H5N1 Subtype Research Articles

Swine Influenza Viruses Isolated from 2019 to 2022 in Shandong Province, China, Exemplify the Dominant Genotype.

Swine influenza viruses (SIVs) have been circulating in swine globally and are potential threats to human health. During the surveillance of SIVs in Shandong Province, China, from 2019 to 2022, 21 reassortant G4 genotype Eurasian avian-like (EA) H1N1 subtypes containing genes from the EA H1N1 (HA and NA), 2009 pandemic (pdm/09) H1N1 virus (PB2, PB1, PA, NP, and M), and classical swine (CS) H1N1 (NS) lineages were isolated. The analysis of the key functional amino acid sites in the isolated viruses showed that two mutation sites (190D and 225E) that preferentially bind to the human α2-6 sialic acid receptor were found in HA. In PB2, three mutation sites (271A, 590S, and 591R) that may increase mammalian fitness and a mutation site (431M) that increases pathogenicity in mice were found. A typical human signature marker that may promote infection in humans, 357K, was found in NP. The viruses could replicate efficiently in mouse lungs and turbinates, and one of the H1N1 isolates could replicate in mouse kidneys and brains without prior adaption, which indicates that the viruses potentially pose a threat to human health. Histopathological results showed that the isolated viruses caused typical bronchopneumonia and encephalitis in mice. The results indicate that G4 genotype H1N1 has potential transmissibility to humans, and surveillance should be enhanced, which could provide important information for assessing the pandemic potential of the viruses.

Pinnipeds and avian influenza: a global timeline and review of research on the impact of highly pathogenic avian influenza on pinniped populations with particular reference to the endangered Caspian seal (Pusa caspica).

This study reviews chronologically the international scientific and health management literature and resources relating to impacts of highly pathogenic avian influenza (HPAI) viruses on pinnipeds in order to reinforce strategies for the conservation of the endangered Caspian seal (Pusa caspica), currently under threat from the HPAI H5N1 subtype transmitted from infected avifauna which share its haul-out habitats. Many cases of mass pinniped deaths globally have occurred from HPAI spill-overs, and are attributed to infected sympatric aquatic avifauna. As the seasonal migrations of Caspian seals provide occasions for contact with viruses from infected migratory aquatic birds in many locations around the Caspian Sea, this poses a great challenge to seal conservation. These are thus critical locations for the surveillance of highly pathogenic influenza A viruses, whose future reassortments may present a pandemic threat to humans.

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.

Co-infection of H9N2 subtype avian influenza virus and QX genotype live attenuated infectious bronchitis virus increase the pathogenicity in SPF chickens

Avian influenza virus (AIV) infection and vaccination against live attenuated infectious bronchitis virus (aIBV) are frequent in poultry worldwide. Here, we evaluated the clinical effect of H9N2 subtype AIV and QX genotype aIBV co-infection in specific-pathogen-free (SPF) white leghorn chickens and explored the potential mechanisms underlying the observed effects using by 4D-FastDIA-based proteomics. The results showed that co-infection of H9N2 AIV and QX aIBV increased mortality and suppressed the growth of SPF chickens. In particular, severe lesions in the kidneys and slight respiratory signs similar to the symptoms of virulent QX IBV infection were observed in some co-infected chickens, with no such clinical signs observed in single-infected chickens. The replication of H9N2 AIV was significantly enhanced in both the trachea and kidneys, whereas there was only a slight effect on the replication of the QX aIBV. Proteomics analysis showed that the IL-17 signaling pathway was one of the unique pathways enriched in co-infected chickens compared to single infected-chickens. A series of metabolism and immune response-related pathways linked with co-infection were also significantly enriched. Moreover, co-infection of the two pathogens resulted in the enrichment of the negative regulation of telomerase activity. Collectively, our study supports the synergistic effect of the two pathogens, and pointed out that aIBV vaccines might increased IBV-associated lesions due to pathogenic co-infections. Exacerbation of the pathogenicity and mortality in H9N2 AIV and QX aIBV co-infected chickens possibly occurred because of an increase in H9N2 AIV replication, the regulation of telomerase activity, and the disturbance of cell metabolism and the immune system.

Large-Scale Serological Survey of Influenza A Virus in South Korean Wild Boar (Sus scrofa).

In this comprehensive large-scale study, conducted from 2015 to 2019, 7,209 wild boars across South Korea were sampled to assess their exposure to influenza A viruses (IAVs). Of these, 250 (3.5%) were found to be IAV-positive by ELISA, and 150 (2.1%) by the hemagglutination inhibition test. Detected subtypes included 23 cases of pandemic 2009 H1N1, six of human seasonal H3N2, three of classical swine H1N1, 13 of triple-reassortant swine H1N2, seven of triple-reassortant swine H3N2, and seven of swine-origin H3N2 variant. Notably, none of the serum samples tested positive for avian IAV subtypes H3N8, H5N3, H7N7, and H9N2 or canine IAV subtype H3N2. This serologic analysis confirmed the exposure of Korean wild boars to various subtypes of swine and human influenza viruses, with some serum samples cross-reacting between swine and human strains, indicating potential infections with multiple IAVs. The results highlight the potential of wild boar as a novel mixing vessel, facilitating the adaptation of IAVs and their spillover to other hosts, including humans. In light of these findings, we recommend regular and frequent surveillance of circulating influenza viruses in the wild boar population as a proactive measure to prevent potential human influenza pandemics and wild boar influenza epizootics.

Inhibitory effects of Belamcanda extract on inflammatory response and antiviral mechanism in H9N2 Avian influenza virus: insights from in vitro and in vivo studies

Avian influenza, particularly the H9N2 subtype, presents significant challenges to poultry health, underscoring the need for effective antiviral interventions. This study explores the antiviral capabilities of Belamcanda extract, a traditional Chinese medicinal herb, against H9N2 Avian Influenza Virus (AIV) in Specific Pathogen-Free (SPF) chicks. Through a comprehensive approach, we evaluated the impact of the extract on cytokine modulation and crucial immunological signaling pathways, essential for understanding the host-virus interaction. Our findings demonstrate that Belamcanda extract significantly modulates the expression of key inflammatory cytokines, including Tumor Necrosis Factor Alpha (TNF-α), Interleukin-1 (IL-1), Interleukin-2 (IL-2), and Interleukin-6 (IL-6), which are pivotal to the host's response to H9N2 AIV infection. Western blot analysis further revealed that the extract markedly reduces the expression of critical immune signaling molecules such as Toll-Like Receptor 3 (TLR3), TIR-domain-containing adapter-inducing interferon-β (TRIF), and Nuclear Factor Kappa B (NF-κB). These insights into the mechanisms by which Belamcanda extract influences host immune responses and hinders viral replication highlight its potential as an innovative antiviral agent for poultry health management. The study advances our comprehension of natural compounds' antiviral mechanisms and lays the groundwork for developing strategies to manage viral infections in poultry. The demonstrated ability of Belamcanda extract to modulate immune responses and inhibit viral replication establishes it as a promising candidate for future antiviral therapy development, especially in light of the need for effective treatments against evolving influenza virus strains and the critical demand for enhanced poultry health management strategies.

Effects of the Glycosylation of the HA Protein of H9N2 Subtype Avian Influenza Virus on the Pathogenicity in Mice and Antigenicity

As the H9N2 subtype avian influenza virus (H9N2 AIV) evolves naturally, mutations in the hemagglutinin (HA) protein still occur, which involves some sites with glycosylations. It is widely established that glycosylation of the H9N2 AIV HA protein has a major impact on the antigenicity and pathogenicity of the virus. However, the biological implications of a particular glycosylation modification site (GMS) have not been well investigated. In this study, we generated viruses with different GMSs based on wild-type (WT) viruses. Antigenicity studies revealed that the presence of viruses with a 200G+/295G− mutation (with glycosylation at position 200 and deletion of glycosylation at position 295 in the HA protein) combined with a single GMS, such as 87G+, 127G+, 148G+, 178G+, or 265G+, could significantly affect the antigenicity of the virus. Pathogenicity assays revealed that the addition of GMS, such as 127G+, 188G+, 148G+, 178G+, or 54G+, decreased the virulence of the virus in mice, except for 87G+. The removal of GMS, such as 280G− or 295G−, increased the pathogenicity of the virus in mice. Further studies on pathogenicity revealed that 87G+/295G− could also enhance the pathogenicity of the virus. Finally, we selected the WT, WT-87G+, WT-295G−, and WT-87G+/295G− strains as our further research targets to investigate the detailed biological properties of the viruses. GMS, which can enhance viral pathogenicity, did not significantly affect replication or viral stability in vitro but significantly promoted the expression of proinflammatory factors to enhance inflammatory responses in mouse lungs. These findings further deepen our understanding of the influence of the glycosylation of the HA protein of H9N2 AIV on the pathogenicity and antigenicity of the virus in mice.

Spatial and Temporal Characteristic Analysis and Risk Assessment of Global Highly Pathogenic Avian Influenza H5N8 Subtype

H5N8 HPAI is a highly infectious avian disease that now poses a serious threat and potential risk to poultry farming, wild birds, and public health. In this study, to investigate the seasonality and transmission directionality of global H5N8 HPAI, the spatial and temporal analysis of H5N8 HPAI was conducted using time series decomposition and directional distribution analysis. An ecological niche model was developed for H5N8 HPAI in poultry to identify areas at high risk of H5N8 HPAI in poultry and associated risk factors. The results indicated that three global pandemics of H5N8 HPAI emerged from 2014 to 2022, all showing a southeast–northwest distribution direction. H5N8 HPAI occurred more frequently in winter and less frequently in summer. The southwestern border region and the southeastern region of North America, the southern region of South America, most of Europe, the southern border region and the northern border region of Africa, and the southwestern region and the southeastern region of Asia provide the suitable environment for the occurrence of H5N8 HPAI in poultry. Chicken density, duck density, population density, bio1 (annual mean temperature), and land cover were considered important variables for the occurrence of H5N8 HPAI in poultry. This study can help optimize the use of resources and provide new information for policymakers to carry out prevention and control efforts.

Dual N-linked glycosylation at residues 133 and 158 in the hemagglutinin are essential for the efficacy of H7N9 avian influenza virus like particle vaccine in chickens and mice

H7N9 subtype avian influenza virus (AIV) poses a great challenge to poultry industry. Virus-like particle (VLP) is a prospective alternative for the traditional egg-based influenza vaccines. N-linked glycosylation (NLG) regulates the efficacy of influenza vaccines, whereas the impact of NLG modifications on the efficacy of influenza VLP vaccines remains unclear. Here, H7N9 VLPs were assembled in insect cells through co-infection with the baculoviruses expressing the NLG-modified hemagglutinin (HA), neuraminidase and matrix proteins, and the VLP vaccines were assessed in chickens and mice. NLG modifications significantly enhanced hemagglutination-inhibition and virus neutralization antibody responses in mice, rather than in chickens, because different immunization strategies were used in these animal models. The presence of dual NLG at residues 133 and 158 significantly elevated HA-binding IgG titers in chickens and mice. The VLP vaccines conferred complete protection and significantly suppressed virus replication and lung pathology post challenge with H7N9 viruses in chickens and mice. VLP immunization activated T cell immunity-related cytokine response and inhibited inflammatory cytokine response in mouse lung. Of note, the presence of dual NLG at residues 133 and 158 optimized the capacity of the VLP vaccine to stimulate interleukin-4 expression, inhibit virus shedding or alleviate lung pathology in chickens or mice. Intriguingly, the VLP vaccine with NLG addition at residue 133 provided partial cross-protection against the H5Nx subtype AIVs in chickens and mice. In conclusion, dual NLG at residues 133 and 158 in HA can be potentially used to enhance the efficacy of H7N9 VLP vaccines in chickens and mammals.

Identification of B-cell epitopes located on the surface in the PB2 protein of the H9N2 subtype avian influenza virus

ABSTRACT Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

Novel Genotype of HA Clade 2.3.4.4b H5N8 Subtype High Pathogenicity Avian Influenza Virus Emerged at a Wintering Site of Migratory Birds in Japan, 2021/22 Winter.

Surveillance of avian influenza virus (AIV) was conducted in the 2021-2022 winter season at a wintering site of migratory Anatidae in Japan. An H5N8 subtype high pathogenicity AIV (HPAIV) with a unique gene constellation and four low pathogenicity AIVs (LPAIVs) were isolated from environmental samples. The genetic origin of the HPAIV (NK1201) was determined with whole-genome sequencing and phylogenetic analyses. Six of NK1201's eight genes were closely related to HA clade 2.3.4.4b H5N8 subtype HPAIVs, belonging to the G2a group, which was responsible for outbreaks in poultry farms in November 2021 in Japan. However, the remaining two genes, PB1 and NP, most closely matched those of the LPAIVs H7N7 and H1N8, which were isolated at the same place in the same 2021-2022 winter. No virus of the NK1201 genotype had been detected prior to the 2021-2022 winter, indicating that it emerged via genetic reassortment among HPAIV and LPAIVs, which were prevalent at the same wintering site. In addition, experimental infection in chickens indicated that NK1201 had slightly different infectivity compared to the reported infectivity of the representative G2a group H5N8 HPAIV, suggesting that the PB1 and NP genes derived from LPAIVs might have affected the pathogenicity of the virus in chickens. Our results directly demonstrate the emergence of a novel genotype of H5N8 HPAIV through gene reassortment at a wintering site. Analyses of AIVs at wintering sites can help to identify the emergence of novel HPAIVs, which pose risks to poultry, livestock, and humans.

Sequence-based epitope mapping of high pathogenicity avian influenza H5 clade 2.3.4.4b in Latin America.

High Pathogenicity Avian Influenza (HPAI) poses a significant threat to public and animal health. Clade 2.3.4.4b recently emerged from the Eastern hemisphere and disseminated globally, reaching the Latin American (LATAM) region in late 2022 for the first time. HPAI in LATAM has resulted in massive mortalities and culling of poultry and wild birds, causing infection in mammals and humans. Despite its meaningful impact in the region, only occasional evidence about the genetic and epitope characteristics of the introduced HPAI is reported. Hence, this study seeks to phylogenetically characterize the molecular features and the source of HPAI in LATAM by evaluating potential antigenic variations. For such a purpose, we analyzed 302 whole genome sequences. All Latin American viruses are descendants of the 2.3.4.4b clade of the European H5N1 subtype. According to genomic constellations deriving from European and American reassortments, the identification of three major subtypes and eight sub-genotypes was achievable. Based on the variation of antigenic motifs at the HA protein in LATAM, we detected three potential antigenic variants, indicating the HA-C group as the dominant variant. This study decidedly contributes to unraveling the origin of the 2.3.4.4b clade in LATAM, its geographic dissemination, and evolutionary dynamics within Latin American countries. These findings offer useful information for public health interventions and surveillance initiatives planned to prevent and manage the transmission of avian influenza viruses.

Genetic drift and purifying selection shape within-host influenza A virus populations during natural swine infections.

Patterns of within-host influenza A virus (IAV) diversity and evolution have been described in natural human infections, but these patterns remain poorly characterized in non-human hosts. Elucidating these dynamics is important to better understand IAV biology and the evolutionary processes that govern spillover into humans. Here, we sampled an IAV outbreak in pigs during a week-long county fair to characterize viral diversity and evolution in this important reservoir host. Nasal wipes were collected on a daily basis from all pigs present at the fair, yielding up to 421 samples per day. Subtyping of PCR-positive samples revealed the co-circulation of H1N1 and H3N2 subtype swine IAVs. PCR-positive samples with robust Ct values were deep-sequenced, yielding 506 sequenced samples from a total of 253 pigs. Based on higher-depth re-sequenced data from a subset of these initially sequenced samples (260 samples from 168 pigs), we characterized patterns of within-host IAV genetic diversity and evolution. We find that IAV genetic diversity in single-subtype infected pigs is low, with the majority of intrahost Single Nucleotide Variants (iSNVs) present at frequencies of <10%. The ratio of the number of nonsynonymous to the number of synonymous iSNVs is significantly lower than under the neutral expectation, indicating that purifying selection shapes patterns of within-host viral diversity in swine. The dynamic turnover of iSNVs and their pronounced frequency changes further indicate that genetic drift also plays an important role in shaping IAV populations within pigs. Taken together, our results highlight similarities in patterns of IAV genetic diversity and evolution between humans and swine, including the role of stochastic processes in shaping within-host IAV dynamics.

Phylogenetic and Molecular Characteristics of Wild Bird-Origin Avian Influenza Viruses Circulating in Poland in 2018−2022: Reassortment, Multiple Introductions, and Wild Bird–Poultry Epidemiological Links

Since 2020, a significant increase in the severity of H5Nx highly pathogenic avian influenza (HPAI) epidemics in poultry and wild birds has been observed in Poland. To further investigate the genetic diversity of HPAI H5Nx viruses of clade 2.3.4.4b, HPAIV-positive samples collected from dead wild birds in 2020–2022 were phylogenetically characterized. In addition, zoonotic potential and possible reassortment between HPAIVs and LPAIVs circulating in the wild avifauna in Poland have been examined. The genome-wide phylogenetic analysis revealed the presence of three different avian influenza virus (AIV) subtypes (H5N8, H5N5, and H5N1) during the HPAI 2020/2021 season, while in the next HPAI 2021/2022 epidemic only one H5N1 subtype encompassing seven various genotypes (G1–G7) was confirmed. No reassortment events between LPAIVs (detected in the framework of active surveillance) and HPAIVs circulating in Poland have been captured, but instead, epidemiological links between wild birds and poultry due to bidirectional, i.e., wild bird-to-poultry and poultry-to-wild bird HPAIV transmission were evident. Furthermore, at least five independent H5N8 HPAIV introductions into the Baltic Sea region related to unprecedented mass mortality among swans in February–March 2021 in Poland, as well as a general tendency of current H5Nx viruses to accumulate specific mutations associated with the ability to break the interspecies barrier were identified. These results highlight the importance of continuous active and passive surveillance for AI to allow a rapid response to emerging viruses.

Mapping Genetic Markers Associated with Antigenicity and Host Range in H9N2 Influenza A Viruses Infecting Poultry in Pakistan.

The aim of the current study was to map the genetic diversity in the haemagglutinin (HA) glycoprotein of influenza A viruses (IAVs) of the H9N2 subtype. Twenty-five H9N2 IAVs were isolated from broiler chickens from March to July 2019. The HA gene was amplified, and phylogenetic analysis was performed to determine the evolutionary relationship. Important antigenic amino acid residues of HA attributed to immune escape and zoonotic potential were compared among H9N2 IAVs. Phylogenetic analysis revealed that sublineage B2 under the G1 lineage in Pakistan was found to be diversified, and newly sequenced H9N2 isolates were nested into two clades (A and B). Mutations linked to the antigenic variation and potential immune escape were observed as G72E (1/25, 4%), A180T (3/25, 12%), and A180V (1/25, 4%). A twofold significant reduction (P < 0.01) in log2 hemagglutination inhibition titers was observed with H9N2 IAV naturally harboring amino acid V180 instead of A180 in HA protein. Moreover, in the last 20 years, complete substitution at residues (T127D, D135N, and L150N) and partial substitution at residues (72, 74, 131, 148, 180, 183, 188, 216, 217, and 249, mature H9 HA numbering) associated with changes in antigenicity were observed. The presence of L216 in all H9N2 IAV isolates and T/V180 in four isolates in the receptor-binding site reveals the potential of these viruses to cross the species barrier to infect human or mammals. The current study observed the circulation of antigenically diverse H9N2 IAV variants that possess potential mutations that can escape the host immune system.

HiBiT-tagged influenza A virus: a stable and efficient tool for antiviral reagent screening and vaccine evaluation

Influenza A viruses (IAVs) possess variable pathogenic potency causing great economic losses in the poultry industry worldwide and threatening public health. The control of IAV epidemics desperately necessitates an efficient platform for screening antiviral compounds and evaluating vaccine efficacy. In this study, we utilized the H9N2 subtype IAV as the working model. An 11-amino-acid HiBiT tag, derived from NanoLuc luciferase, was incorporated into the flexible linker region of the NS1 protein. Subsequently, the recombinant HiBiT-tagged virus was rescued. The recombinant virus exhibited high genetic stability and similar virological characteristics to the parental virus, both in vitro and in vivo. Of particular significance, the replication profile of the HiBiT-tagged virus can be easily measured using the Nano-Glo assay system, achieving an efficient screening platform. Based on this platform, we have developed assays with both convenience and efficiency for screening antiviral reagents, evaluating immunization efficacy, and measuring neutralizing antibodies.

Evolutional dynamics of highly pathogenic avian influenza H5N8 genotypes in wintering bird habitats: Insights from South Korea's 2020–2021 season

The winter of 2020–2021 in South Korea witnessed severe outbreaks of Highly Pathogenic Avian Influenza (HPAI) viruses, specifically multiple genotypes of the H5N8 subtype. These outbreaks prompted an extensive investigation into the genetic characteristics and evolutionary dynamics of these viruses. Under the auspices of the National Institute of Wildlife Disease Control and Prevention (NIWDC), we conducted a nationwide surveillance program, collecting 7588 specimens from diverse wild bird habitats. Influenza A viruses were isolated at a rate of 5.0%, with HPAI H5N8 viruses accounting for 38.5% of isolates, predominantly found in wild bird carcasses (97.3%). Genetic analysis revealed the emergence of novel HPAI genotypes due to genetic reassortment events. G1 and G2 viruses were separately introduced into Korea, with G1 viruses displaying dynamic behavior, resulting in diverse sub-genotypes (G1–1 to G1–5) and mainly isolated from clinical specimens. Conversely, the G2 virus, introduced later, became the dominant strain consistently isolated mainly from bird carcasses (88.9%). These findings underscore the emergence of numerous novel HPAI genotypes shaped by multiple reassortment events in high-density wintering grounds of migratory birds. These sites act as hotspots for genetic exchanges, significantly influencing avian ecology, including resident bird species, and contributing to HPAI H5N8 evolution. The genetic diversity and ongoing evolution of these viruses highlight the need for vigilant surveillance and adaptive control measures. Recognizing the potential spillover to human populations, a One Health approach is essential to mitigate the evolving threats posed by avian influenza.

Evaluation of immunogenicity and protective efficacy of bacteriophage conjugated haemagglutinin based subunit vaccine against equine influenza virus in a murine model.

Equine influenza (EI) is a highly contagious acute respiratory disease of equines caused by the H3N8 subtype of Influenza A virus i.e. equine influenza virus (EIV). Vaccination is an important and effective tool for the control of EI in equines. Most of the commercial influenza vaccines are produced in embryonated hen's eggs which has several inherent disadvantages. Hence, subunit vaccine based on recombinant haemagglutinin (HA) antigen, being the most important envelope glycoprotein has been extensively exploited for generating protective immune responses, against influenza A and B viruses. We hypothesized that novel vaccine formulation using baculovirus expressed recombinant HA1 (rHA1) protein coupled with bacteriophage will generate strong protective immune response against EIV. In the present study, the recombinant HA1 protein was produced in insect cells using recombinant baculovirus having cloned HA gene of EIV (Florida clade 2 sublineage) and the purified rHA1 was chemically coupled with bacteriophage using a crosslinker to produce rHA1-phage vaccine candidate. The protective efficacy of vaccine preparations of rHA1-phage conjugate and only rHA1 proteins were evaluated in mouse model through assessing serology, cytokine profiling, clinical signs, gross and histopathological changes, immunohistochemistry, and virus quantification. Immunization of vaccine preparations have stimulated moderate antibody response (ELISA titres-5760 ± 640 and 11,520 ± 1280 for rHA1 and rHA1-phage, respectively at 42 dpi) and elicited strong interferon (IFN)-γ expression levels after three immunizations of vaccine candidates. The immunized BALB/c mice were protected against challenge with wild EIV and resulted in reduced clinical signs and body weight loss, reduced pathological changes, decreased EIV antigen distribution, and restricted EIV replication in lungs and nasopharynx. In conclusion, the immune responses with moderate antibody titer and significantly higher cytokine responses generated by the rHA1-phage vaccine preparation without any adjuvant could be a novel vaccine candidate for quick vaccine preparation through further trials of vaccine in the natural host.

Genetic and biological properties of H9N2 avian influenza viruses isolated in central China from 2020 to 2022

The H9N2 subtype of avian influenza virus (AIV) is widely prevalent in poultry and wild birds globally, and has become the predominant subtype circulating in poultry in China. The H9N2 AIV can directly or indirectly (by serving as a “donor virus”) infect humans, posing a significant threat to public health. Currently, there is a lack of in-depth research on the prevalence of H9N2 viruses in Shanxi Province, central China. In this study, we isolated 14 H9N2 AIVs from October 2020 to April 2022 in Shanxi Province, and genetic analysis revealed that these viruses belonged to 7 different genotypes. Our study on animals revealed that the H9N2 strains we identified displayed high transmission efficiency among chicken populations, and exhibited diverse replication abilities within these birds. These viruses could replicate efficiently in the lungs of mice, with one strain also demonstrating the capacity to reproduce in organs like the brain and kidneys. At the cellular level, the replication ability of different H9N2 strains was evaluated using plaque formation assays and multi-step growth curve assays, revealing significant differences in the replication and proliferation efficiency of the various H9N2 viruses at the cellular level. The antigenicity analysis suggested that these isolates could be classified into 2 separate antigenic clusters. Our research provides crucial data to help understand the prevalence and biological characteristics of H9N2 AIVs in central China. It also highlights the necessity of enhancing the surveillance of H9N2 AIVs.

Recent H9N2 avian influenza virus lost hemagglutination activity due to a K141N substitution in hemagglutinin.

The H9N2 avian influenza virus (AIV) represents a significant risk to both the poultry industry and public health. Our surveillance efforts in China have revealed a growing trend of recent H9N2 AIV strains exhibiting a loss of hemagglutination activity at 37°C, posing challenges to detection and monitoring protocols. This study identified a single K141N substitution in the hemagglutinin (HA) glycoprotein as the culprit behind this diminished hemagglutination activity. The study evaluated the evolutionary dynamics of residue HA141 and studied the impact of the N141K substitution on aspects such as virus growth, thermostability, receptor-binding properties, and antigenic properties. Our findings indicate a polymorphism at residue 141, with the N variant becoming increasingly prevalent in recent Chinese H9N2 isolates. Although both wild-type and N141K mutant strains exclusively target α,2-6 sialic acid receptors, the N141K mutation notably impedes the virus's ability to bind to these receptors. Despite the mutation exerting minimal influence on viral titers, antigenicity, and pathogenicity in chicken embryos, it significantly enhances viral thermostability and reduces plaque size on Madin-Darby canine kidney (MDCK) cells. Additionally, the N141K mutation leads to decreased expression levels of HA protein in both MDCK cells and eggs. These findings highlight the critical role of the K141N substitution in altering the hemagglutination characteristics of recent H9N2 AIV strains under elevated temperatures. This emphasizes the need for ongoing surveillance and genetic analysis of circulating H9N2 AIV strains to develop effective control and prevention measures.IMPORTANCEThe H9N2 subtype of avian influenza virus (AIV) is currently the most prevalent low-pathogenicity AIV circulating in domestic poultry globally. Recently, there has been an emerging trend of H9N2 AIV strains acquiring increased affinity for human-type receptors and even losing their ability to bind to avian-type receptors, which raises concerns about their pandemic potential. In China, there has been a growing number of H9N2 AIV strains that have lost their ability to agglutinate chicken red blood cells, leading to false-negative results during surveillance efforts. In this study, we identified a K141N mutation in the HA protein of H9N2 AIV to be responsible for the loss of hemagglutination activity. This finding provides insight into the development of effective surveillance, prevention, and control strategies to mitigate the threat posed by H9N2 AIV to both animal and human health.