Influenza viruses rapidly evolve to evade previously acquired human immunity. Maintaining vaccine efficacy necessitates continuous monitoring of antigenic differences among strains. Traditional serological methods for assessing these differences are labor-intensive and time-consuming, highlighting the need for efficient computational approaches. This paper proposes MetaFluAD, a meta-learning-based method designed to predict quantitative antigenic distances among strains. This method models antigenic relationships between strains, represented by their hemagglutinin (HA) sequences, as a weighted attributed network. Employing a graph neural network (GNN)-based encoder combined with a robust meta-learning framework, MetaFluAD learns comprehensive strain representations within a unified space encompassing both antigenic and genetic features. Furthermore, the meta-learning framework enables knowledge transfer across different influenza subtypes, allowing MetaFluAD to achieve remarkable performance with limited data. MetaFluAD demonstrates excellent performance and overall robustness across various influenza subtypes, including A/H3N2, A/H1N1, A/H5N1, B/Victoria, and B/Yamagata. MetaFluAD synthesizes the strengths of GNN-based encoding and meta-learning to offer a promising approach for accurate antigenic distance prediction. Additionally, MetaFluAD can effectively identify dominant antigenic clusters within seasonal influenza viruses, aiding in the development of effective vaccines and efficient monitoring of viral evolution.

Influenza virus infection poses a continual menace to public health. Here, we developed soluble trimeric HA ectodomain vaccines by establishing interprotomer disulfide bonds in the stem region, which effectively preserve the native antigenicity of stem epitopes. The stable trimeric H1 ectodomain proteins exhibited higher thermal stabilities in comparison with unmodified HAs and showed strong binding activities towards a panel of anti-stem cross-reactive antibodies that recognize either interprotomer or intraprotomer epitopes. Negative stain transmission electron microscopy (TEM) analysis revealed the stable trimer architecture of the interprotomer disulfide-stapled WA11#5, NC99#2, and FLD#1 proteins as well as the irregular aggregation of unmodified HA molecules. Immunizations of mice with those trimeric HA ectodomain vaccines formulated with incomplete Freund\'s adjuvant elicited significantly more potent cross-neutralizing antibody responses and offered broader immuno-protection against lethal infections with heterologous influenza strains compared to unmodified HA proteins. Additionally, the findings of our study indicate that elevated levels of HA stem-specific antibody responses correlate with strengthened cross-protections. Our design strategy has proven effective in trimerizing HA ectodomains derived from both influenza A and B viruses, thereby providing a valuable reference for designing future influenza HA immunogens.

BACKGROUND: Non-pharmaceutical measures and travel restrictions have halted the spread of coronavirus disease 2019 (COVID-19) and influenza. Nonetheless, with COVID-19 restrictions lifted, an unanticipated outbreak of the influenza B/Victoria virus in late 2021 and another influenza H3N2 outbreak in mid-2022 occurred in Guangdong, southern China. The mechanism underlying this phenomenon remains unknown. To better prepare for potential influenza outbreaks during COVID-19 pandemic, we studied the molecular epidemiology and phylogenetics of influenza A(H3N2) and B/Victoria that circulated during the COVID-19 pandemic in this region.
METHODS: From January 1, 2018 to December 31, 2022, we collected throat swabs from 173,401 patients in Guangdong who had acute respiratory tract infections. Influenza viruses in the samples were tested using reverse transcription-polymerase chain reaction, followed by subtype identification and sequencing of hemagglutinin (HA) and neuraminidase (NA) genes. Phylogenetic and genetic diversity analyses were performed on both genes from 403 samples. A rigorous molecular clock was aligned with the phylogenetic tree to measure the rate of viral evolution and the root-to-tip distance within strains in different years was assessed using regression curve models to determine the correlation.
RESULTS: During the early period of COVID-19 control, various influenza viruses were nearly undetectable in respiratory specimens. When control measures were relaxed in January 2020, the influenza infection rate peaked at 4.94% (39/789) in December 2021, with the influenza B/Victoria accounting for 87.18% (34/39) of the total influenza cases. Six months later, the influenza infection rate again increased and peaked at 11.34% (255/2248) in June 2022; influenza A/H3N2 accounted for 94.51% (241/255) of the total influenza cases in autumn 2022. The diverse geographic distribution of HA genes of B/Victoria and A/H3N2 had drastically reduced, and most strains originated from China. The rate of B/Victoria HA evolution (3.11 × 10-3, P < 0.05) was 1.7 times faster than before the COVID-19 outbreak (1.80 × 10-3, P < 0.05). Likewise, the H3N2 HA gene\'s evolution rate was 7.96 × 10-3 (P < 0.05), which is 2.1 times faster than the strains\' pre-COVID-19 evolution rate (3.81 × 10-3, P < 0.05).
CONCLUSIONS: Despite the extraordinarily low detection rate of influenza infection, concealed influenza transmission may occur between individuals during strict COVID-19 control. This ultimately leads to the accumulation of viral mutations and accelerated evolution of H3N2 and B/Victoria viruses. Monitoring the evolution of influenza may provide insights and alerts regarding potential epidemics in the future.

H9N2 avian influenza virus (AIV), one of the predominant subtypes circulating in the poultry industry, inflicts substantial economic damage. Mutations in the hemagglutinin (HA) and neuraminidase (NA) proteins of H9N2 frequently alter viral antigenicity and replication. In this paper, we analyzed the HA genetic sequences and antigenic properties of 26 H9N2 isolates obtained from chickens in China between 2012 and 2019. The results showed that these H9N2 viruses all belonged to h9.4.2.5, and were divided into two clades. We assessed the impact of amino acid substitutions at HA sites 145, 149, 153, 164, 167, 168, and 200 on antigenicity, and found that a mutation at site 164 significantly modified antigenic characteristics. Amino acid variations at sites 145, 153, 164 and 200 affected virus\'s hemagglutination and the growth kinetics in mammalian cells. These results underscore the critical need for ongoing surveillance of the H9N2 virus and provide valuable insights for vaccine development.

As a highly pathogenic avian virus, H5 influenza poses a serious threat to livestock, the poultry industry, and public health security. Hemagglutinin (HA) is both the dominant epitope and the main target of influenza-neutralizing antibodies. Here, we designed a nanoparticle hemagglutinin influenza vaccine to improve the immunogenicity of the influenza vaccine. In this study, HA5 subtype influenza virus was used as the candidate antigen and was combined with the artificially designed double-branch scaffold protein I53_dn5 A and B. A structurally correct and bioactive trimer HA5-I53_dn5B/Y98F was obtained through secretion and purification using an insect baculovirus expression system; I53_dn5A was obtained by purification using a prokaryotic expression system. HA5-I53_dn5B/Y98F and I53_dn5A self-assembled into spherical nanoparticles (HA5-I53_dn5) in vitro with a diameter of about 45 nm. Immunization and serum test results showed that both HA5-I53_dn5B/Y98F and HA5-I53_dn5 could induce HA5-specific antibodies; however, the immunogenicity of HA5-I53_dn5 was better than that of HA5-I53_dn5B/Y98F. Groups treated with HA5-I53_dn5B and HA5-I53_dn5 nanoparticles produced IgG antibody titers that were not statistically different from those of the nanoparticle-containing adjuvant group. This production of trimerized HA5-I53_dn5B and HA5-I53_dn5 nanoparticles using baculovirus expression provides a reference for the development of novel, safe, and efficient influenza vaccines.

In recent years, the avian influenza virus has emerged as a significant threat to both human and public health. This study focuses on a patient infected with the H10N3 subtype of avian influenza virus, admitted to the Third People\'s Hospital of Kunming City on March 6, 2024. Metagenomic RNA sequencing and polymerase chain reaction (PCR) analysis were conducted on the patient\'s sputum, confirming the H10N3 infection. The patient presented severe pneumonia symptoms such as fever, expectoration, chest tightness, shortness of breath, and cough. Phylogenetic analysis of the Haemagglutinin (HA) and neuraminidase (NA) genes of the virus showed that the virus was most closely related to a case of human infection with the H10N3 subtype of avian influenza virus found in Zhejiang Province, China. Analysis of amino acid mutation sites identified four mutations potentially hazardous to human health. Consequently, this underscores the importance of continuous and vigilant monitoring of the dynamics surrounding the H10N3 subtype of avian influenza virus, utilizing advanced genomic surveillance techniques.

The H5N1 avian influenza virus seriously affects the health of poultry and humans. Once infected, the mortality rate is very high. Therefore, accurate and timely detection of the H5N1 avian influenza virus is beneficial for controlling its spread. This article establishes a dual gene detection method based on dual RPA for simultaneously detecting the HA and M2 genes of H5N1 avian influenza virus, for the detection of H5N1 avian influenza virus. Design specific primers for the conserved regions of the HA and M2 genes. The sensitivity of the dual RT-RPA detection method for HA and M2 genes is 1 × 10-7 ng/μL. The optimal primer ratio is 1:1, the optimal reaction temperature is 40 °C, and the optimal reaction time is 20 min. Dual RT-RPA was used to detect 72 samples, and compared with RT-qPCR detection, the Kappa value was 1 (p value < 0.05), and the clinical sample detection sensitivity and specificity were both 100%. The dual RT-RPA method is used for the first time to simultaneously detect two genes of the H5N1 avian influenza virus. As an accurate and convenient diagnostic tool, it can be used to diagnose the H5N1 avian influenza virus.

The proportion of human isolates with reduced neuraminidase inhibitors (NAIs) susceptibility in highly pathogenic avian influenza (HPAI) H7N9 virus was high. These drug-resistant strains showed good replication capacity without serious loss of fitness. In the presence of oseltamivir, R229I substitution were found in HA1 region of the HPAI H7N9 virus before NA R292K appeared. HPAI H7N9 or H7N9/PR8 recombinant viruses were developed to study whether HA R229I could increase the fitness of the H7N9 virus bearing NA 292K. Replication efficiency was assessed in MDCK or A549 cells. Neuraminidase enzyme activity and receptor-binding ability were analyzed. Pathogenicity in C57 mice was evaluated. Antigenicity analysis was conducted through a two-way HI test, in which the antiserum was obtained from immunized ferrets. Transcriptomic analysis of MDCK infected with HPAI H7N9 24hpi was done. It turned out that HA R229I substitution from oseltamivir induction in HA1 region increased (1) replication ability in MDCK(P < 0.05) and A549(P < 0.05), (2) neuraminidase enzyme activity, (3) binding ability to both α2,3 and α2,6 receptor, (4) pathogenicity to mice(more weight loss; shorter mean survival day; viral titer in respiratory tract, P < 0.05; Pathological changes in pneumonia), (5) transcriptome response of MDCK, of the H7N9 virus bearing NA 292K. Besides, HA R229I substitution changed the antigenicity of H7N9/PR8 virus (>4-fold difference of HI titre). It indicated that through the fine-tuning of HA-NA balance, R229I increased the fitness and changed the antigenicity of H7N9 virus bearing NA 292K. Public health attention to this mechanism needs to be drawn.

The receptor-binding site of influenza A virus hemagglutinin partially overlaps with major antigenic sites and constantly evolves. In this study, we observe that mutations G186D and D190N in the hemagglutinin receptor-binding site have coevolved in two recent human H3N2 clades. X-ray crystallography results show that these mutations coordinately drive the evolution of the hemagglutinin receptor binding mode. Epistasis between G186D and D190N is further demonstrated by glycan binding and thermostability analyses. Immunization and neutralization experiments using mouse and human samples indicate that the evolution of receptor binding mode is accompanied by a change in antigenicity. Besides, combinatorial mutagenesis reveals that G186D and D190N, along with other natural mutations in recent H3N2 strains, alter the compatibility with a common egg-adaptive mutation in seasonal influenza vaccines. Overall, our findings elucidate the role of epistasis in shaping the recent evolution of human H3N2 hemagglutinin and substantiate the high evolvability of its receptor-binding mode.

Influenza virus infection is initiated by the attachment of the viral haemagglutinin (HA) protein to sialic acid receptors on the host cell surface. Most virus particles enter cells through clathrin-mediated endocytosis (CME). However, it is unclear how viral binding signals are transmitted through the plasma membrane triggering CME. Here we found that metabotropic glutamate receptor subtype 2 (mGluR2) and potassium calcium-activated channel subfamily M alpha 1 (KCa1.1) are involved in the initiation and completion of CME of influenza virus using an siRNA screen approach. Influenza virus HA directly interacted with mGluR2 and used it as an endocytic receptor to initiate CME. mGluR2 interacted and activated KCa1.1, leading to polymerization of F-actin, maturation of clathrin-coated pits and completion of the CME of influenza virus. Importantly, mGluR2-knockout mice were significantly more resistant to different influenza subtypes than the wild type. Therefore, blocking HA and mGluR2 interaction could be a promising host-directed antiviral strategy.