H1 Subtypes Research Articles

Influenza A Virus H7 nanobody recognizes a conserved immunodominant epitope on hemagglutinin head and confers heterosubtypic protection

Influenza remains a persistent global health challenge, largely due to the virus’ continuous antigenic drift and occasional shift, which impede the development of a universal vaccine. To address this, the identification of broadly neutralizing antibodies and their epitopes is crucial. Nanobodies, with their unique characteristics and binding capacity, offer a promising avenue to identify such epitopes. Here, we isolate and purify a hemagglutinin (HA)-specific nanobody that recognizes an H7 subtype of influenza A virus. The nanobody, named E10, exhibits broad-spectrum binding, cross-group neutralization and in vivo protection across various influenza A subtypes. Through phage display and in vitro characterization, we demonstrate that E10 specifically targets an epitope on HA head which is part of the conserved lateral patch and is highly immunodominant upon H7 infection. Importantly, immunization with a peptide including the E10 epitope elicits cross-reactive antibodies and mediates partial protection from lethal viral challenge. Our data highlights the potential of E10 and its associated epitope as a candidate for future influenza prevention strategies.

Evidence of avian and human influenza A virus infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

Crocodilians are susceptible to a range of virus infection including influenza A virus (IAV). However, little is known about the ecology and epidemiology of IAV in crocodile species. This study aimed to investigate IAV infection in farmed Siamese crocodiles in central Thailand. We collected plasma samples and pharyngeal swab samples from Siamese crocodiles residing in 13 crocodile farms in 9 provinces of central Thailand during 2019. Additional archival plasma samples of Siamese crocodiles collected in 2012 and 2018 were also included in the study. Plasma samples were screened for influenza A antibodies by a hemagglutination inhibition (HI) assay and positive were evaluated by a cytopathic effect/hemagglutination based-microneutralization (MN) assay. Swab samples were tested for influenza viral RNA by a real-time RT-PCR assay targeting the influenza matrix (M) gene. Among 246 tested plasma samples, the overall seroprevalence of antibodies against IAV in farmed Siamese crocodiles was 17.5% (43/246). The most common hemagglutinin (HA) subtype was H2 (46.5%, 20/43) followed by H9 (39.5%, 17/43), human H1 (14%, 6/43) and H1 (7%, 3/43). Multiple HA subtypes were also detected in 7% (3/43) of infected crocodiles with combination of H1 and H2 subtypes. All 126 tested swab samples were negative for influenza viral RNA. In addition, we demonstrated the ability of wild-type IAV subtypes (H1, H2, H9 and human H1) to infect primary Siamese crocodile fibroblast cells. To our knowledge, this is the first report of serological evidences of avian and human IAV infection in Siamese crocodiles. Our findings highlighted the role of crocodile species in the ecology of IAV particularly the potential to serve as the reservoir or mixing vessel for the viruses that significantly threaten both human and animal health.

Evidence of an emerging triple-reassortant H3N3 avian influenza virus in China

The H3 subtype of avian influenza virus (AIV) stands out as one of the most prevalent subtypes, posing a significant threat to public health. In this study, a novel triple-reassortant H3N3 AIV designated A/chicken/China/16/2023 (H3N3), was isolated from a sick chicken in northern China. The complete genome of the isolate was determined using next-generation sequencing, and the AIV-like particles were confirmed via transmission electron microscopy. Subsequent phylogenetic analyses revealed that HA and NA genes of the H3N3 isolate clustered within the Eurasian lineage of AIVs, exhibiting the closest genetic relationship with other H3N3 AIVs identified in China during 2023. Interestingly, the HA and NA genes of the nove H3N3 isolate were originated from H3N8 and H10N3 AIVs, respectively, and the six internal genes originated from prevalent H9N2 AIVs. These findings indicated the novel H3N3 isolate possesses a complex genetic constellation, likely arising from multiple reassortment events involving H3N8, H9N2, and H10N3 subtype influenza viruses. Additionally, the presence of Q226 and T228 in the HA protein suggests the H3N3 virus preferentially binds to α-2,3-linked sialic acid receptors. The HA cleavage site motif (PEKQTR/GIF) and the absence of E627K and D701N mutations in PB2 protein classify the virus as a characteristic low pathogenicity AIV. However, several mutations in internal genes raise concerns about potential increases in viral resistance, virulence, and transmission in mammalian hosts. Overall, this study provides valuable insights into the molecular and genetic characterization of the emerging triple-reassortant H3N3 AIVs, and continued surveillance of domestic poultry is essential for monitoring the H3N3 subtype evolution and potential spread.

Combination Adjuvants Enhance Recombinant H5 Hemagglutinin Vaccine Protection Against High-Dose Viral Challenge in Chickens.

Recombinant avian influenza subunit vaccines often require adjuvants to enhance immune responses. This study aims to evaluate the immune-enhancing potential of seven combination adjuvants in specific pathogen-free (SPF) chickens. SPF chickens were vaccinated with combinations of ISA78VG and adjuvants, including Quil-A, CpG, and monophosphoryl lipid A (MPLA). Their immune responses were assessed using a vaccination and viral challenge protection model. The combinations of ISA78VG with Quil-A, CpG&MPLA or CpG&Quil-A significantly enhanced antibody responses and provided cross-protection against the H5N8-20135 strain. The ISA78VG&MPLA and ISA78VG&CpG&MPLA combinations induced the stronger IFN-γ production, with CpG further amplifying the immune response. The ISA78VG&Quil-A formulation, in particular, stimulated rapid antibody responses, achieving a 100% seroconversion by day 14 and high titers of hemagglutination inhibition (HI) antibodies against both the recombinant HA antigen and the H5N6-20053 virus. The ISA78VG&Quil-A combination is an ideal adjuvant for enhancing the immunogenicity of avian influenza rHA subunit vaccines, offering a promising strategy for H5 subtype vaccine development.

Surveillance of Highly Pathogenic Avian Influenza Virus in Wild Canids from Pennsylvania, USA.

The avian influenza virus is a global pathogen with significant health and economic implications. While primarily a pathogen of wild and domestic birds, recent outbreaks of the H5N1 highly pathogenic avian influenza virus (HPAIV) clade 2.3.4.4b have caused mortality in a wide variety of mammals, including members of the Canidae family, on multiple continents. Despite sporadic mortality events globally, the epidemiology and pathobiology of H5N1 HPAIV in wild canids remains poorly defined. During 2022-2024, 41 wild canid carcasses (diagnostic cases), including 23 red foxes and 18 gray foxes, were tested for the influenza A virus (IAV) via PCR, with five red fox kits testing positive (12%). Infected animals had variably severe encephalitis, pneumonia, and occasionally myocarditis associated with strong immunolabeling for IAV. Serum from 269 wild canids in Pennsylvania was tested for antibodies to IAV, including 133 samples collected prior to 2021 (pre-H5N1 HPAIV 2.3.4.4b introduction) and 136 collected after 2022 (post-H5N1 HPAIV 2.3.4.4b introduction). All samples collected prior to 2021 were seronegative for IAV. Two coyotes from 2024 were seropositive for IAV but were negative for antibodies to the H5 and N1 subtypes. Collectively, these data suggest that while sporadic H5N1 HPAIV infection and mortality can occur in wild canids, particularly juvenile red foxes, infection was limited in these outwardly healthy and opportunistically sampled animals. Future studies should utilize a risk-based approach to target sampling of wild canids at increased risk for H5N1 HPAIV infection, such as those around waterfowl habitats or spatially around wild bird or domestic animal outbreaks.

The novel H10N3 avian influenza virus acquired airborne transmission among chickens: an increasing threat to public health.

Following two human infections with the H10N3 avian influenza virus (AIV) in 2021 and 2022, a third case was discovered in Yunnan, China, in 2024, raising concerns about the potential for future pandemics. Recent studies have indicated that novel H10N3 viruses are highly pathogenic in mice and can be transmitted between guinea pigs via respiratory droplets without prior adaptation. However, the biological characteristics of novel H10N3 in poultry have not been fully elucidated. Our findings revealed that H10 subtype AIVs are predominantly prevalent in waterfowl. Notably, H10N8 and H10N3 viruses that have infected humans were primarily isolated from chickens. For the first time, double basic hemagglutinin cleavage sites (motif PEIKQGR↓GL) were identified in novel H10N3 AIVs, which exhibit enhanced replication in chickens, and can be transmitted between chickens through direct contact and respiratory droplets. Animal experimental studies demonstrated that ducks are also susceptible to H10N3 viruses and that the virus is transmissible through direct contact, suggesting a greater risk of transmission and recombination. Serological studies conducted among poultry workers suggest that while the human population was largely naïve to H10N3 infection, sporadic and undetected human infections did occur, indicating a potential increasing trend. These data further emphasize the growing threat to public health posed by zoonotic H10N3 subtype AIVs.IMPORTANCEExposure to poultry in live poultry markets (LPMs) is strongly associated with human infection with avian influenza viruses (AIVs), with chickens being the most common species found in these markets in China. The prevalence of AIVs in chickens, therefore, increases the risk of human infection. Notably, the main host of the novel H10N3 virus has shifted from waterfowl to chickens, and the virus can be transmitted between chickens via respiratory droplets, posing a potential risk of a pandemic within poultry populations. The novel H10N3 virus also remains sensitive to ducks and can be transmitted through direct contact, which means a greater risk of transmission and recombination. Significantly, the human population remains largely naïve to H10N3 infection, but sporadic seropositivity among poultry workers indicates previous exposure to H10 subtype AIVs. Therefore, a comprehensive surveillance of the novel H10N3 viruses in poultry is imperative. Effective control of the virus within poultry populations could significantly reduce the risk of emerging human infections.

Detection of antibodies against H5 subtype highly pathogenic avian influenza viruses in multiple raccoons in Tokachi District, Hokkaido, Japan, from 2022 to 2023.

In recent years, infection cases of H5 subtype highly pathogenic avian influenza viruses (HPAIVs) in wild mammals have increased globally. To obtain recent epidemiological information regarding influenza A virus (IAV) infection in raccoons (Procyon lotor), the prevalence of anti-IAV antibodies in sera was analyzed among raccoons captured in Tokachi District, Hokkaido, Japan, from 2019 to 2023. Screening of serum samples using enzyme-linked immunosorbent assay and agar gel precipitation test detected anti-IAV antibodies in 5 of 114 (4.4%) raccoons. All positive sera were from raccoons captured from 2022 to 2023. The hemagglutination inhibition test revealed that all five serum samples contained anti-H5 subtype HPAIV antibodies, and one also contained anti-H1 subtype antibodies. The neuraminidase inhibition test revealed that all five sera contained anti-N1 subtype antibodies, and one also contained anti-N8 subtype antibodies. In the virus neutralization test, these five sera showed stronger neutralization activity against the H5 subtype clade 2.3.4.4b HPAIV strain recently circulating worldwide compared to the old H5 HPAIV strain isolated in Japan in 2007. Findings suggested that raccoons could be involved in the circulation of H5 HPAIVs in nature.

H5 subtype avian influenza virus induces Golgi apparatus stress response via TFE3 pathway to promote virus replication.

During infection, avian influenza virus (AIV) triggers endoplasmic reticulum (ER) stress, a well-established phenomenon in previous research. The Golgi apparatus, situated downstream of the ER and crucial for protein trafficking, may be impacted by AIV infection. However, it remains unclear whether this induces Golgi apparatus stress (GAS) and its implications for AIV replication. We investigated the morphological changes in the Golgi apparatus and identified GAS response pathways following infection with the H5 subtype AIV strain A/Mallard/Huadong/S/2005. The results showed that AIV infection induced significant swelling and fragmentation of the Golgi apparatus in A549 cells, indicating the presence of GAS. Among the analyzed GAS response pathways, TFE3 was significantly activated during AIV infection, while HSP47 was activated early in the infection process, and CREB3-ARF4 remained inactive. The blockade of the TFE3 pathway effectively inhibited AIV replication in A549 cells and attenuated AIV virulence in mice. Additionally, activation of the TFE3 pathway promoted endosome acidification and upregulated transcription levels of glycosylation enzymes, facilitating AIV replication. These findings highlight the crucial role of the TFE3 pathway in mediating GAS response during AIV infection, shedding light on its significance in viral replication.

Development and evaluation of three multienzyme isothermal rapid amplification assays for fowl adenovirus serotype 4

Fowl adenovirus serotype 4 (FAdV-4) is the main causative agent of hydropericardium hepatitis syndrome (HHS), which has resulted in huge economic losses to the poultry industry in recent years. Hence, a rapid and simple visual detection method is needed for identification of FAdV-4. In this study, three multienzyme isothermal rapid amplification (MIRA) assays, basic MIRA, MIRA-qPCR and MIRA-LFD were developed for detection of FAdV-4. The amplification primers and reaction conditions were optimized, and the specificity and sensitivity of the assays were evaluated. The MIRA assays were specific for FAdV-4 with no cross-reaction with novel goose astrovirus, H9 subtype avian influenza virus, duck enteritis virus, Muscovy duck reovirus, or duck circovirus. The basic MIRA assay required only one primer pair and the reaction can be completed within 30 min at 36 °C. The MIRA-qPCR and MIRA-LFD assays were completed in 20 min with a minimum detection limit of 1 × 101 copies/μL and 1 × 102 copies/μL, respectively. The results of the MIRA-LFD assay can be observed directly with the naked eye, omitting the need for specialized instruments. The positive rate of three proposed MIRA assays were consistent with that of the conventional PCR assay. The MIRA assays are simple, rapid, and effective diagnostic tools for field detection of FAdV-4.

Neuraminidase Antibody Response to Homologous and Drifted Influenza A Viruses After Immunization with Seasonal Influenza Vaccines.

Humoral immunity directed against neuraminidase (NA) of the influenza virus may soften the severity of infection caused by new antigenic variants of the influenza viruses. Evaluation of NA-inhibiting (NI) antibodies in combination with antibodies to hemagglutinin (HA) may enhance research on the antibody response to influenza vaccines. The study examined 64 pairs of serum samples from patients vaccinated with seasonal inactivated trivalent influenza vaccines (IIVs) in 2018 according to the formula recommended by the World Health Organization (WHO) for the 2018-2019 flu season. Antibodies against drift influenza viruses A/Guangdong-Maonan/SWL1536/2019(H1N1)pdm09 and A/Brisbane/34/2018(H3N2) were studied before vaccination and 21 days after vaccination. To assess NI antibodies, we used an enzyme-linked lectin assay (ELLA) with pairs of reassortant viruses A/H6N1 and A/H6N2. Anti-HA antibodies were detected using a hemagglutination inhibition (HI) test. The microneutralization (MN) test was performed in the MDCK cell line using viruses A/H6N1 and A/H6N2. Seasonal IIVs induce a significant immune response of NI antibodies against influenza A/H1N1pdm09 and A/H3N2 viruses. A significantly reduced 'herd' immunity to drift influenza A/H1N1pdm09 and A/H3N2 viruses was shown, compared with previously circulating strains. This reduction was most pronounced in strains possessing neuraminidase N2. Seasonal IIVs caused an increase in antibodies against homologous and drifted viruses; however, an increase in antibodies to drifting viruses was observed more often among older patients. The level of NI antibodies for later A/H1N1pdm09 virus in response to IIVs was statistically significantly lower among younger people. After IIV vaccination, the percentage of individuals with HI antibody levels ≥ 1:40 and NI antibody levels ≥ 1:20 was 32.8% for drift A/H1N1pdm09 virus and 17.2% for drift A/H3N2 virus. Antisera containing HI and NI antibodies exhibited neutralizing properties in vitro against viruses with unrelated HA of the H6 subtype. Drift A/H1N1pdm09 and A/H3N2 viruses demonstrated significantly lower reactivity to HI and NI antibodies against early influenza viruses. In response to seasonal IIVs, the level of seroprotection has increased, including against drift influenza A viruses, but protective antibody levels against A/H1N1pdm09 have risen to a greater extent. A reduced immune response to the N1 protein of the A/H1N1pdm09 drift virus was obtained in individuals under 60 years of age. Based on our findings, it is hypothesized that in the cases of a HA mismatch, vaccination against N1-containing influenza viruses may be necessary for individuals under 60, while broader population-level vaccination against N2-containing viruses may be required.

Highly Pathogenic H5N1 Influenza A Virus (IAV) in Blue-Winged Teal in the Mississippi Flyway Is Following the Historic Seasonal Pattern of Low-Pathogenicity IAV in Ducks.

Highly pathogenic H5N1 (HP H5N1) influenza A virus (IAV) has been detected annually in North American ducks since its introduction during 2021, but it is unknown if this virus will follow the same seasonal and geographic patterns that have been observed with low-pathogenicity (LP) IAV in this reservoir. We monitored blue-winged teal in the Mississippi flyway prior to the detection of HP H5N1 and during two post-introduction migration cycles from spring 2022 to spring 2024, testing birds for infection and antibodies to IAV nucleoprotein (NP), hemagglutinin subtype H5, and neuraminidase subtype N1. Antigens representing clade 2.3.4.4b HP H5 and LP North American H5 were used for hemagglutination inhibition (HI) and virus neutralization (VN) tests for H5 antibodies. Virologic results were consistent with historic seasonal and geographic patterns reported for LP IAV with peak infections occurring in pre-migration staging areas in Minnesota during fall 2022. However, the high prevalence of the H5 subtype was exceptional compared to historic prevalence estimates at this same site and for the Mississippi flyway. HP H5N1 was detected on wintering areas in Louisiana and Texas during the fall of that same year and this was followed by an increase in estimated antibody prevalence to NP, H5, and N1 with no HP H5N1 detections during the wintering or spring migration periods of 2022/2023. HP H5N1 was not detected in Minnesota during fall 2023 but was detected from a single bird in Louisiana. However, a similar increase in antibody prevalence was observed during the winter and spring period of 2023 and 2024. Over the two migration cycles, there was a temporal shift in observed prevalence and relative titers against the H5 antigens with a higher proportion of ducks testing positive to the 2.3.4.4b H5 antigen and higher relative titer to that antigen compared to the representative LP North American H5 antigen. The seasonal and geographic patterns observed appear to be driven by population immunity during the migration cycle. Results support an initial high infection rate of HP H5N1 in blue-winged teal in the Mississippi flyway followed by a high prevalence of antibodies to NP, H5, and N1. Although prevalence was much reduced in the second migration cycle following introduction, it is not known if this pattern will persist in the longer term or affect historic patterns of subtype diversity in this reservoir.

Research note: Simultaneous detection of GPV, H5 AIV, and GoAstV via TaqMan probe-based multiplex qPCR

The endemic status of goose parvovirus (GPV), H5 subtype avian influenza virus (AIV), and goose astrovirus (GoAstV) infections continues to devastate the poultry industry in China. Despite this, there exists a notable gap in the application of molecular diagnostic techniques. This investigation described the development of a multiplex qualitative polymerase chain reaction (qPCR) assay capable of concurrently detecting GPV, H5 AIV, and GoAstV, with no cross-reactivity observed with other avian viral pathogens. The assay exhibited a detection threshold of 10 copies/μL for both GPV and GoAstV, and 1 copy/μL for H5 AIV. The intra- and inter-assay coefficients of variation were < 3.0%, signifying high repeatability within and across assay batches. Utilizing this multiplex qPCR assay, a batch of 60 clinical samples was analyzed to assess its practical utility. The detected prevalence rates for GoAstV, GPV, and H5 AIV were 35.0% (21/60), 21.7% (13/60), and 15.0% (9/60), respectively. Concurrent infections were also identified, with rates for GPV + GoAstV, GPV + H5 AIV, GoAstV + H5 AIV, and GPV + GoAstV + H5 AIV being 6.7% (4/60), 3.3% (2/60), 3.3% (2/60), and 3.3% (2/60), respectively. The developed multiplex qPCR assay exhibited a diagnostic concordance rate equivalent to that of traditional PCR techniques. This novel assay serves as a rapid, efficient, specific, and sensitive tool for the detection of prevalent goose viruses, thereby enhancing disease management strategies and epidemiological monitoring efforts.

Active surveillance of avian influenza in the southwestern Poyang Lake area, China: Analyzing changes in wholesale and frozen fresh retail markets post-policy implementation

This study aims to conduct active surveillance of avian influenza in the southwestern Poyang Lake area of China and to analyze the changes in avian influenza prevalence in wholesale poultry markets and frozen fresh retail markets following the implementation of policies regulating frozen fresh poultry products. The type A avian influenza virus nucleic acids were detected using real-time reverse transcription polymerase chain reaction (real-time RT-PCR), and a triplex real-time RT-PCR assay kit specific for H5/H7/H9 RNA was utilized on the influenza A-positive samples to differentiate among the avian influenza virus subtypes. From October 2020 to June 2024, the positivity rate of the live poultry wholesale market was 59.2 %. The positivity rate of frozen fresh retail markets was 45.4 %. In August 2023, the H9 subtype had the highest positivity rate in both markets. However, after that, the rate of untyped positives began to rise, particularly in the live poultry wholesale market where the positivity rate of the H5 subtype also showed an increasing trend. Implementing a frozen fresh poultry products policy has effectively reduced the avian influenza positivity rate in frozen fresh retail markets over the first two years. However, the positivity rate showed a rebound trend in the last two years. The live poultry wholesale market may be the source of the spread of avian influenza in frozen fresh retail markets, so managing the live poultry wholesale market and surveillance avian influenza should be strengthened. Recent surveillance indicates a significant uptick in the positivity rates of the H5 subtype and untyped strains of avian influenza, underscoring the importance of continued vigilance and strengthened prevention and control measures.

Antimicrobial and antibiofilm activity of human recombinant H1 histones against bacterial infections.

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombinant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.IMPORTANCEThe constant increase of multidrug-resistant bacteria is a critical global concern. The inefficacy of current therapies to treat bacterial infections is attributed to multiple mechanisms of resistance, including the capacity to form biofilms. Therefore, the identification of novel and safe therapeutic strategies is imperative. This study confirms the antimicrobial potential of three histone H1 subtypes against both Gram-negative and Gram-positive bacteria. Furthermore, histones H1.0 and H1.4 demonstrated in vivo efficacy without associated toxicity in an acute infection model of Pseudomonas aeruginosa PAO1 in Galleria mellonella larvae. The bactericidal effect of these proteins also resulted in biomass reduction of P. aeruginosa PAO1 biofilms. Given the clinical significance of this opportunistic pathogen, our research provides a comprehensive initial evaluation of the efficacy, toxicity, and mechanism of action of a potential new therapeutic approach against acute and chronic bacterial infections.

Transmission of a human isolate of clade 2.3.4.4b A(H5N1) virus in ferrets.

Since 2020, there has been unprecedented global spread of highly pathogenic avian influenza A(H5N1) in wild bird populations with spillover into a variety of mammalian species and sporadically humans1. In March 2024, clade 2.3.4.4b A(H5N1) virus was first detected in dairy cattle in the USA, with subsequent detection in numerous states2, leading to more than a dozen confirmed human cases3,4. In this study, we used the ferret, a well-characterized animal model that permits concurrent investigation of viral pathogenicity and transmissibility5, in the evaluation of A/Texas/37/2024 (TX/37) A(H5N1) virus isolated from a dairy farm worker in Texas6. Here we show that the virus has a remarkable ability for robust systemic infection in ferrets, leading to high levels of virus shedding and spread to naive contacts. Ferrets inoculated with TX/37 rapidly exhibited a severe and fatal infection, characterized by viraemia and extrapulmonary spread. The virus efficiently transmitted in a direct contact setting and was capable of indirect transmission through fomites. Airborne transmission was corroborated by the detection of infectious virus shed into the air by infected animals, albeit at lower levels compared to those of the highly transmissible human seasonal and swine-origin H1 subtype strains. Our results show that despite maintaining anavian-like receptor-binding specificity, TX/37 exhibits heightened virulence, transmissibility and airborne shedding relative to other clade 2.3.4.4b virus isolated before the 2024 cattle outbreaks7, underscoring the need for continued public health vigilance.

Epidemiology and biological characteristics of influenza A (H4N6) viruses from wild birds

ABSTRACT During the active surveillance, we isolated nine H4N6 subtype influenza A viruses from wild birds in China. To reveal the epidemiology and biology characteristics of H4 subtype influenza A virus from wild birds, we investigated H4 subtype viruses available in the public source, and found that the H4 viruses have been detected in at least 37 countries to date, and more than 73.6% of the viruses were from wild Anseriformes. Bayesian phylogeographic analysis showed that Mongolia worked as the important transmission centre for Eurasian lineage H4 viruses spreading. Phylogenetic analysis of HA genes indicated that global H4 influenza A viruses were divided into Eurasian and North American lineage, our nine H4N6 isolates fell into the Eurasian lineage. Recombination analysis suggested that nine H4N6 isolates underwent complex gene recombination with various subtypes of influenza A viruses and formed two genotypes. Notably, nine H4N6 isolates acquired mammalian virulence-increasing residues. Two representative H4N6 viruses possessed dual receptor binding specificity, they could efficiently replicate in MDCK and 293 T cells in vitro infection, also could cross the species barrier to infect mice directly without prior adaption in vivo experiments. These findings emphasize the public health issues represented by H4 viruses, and highlight the need to strengthen the active surveillance of H4 viruses from wild birds.

Application of lentinan in suppression of Marek's disease virus infection

Marek's disease virus (MDV) is an extremely widespread avian immunosuppressive virus. In recent years, many reports have shown that there are still cases of MDV infection and immunosuppression after immunization with the vaccine. Consequently, there is a need to develop alternative complementary approaches for strengthening the efficacy of MDV prevention and control measures. Lentinan (LNT) is a macromolecular compound with immune-enhancing activity extracted from shiitake mushrooms. To explore the value and effectiveness of administering LNT through drinking water in the prevention and control of MDV, this study first observed the effects of high and low doses of LNT on weight gain, organ development, viral replication, and antibody titer of an avian influenza virus subtype H9 (AIV-H9) inactivated vaccine in specific pathogen-free (SPF) chicks infected with the wild strain of MDV. The results showed that both high and low doses of LNT significantly alleviated the weight gain retardation and liver and spleen enlargement caused by MDV infection, and significantly inhibited the replication of MDV in SPF chicks (P < 0.05), compared with the MDV-positive control group, both high and low doses of LNT significantly increased the antibody titer of AIV-H9 after immunization with inactivated AIV-H9 vaccine (P < 0.0001). On this basis, we also observed the effects of a chicken Marek's disease meq gene deletion live vaccine (SC9-1 strain), administered alone or in combination with LNT, on MDV infections of varying virulence in Hy-Line Brown chicks. The results showed that combined administration of LNT and the SC9-1 vaccine resulted in a significant alleviation of weight gain retardation and liver and spleen enlargement due to MDV infection (P < 0.05), as well as a significant inhibition of MDV replication and release in Hy-Line Brown chicks compared to the vaccine alone (P < 0.0001). These findings suggest that LNT not only alleviates the adverse effects of MDV infection in chickens but also enhances the efficacy of MDV vaccination, offering a potential auxiliary measure for controlling MDV infection.

Molecular Characterization of a Clade 2.3.4.4b H5N1 High Pathogenicity Avian Influenza Virus from a 2022 Outbreak in Layer Chickens in the Philippines.

H5 subtype high-pathogenicity avian influenza (HPAI) viruses continue to devastate the poultry industry and threaten food security and public health. The first outbreak of H5 HPAI in the Philippines was reported in 2017. Since then, H5 HPAI outbreaks have been reported in 2020, 2022, and 2023. Here, we report the first publicly available complete whole-genome sequence of an H5N1 high-pathogenicity avian influenza virus from a case in Central Luzon. Samples were collected from a flock of layer chickens exhibiting signs of lethargy, droopy wings, and ecchymotic hemorrhages in trachea with excessive mucus exudates. A high mortality rate of 96-100% was observed within the week. Days prior to the high mortality event, migratory birds were observed around the chicken farm. Lungs, spleen, cloacal swabs, and oropharyngeal-tracheal swabs were taken from two chickens from this flock. These samples were positive in quantitative RT-PCR assays for influenza matrix and H5 hemagglutinin (HA) genes. To further characterize the virus, the same samples were subjected to whole-virus-genome amplification and sequencing using the Oxford Nanopore method with mean coverages of 19,190 and 2984, respectively. A phylogenetic analysis of the HA genes revealed that the H5N1 HPAI virus from Central Luzon belongs to the Goose/Guangdong lineage clade 2.3.4.4b viruses. Other segments also have high sequence identity and the same genetic lineages as other clade 2.3.4.4b viruses from Asia. Collectively, these data indicate that wild migratory birds are the likely source of H5N1 viruses from the 2022 outbreaks in the Philippines. Thus, biosecurity practices and surveillance for HPAI viruses in both domestic and wild birds should be increased to prevent and mitigate HPAI outbreaks.

Quantification of Histone H1 Subtypes Using Targeted Proteomics.

Histone H1 is involved in the regulation of chromatin structure. Human somatic cells express up to seven subtypes. The variability in the proportions of somatic H1s (H1 complement) is one piece of evidence supporting their functional specificity. Alterations in the protein levels of different H1 subtypes have been observed in cancer, suggesting their potential as biomarkers and that they might play a role in disease development. We have developed a mass spectrometry-based (MS) parallel reaction monitoring (PRM) assay suitable for the quantification of H1 subtypes. Our PRM method is based on the quantification of unique peptides for each subtype, providing high specificity. Evaluation of the PRM performance on three human cell lines, HeLa, K562, and T47D, showed high reproducibility and sensitivity. Quantification values agreed with the electrophoretic and Western blot data, indicating the accuracy of the method. We used PRM to quantify the H1 complement in peripheral blood samples of healthy individuals and chronic myeloid leukemia (CML) patients. In CML, the first line of therapy is a tyrosine kinase inhibitor, imatinib. Our preliminary data revealed differences in the H1 complement in CML patients between imatinib responders and non-responders. These results support further research to determine if the H1 content or subtype composition could help predict imatinib response.

Immunity and Protective Efficacy of a Plant-Based Tobacco Mosaic Virus-like Nanoparticle Vaccine against Influenza a Virus in Mice.

The rapid production of influenza vaccines is crucial to meet increasing pandemic response demands. Here, we developed plant-made vaccines comprising centralized consensus influenza hemagglutinin (HA-con) proteins (H1 and H3 subtypes) conjugated to a modified plant virus, tobacco mosaic virus (TMV) nanoparticle (TMV-HA-con). We compared immune responses and protective efficacy against historical H1 or H3 influenza A virus infections among TMV-HA-con, HA-con protein combined with AddaVax™ adjuvant, and whole-inactivated virus vaccine (Fluzone®). Immunogenicity studies demonstrated robust IgG, IgM, and IgA responses in the TMV-HA-con and HA-con protein vaccinated groups, with relatively low induction of interferon (IFN)-γ+ T-cell responses across all vaccinated groups. The TMV-HA-con and HA-con protein groups displayed partial protection (100% and 80% survival) with minimal weight loss following challenge with two H1N1 strains. The HA-con protein group exhibited 80% and 100% survival against two H3 strains, whereas the TMV-HA-con groups showed reduced protection (20% survival). The Fluzone® group conferred 20-100% survival against two H1N1 strains and one H3N1 strain, but did not protect against H3N2 infection. Our findings indicate that TMV-HA and HA-con protein vaccines with adjuvant induce protective immune responses against influenza A virus infections. Furthermore, our results underscore the potential of plant-based production using TMV-like nanoparticles for developing influenza A virus candidate vaccines.