale economic and livestock loss and, in some cases raising concerns about their zoonotic potential. The virulence and pathogenicity of H5Nx (e.g., H5N1, H5N2) AI strains for poultry could be inferred through various approaches, and it has been frequently performed by detecting certain pathogenicity markers in their haemagglutinin (HA) gene. The utilization of predictive modeling methods represents a possible approach to exploring this genotypic-phenotypic relationship for assisting experts in determining the pathogenicity of circulating AI viruses. Therefore, the main objective of this study was to evaluate the predictive performance of different machine learning (ML) techniques for in-silico prediction of pathogenicity of H5Nx viruses in poultry, using complete genetic sequences of the HA gene. We annotated 2137 H5Nx HA gene sequences based on the presence of the polybasic HA cleavage site (HACS) with 46.33% and 53.67% of sequences previously identified as highly pathogenic (HP) and low pathogenic (LP), respectively. We compared the performance of different ML classifiers (e.g., logistic regression (LR) with the lasso and ridge regularization, random forest (RF), K-nearest neighbor (KNN), Naïve Bayes (NB), support vector machine (SVM), and convolutional neural network (CNN)) for pathogenicity classification of raw H5Nx nucleotide and protein sequences using a 10-fold cross-validation technique. We found that different ML techniques can be successfully used for the pathogenicity classification of H5 sequences with ∼99% classification accuracy. Our results indicate that for pathogenicity classification of (1) aligned deoxyribonucleic acid (DNA) and protein sequences, with NB classifier had the lowest accuracies of 98.41% (+/-0.89) and 98.31% (+/-1.06), respectively; (2) aligned DNA and protein sequences, with LR (L1/L2), KNN, SVM (radial basis function (RBF)) and CNN classifiers had the highest accuracies of 99.20% (+/-0.54) and 99.20% (+/-0.38), respectively; (3) unaligned DNA and protein sequences, with CNN's achieved accuracies of 98.54% (+/-0.68) and 99.20% (+/-0.50), respectively. ML methods show potential for regular classification of H5Nx virus pathogenicity for poultry species, particularly when sequences containing regular markers were frequently present in the training dataset.

Discrimination of the H1N1 and H5N2 Variants of Influenza A Virus Using an Isomeric Sialic Acid-Conjugated Graphene Field-Effect Transistor.

There has been a continuous effort to fabricate a fast, sensitive, and inexpensive system for influenza virus detection to meet the demand for effective screening in point-of-care testing. Herein, we report a sialic acid (SA)-conjugated graphene field-effect transistor (SA-GFET) sensor designed using α2,3-linked sialic acid (3'-SA) and α2,6-linked sialic acid (6'-SA) for the detection and discrimination of the hemagglutinin (HA) protein of the H5N2 and H1N1 viruses. 3'-SA and 6'-SA specific for H5 and H1 influenza were used in the SA-GFET to capture the HA protein of the influenza virus. The net charge of the captured viral sample led to a change in the electrical current of the SA-GFET platform, which could be correlated to the concentration of the viral sample. This SA-GFET platform exhibited a highly sensitive response in the range of 101-106 pfu mL-1, with a limit of detection (LOD) of 101 pfu mL-1 in buffer solution and a response time of approximately 10 s. The selectivity of the SA-GFET platform for the H1N1 and H5N2 influenza viruses was verified by testing analogous respiratory viruses, i.e., influenza B and the spike protein of SARS-CoV-2 and MERS-CoV, on the SA-GFET. Overall, the results demonstrate that the developed dual-channel SA-GFET platform can potentially serve as a highly efficient and sensitive sensing platform for the rapid detection of infectious diseases.

Reverse genetics based H5N2 vaccine provides clinical protection against H5N1, H5N8 and H9N2 avian influenza infection in chickens.

The current study aimed to develop broadly protective vaccines for avian influenza. In an earlier study, HA stalk (universal flu vaccine) was found to be broadly protective against different subtypes of influenza virus in mice. Hence, we were interested to know its breadth of protective efficacy either alone or combined with inactivated rgH5N2 (clade 2.3.2.1a) vaccine against challenge viruses of homologous H5N1, heterologous H5N8 (clade 2.3.4.4) and heterosubtypic H9N2 virus in specific pathogen-free chickens. The rgH5N2 vaccine alone or in combination with HA stalk elicited sufficient pre-challenge immunity in the form of haemagglutination inhibiting (HI) antibodies and neutralizing antibodies (MNT) against H5N1, H5N8, and H9N2 in chickens. The rgH5N2 vaccine alone or in combination with HA stalk also attenuated the shedding of H5N1, H5N8 and H9N2 in chickens and protected against the lethal challenge of H5N1 or H5N8. In contrast, all HA stalk immunised chickens died upon H5N1 or H5N8 challenge and H9N2 challenged chickens survived. Our study suggests that the rgH5N2 vaccine can provide clinical protection against H5N1, H5N8 and can attenuate the viral shedding of H9N2 in chickens.

Cross-Reactive Fc-Fused Single-Domain Antibodies to Hemagglutinin Stem Region Protect Mice from Group 1 Influenza a Virus Infection.

The continued evolution of influenza viruses reduces the effectiveness of vaccination and antiviral drugs. The identification of novel and universal agents for influenza prophylaxis and treatment is an urgent need. We have previously described two potent single-domain antibodies (VHH), G2.3 and H1.2, which bind to the stem domain of hemagglutinin and efficiently neutralize H1N1 and H5N2 influenza viruses in vivo. In this study, we modified these VHHs with Fc-fragment to enhance their antiviral activity. Reformatting of G2.3 into bivalent Fc-fusion molecule increased its in vitro neutralizing activity against H1N1 and H2N3 viruses up to 80-fold and, moreover, resulted in obtaining the ability to neutralize H5N2 and H9N2 subtypes. We demonstrated that a dose as low as 0.6 mg/kg of G2.3-Fc or H1.2-Fc administered systemically or locally before infection could protect mice from lethal challenges with both H1N1 and H5N2 viruses. Furthermore, G2.3-Fc reduced the lung viral load to an undetectable level. Both VHH-Fc antibodies showed in vivo therapeutic efficacy when delivered via systemic or local route. The findings support G2.3-Fc as a potential therapeutic agent for both prophylaxis and therapy of Group 1 influenza A infection.

Wild Bird Surveillance in the Gauteng Province of South Africa during the High-Risk Period for Highly Pathogenic Avian Influenza Virus Introduction.

Migratory birds carried clade 2.3.4.4B H5Nx highly pathogenic avian influenza (HPAI) viruses to South Africa in 2017, 2018 and 2021, where the Gauteng Province is a high-risk zone for virus introduction. Here, we combined environmental faecal sampling with sensitive rRT-PCR methods and direct Ion Torrent sequencing to survey wild populations between February and May 2022. An overall IAV incidence of 42.92% (100/231) in water bird faecal swab pools or swabs from moribund or dead European White Storks (Ciconia ciconia) was detected. In total, 7% of the IAV-positive pools tested H5-positive, with clade 2.3.4.4B H5N1 HPAI confirmed in the storks; 10% of the IAV-positive samples were identified as H9N2, and five complete H9N2 genomes were phylogenetically closely related to a local 2021 wild duck H9N2 virus, recent Eurasian LPAI viruses or those detected in commercial ostriches in the Western and Eastern Cape Provinces since 2018. H3N1, H4N2, H5N2 and H8Nx subtypes were also identified. Targeted surveillance of wild birds using environmental faecal sampling can thus be effectively applied under sub-Saharan African conditions, but region-specific studies should first be used to identify peak prevalence times which, in southern Africa, is linked to the peak rainfall period, when ducks are reproductively active.

A variant NS1 protein from H5N2 avian influenza virus suppresses PKR activation and promotes replication and virulence in mammals.

Highly pathogenic avian influenza viruses (HPAIVs) frequently receive global attention as threats to public health. The NS1 protein is a key virulence factor known to impair host antiviral responses. The study herein revealed HPAIV H5N2 NS gene encoded additional protein; a truncated NS1 variant, designated NS3, produced by alternative splicing of the NS transcript. To examine the function of NS3 during infection, we generated recombinant viruses expressing either full-length NS1 (RG-AIV-T375G) or NS3 (RG-AIV-NS3). Interestingly, RG-AIV-NS3 virus produced higher titres than RG-AIV-T375G in multiple mammalian cell lines. However, RG-AIV-T375G exhibited a replication advantage over RG-AIV-NS3 in chicken DF-1 cells, indicating that host cell identity dictates the effect of NS3 on viral replication. In mice and mammalian cells, RG-AIV-NS3 infection elicited higher level of cytokines, including IFN-ß, MX and TNF-α, potentially due to its higher replication activity. Based on mini-genome assay, NS3 had pronounced effects on viral replication machinery. Surprisingly, NS3 retained an interaction with PKR and suppressed PKR activation despite its lack of amino-acid residues 126-167. The poor replication ability of RG-AIV-T375G was partially restored in cells deficient in PKR suggesting that full-length NS1 may be insufficient to suppress PKR function. Notably, virulence of the full-length NS1-expressing RG-AIV-T375G virus was highly attenuated in mice when compared to RG-AIV-NS3. In summary, our study reveals the existence and function of a previously unidentified H5N2 viral protein, NS3. We found that NS3 is functionally distinct from NS1 protein, as it enhances viral replication and pathogenicity in mammalian systems, potentially via suppression of PKR activity.

Fine-tuning of MXene-nickel oxide-reduced graphene oxide nanocomposite bioelectrode: Sensor for the detection of influenza virus and viral protein.

Influenza viruses can cause epidemics through inter-human transmission, and the social consequences of viral transmission are incalculable. Current diagnostics for virus detection commonly relies on antibodies or nucleic acid as recognition reagent. However, a more advanced and general method for the facile development of new biosensors is increasing in demand. In this study, we report the fabrication of an ultra-sensitive peptide-based nanobiosensor using a nickel oxide (NiO)-reduced graphene oxide (rGO)/MXene nanocomposite to detect active influenza viruses (H1N1 and H5N2) and viral proteins. The sensing mechanism is based on the signal inhibition, the specific interaction between H1N1 (QMGFMTSPKHSV) and H5N1 (GHPHYNNPSLQL) binding peptides anchored on the NiO-rGO/MXene/glassy carbon electrode (GCE) surface and the viral surface protein hemagglutinin (HA) is the critical factor for the decrease in the peak current of the sensor. In this strategy, the NiO-rGO/MXene nanocomposite results in synergistic signal effects, including electrical conductivity, porosity, electroactive surface area, and active site availability when viruses are deposited on the electrode. Based on these observations, the results showed that the developed nanobiosensor was capable of highly sensitive and specific detection of their corresponding influenza viruses and viral proteins with a very low detection limit (3.63 nM of H1N1 and 2.39 nM for H5N1, respectively) and good recovery. The findings demonstrate that the proposed NiO-rGO/MXene-based peptide biosensor can provide insights for developing a wide range of clinical screening tools for detecting affected patients.

2,5-Diketopiperazine Derivatives as Potential Anti-Influenza (H5N2) Agents: Synthesis, Biological Evaluation, and Molecular Docking Study.

2,5-Diketopiperazine derivatives, consisting of benzylidene and alkylidene substituents at 3 and 6 positions, have been considered as a core structure for their antiviral activities. Herein, the novel N-substituted 2,5-Diketopiperazine derivatives were successfully prepared and their antiviral activities against influenza virus were evaluated by monitoring viral propagation in embryonated chicken eggs. It was found that (3Z,6Z)-3-benzylidene-6-(2-methyl propylidene)-4-substituted-2,5-Diketopiperazines (13b-d), (3Z,6E)-3-benzylidene-6-(2-methylpropyli dene)-1-(1-ethyl pyrrolidine)-2,5-Diketopiperazine (14c), and Lansai-C exhibited negative results in influenza virus propagation at a concentration of 25 µg/mL. Additionally, molecular docking study revealed that 13b-d and 14c bound in 430-cavity of neuraminidase from H5N2 avian influenza virus and the synthesized derivatives also strongly interacted with the key amino acid residues, including Arg371, Pro326, Ile427, and Thr439.

Evolutionary Dynamics of Mexican Lineage H5N2 Avian Influenza Viruses.

We have demonstrated for the first time a comprehensive evolutionary analysis of the Mexican lineage H5N2 avian influenza virus (AIV) using complete genome sequences (n = 189), from its first isolation in 1993 until 2019. Our study showed that the Mexican lineage H5N2 AIV originated from the North American wild bird gene pool viruses around 1990 and is currently circulating in poultry populations of Mexico, the Dominican Republic, and Taiwan. Since the implementation of vaccination in 1995, the highly pathogenic AIV (HPAIV) H5N2 virus was eradicated from Mexican poultry in mid-1995. However, the low pathogenic AIV (LPAIV) H5N2 virus has continued to circulate in domestic poultry populations in Mexico, eventually evolving into five distinct clades. In the current study, we demonstrate that the evolution of Mexican lineage H5N2 AIVs involves gene reassortments and mutations gained over time. The current circulating Mexican lineage H5N2 AIVs are classified as LPAIV based on the amino acid sequences of the hemagglutinin (HA) protein cleavage site motif as well as the results of the intravenous pathogenicity index (IVPI). The immune pressure from vaccinations most likely has played a significant role in the positive selection of antigenic drift mutants within the Mexican H5N2 AIVs. Most of the identified substitutions in these viruses are located on the critical antigenic residues of the HA protein and as a result, might have contributed to vaccine failures. This study highlights and stresses the need for vaccine updates while emphasizing the importance of continued molecular monitoring of the HA protein for its antigenic changes compared to the vaccines used.

Genetically Diverse Highly Pathogenic Avian Influenza A(H5N1/H5N8) Viruses among Wild Waterfowl and Domestic Poultry, Japan, 2021.

Genetic analyses of highly pathogenic avian influenza H5 subtype viruses isolated from the Izumi Plain, Japan, revealed cocirculation of 2 genetic groups of clade 2.3.4.4b viruses among migratory waterfowl. Our findings demonstrate that both continuous surveillance and timely information sharing of avian influenza viruses are valuable for rapid risk assessment.