Genomic diversity and evolutionary dynamics of Influenza A viruses in Colombian swine: implications for one health surveillance and control.

Influenza A viruses (IAV) impose significant respiratory disease burdens in both swine and humans worldwide, with frequent human-to-swine transmission driving viral evolution in pigs and highlighting the risk at the animal-human interface. Therefore, a comprehensive One Health approach (interconnection among human, animal, and environmental health) is needed for IAV prevention, control, and response. Animal influenza genomic surveillance remains limited in many Latin American countries, including Colombia. To address this gap, we genetically characterized 170 swine specimens from Colombia (2011-2017). Whole genome sequencing revealed a predominance of pandemic-like H1N1 lineage, with a minority belonging to H3N2 and H1N2 human seasonal-like lineage and H1N1 early classical swine lineages. Significantly, we have identified reassortant and recombinant viruses (H3N2, H1N1) not previously reported in Colombia. This suggests a broad genotypic viral diversity, likely resulting from reassortment between classical endemic viruses and new introductions established in Colombia's swine population (e.g. the 2009 H1N1 pandemic). Our study highlights the importance of a One Health approach in disease control, particularly in an ecosystem where humans are a main source of IAV to swine populations, and emphasizes the need for continued surveillance and enhanced biosecurity measures. The co-circulation of multiple subtypes in regions with high swine density facilitates viral exchange, underscoring the importance of monitoring viral evolution to inform vaccine selection and public health policies locally and globally.

Influenza A viral burst size from thousands of infected single cells using droplet quantitative PCR (dqPCR).

An important aspect of how viruses spread and infect is the viral burst size, or the number of new viruses produced by each infected cell. Surprisingly, this value remains poorly characterized for influenza A virus (IAV), commonly known as the flu. In this study, we screened tens of thousands of cells using a microfluidic method called droplet quantitative PCR (dqPCR). The high-throughput capability of dqPCR enabled the measurement of a large population of infected cells producing progeny virus. By measuring the fully assembled and successfully released viruses from these infected cells, we discover that the viral burst sizes for both the seasonal H3N2 and the 2009 pandemic H1N1 strains vary significantly, with H3N2 ranging from 101 to 104 viruses per cell, and H1N1 ranging from 101 to 103 viruses per cell. Some infected cells produce average numbers of new viruses, while others generate extensive number of viruses. In fact, we find that only 10% of the single-cell infections are responsible for creating a significant portion of all the viruses. This small fraction produced approximately 60% of new viruses for H3N2 and 40% for H1N1. On average, each infected cell of the H3N2 flu strain produced 709 new viruses, whereas for H1N1, each infected cell produced 358 viruses. This novel method reveals insights into the flu virus and can lead to improved strategies for managing and preventing the spread of viruses.

The biological characteristics and infection dynamics of a novel H3N2 canine influenza virus genotype in beagles.

BACKGROUND: The canine influenza virus (CIV) outbreak has garnered considerable attention as it poses a significant threat to dog health. During the H3N2 CIV evolution in beagles, the virus formed a new clade after 2019 and gradually became more adaptable to other mammals. Therefore, successfully elucidating the biological characteristics and constructing a canine influenza infection model is required for CIV characterization. METHODS: We performed genetic analyses to examine the biological characteristics and infection dynamics of CIV. RESULTS: The genotype of our H3N2 CIV strain (from 2019 in Shanghai) belonged to the 5.1 clade, which is now prevalent in China. Using MDCK cells, we investigated viral cytopathic effects. Virus size and morphology were observed using transmission electron microscopy. Beagles were also infected with 104, 105, and 106 50% egg-infectious doses (EID50). When compared with the other groups, the 106 EID50 group showed the most obvious clinical symptoms, the highest virus titers, and typical lung pathological changes. Our results suggested that the other two treatments caused mild clinical manifestations and pathological changes. Subsequently, CIV distribution in the 106 EID50 group was detected by hematoxylin and eosin (H&E) and immunofluorescence (IF) staining, which indicated that CIV primarily infected the lungs. CONCLUSIONS: The framework established in this study will guide further CIV prevention strategies.

Characteristics and evolution of hemagglutinin and neuraminidase genes of Influenza A(H3N2) viruses in Thailand during 2015 to 2018.

Background: Influenza A(H3N2) virus evolves continuously. Its hemagglutinin (HA) and neuraminidase (NA) genes have high genetic variation due to the antigenic drift. This study aimed to investigate the characteristics and evolution of HA and NA genes of the influenza A(H3N2) virus in Thailand. Methods: Influenza A positive respiratory samples from 2015 to 2018 were subtyped by multiplex real-time RT-PCR. Full-length HA and NA genes from the positive samples of influenza A(H3N2) were amplified and sequenced. Phylogenetic analysis with the maximum likelihood method was used to investigate the evolution of the virus compared with the WHO-recommended influenza vaccine strain. Homology modeling and N-glycosylation site prediction were also performed. Results: Out of 443 samples, 147 (33.18%) were A(H1N1)pdm09 and 296 (66.82%) were A(H3N2). The A(H3N2) viruses circulating in 2015 were clade 3C.2a whereas sub-clade 3C.2a1 and 3C.2a2 dominated in 2016-2017 and 2018, respectively. Amino acid substitutions were found in all antigenic sites A, B, C, D, and E of HA but the majority of the substitutions were located at antigenic sites A and B. The S245N and N329S substitutions in the NA gene affect the N-glycosylation. None of the mutations associated with resistance to NA inhibitors were observed. Mean evolutionary rates of the HA and NA genes were 3.47 × 10 -3 and 2.98 × 10-3 substitutions per site per year. Conclusion: The influenza A(H3N2) virus is very genetically diverse and is always evolving to evade host defenses. The HA and NA gene features including the evolutionary rate of the influenza A(H3N2) viruses that were circulating in Thailand between 2015 and 2018 are described. This information is useful for monitoring the genetic characteristics and evolution in HA and NA genes of influenza A(H3N2) virus in Thailand which is crucial for predicting the influenza vaccine strains resulting in high vaccine effectiveness.

Epistasis mediates the evolution of the receptor binding mode in recent human H3N2 hemagglutinin.

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.

Outbreak of influenza and SARS-CoV-2 at the Armed Forces of the Philippines Health Service Education and Training Center, September 25-October 10, 2023.

In the last week of September 2023, a surge of influenza-like illness was observed among students of the Armed Forces of the Philippines (AFP) Health Service Education and Training Center, where 48 (27 males and 21 females; age in years: mean 33, range 27-41) of 247 military students at the Center presented with respiratory symptoms. Between September 25 and October 10, 2023, all 48 symptomatic students were evaluated with real-time reverse transcription polymerase chain reaction and sequencing for both influenza and SARS-CoV-2. Thirteen (27%) students were found positive for influenza A/H3 only, 6 (13%) for SARS-CoV-2 only, and 4 (8%) were co-infected with influenza A/H3 and SARS-CoV-2. Seventeen influenza A/ H3N2 viruses belonged to the same clade, 3C.2a1b.2a.2a.3a, and 4 SARSCoV-2 sequences belonged to the JE1.1 lineage, indicating a common source outbreak for both. The influenza A/H3N2 circulating virus belonged to a different clade than the vaccine strain for 2023 (3C.2a1b.2a.2a). Only 4 students had received the influenza vaccine for 2023. In response, the AFP Surgeon General issued a memorandum to all military health institutions on October 19, 2023 that mandated influenza vaccination as a prerequisite for enrollment of students at all education and training centers, along with implementation of non-pharmaceutical interventions and early notification and testing of students exhibiting influenza-like-illness.

Detection and identification of SARS-CoV-2 and influenza a based on microfluidic technology.

As of now, the global COVID-19 pandemic caused by SARS-CoV-2, which began in 2019, has been effectively controlled. However, the symptoms of influenza A virus infection were similar to those of SARS-CoV-2 infection, but they required different treatment approaches. To make the detection more accurate and the treatment more targeted. We developed a system that integrates RPA and CRISPR assays, allowing for the rapid, highly specific, and sensitive detection and differentiation of SARS-CoV-2, H1N1, and H3N2. Under isothermal amplification conditions, the RPA-CRISPR Cas12a detection system achieved a detection limit as low as 5 copies per µL, demonstrating excellent specificity. The measurement time was approximately 30 minutes. The RPA-CRISPR Cas12a detection system combined with the microfluidic chip we designed to simultaneously detect three viruses, providing a potential solution for efficient and reliable diagnosis.

Duration of fever in children infected with influenza A(H1N1)pdm09, A(H3N2) or B virus and treated with baloxavir marboxil, oseltamivir, laninamivir, or zanamivir in Japan during the 2012-2013 and 2019-2020 influenza seasons.

We compared the duration of fever in children infected with A(H1N1)pdm09, A(H3N2), or influenza B viruses following treatment with baloxavir marboxil (baloxavir) or neuraminidase inhibitors (NAIs) (oseltamivir, zanamivir, or laninamivir). This observational study was conducted at 10 outpatient clinics across 9 prefectures in Japan during the 2012-2013 and 2019-2020 influenza seasons. Patients with influenza rapid antigen test positive were treated with one of four anti-influenza drugs. The type/subtype of influenza viruses were identified from MDCK or MDCK SIAT1 cell-grown samples using two-step real-time PCR. Daily self-reported body temperature after treatment were used to evaluate the duration of fever by treatment group and various underlying factors. Among 1742 patients <19 years old analyzed, 452 (26.0%) were A(H1N1)pdm09, 827 (48.0%) A(H3N2), and 463 (26.0%) influenza B virus infections. Among fours treatment groups, baloxavir showed a shorter median duration of fever compared to oseltamivir in univariate analysis for A(H1N1)pdm09 virus infections (baloxavir, 22.0 h versus oseltamivir, 26.7 h, P < 0.05; laninamivir, 25.5 h, and zanamivir, 25.0 h). However, this difference was not significant in multivariable analyses. For A(H3N2) virus infections, there were no statistically significant differences observed (20.3, 21.0, 22.0, and 19.0 h) uni- and multivariable analyses. For influenza B, baloxavir shortened the fever duration by approximately 15 h than NAIs (20.3, 35.0, 34.3, and 34.1 h), as supported by uni- and multivariable analyses. Baloxavir seems to have comparable clinical effectiveness with NAIs on influenza A but can be more effective for treating pediatric influenza B virus infections than NAIs.

Late-Season Influenza Vaccine Effectiveness Against Medically Attended Outpatient Illness, United States, December 2022-April 2023.

BACKGROUND: The 2022-23 US influenza season peaked early in fall 2022. METHODS: Late-season influenza vaccine effectiveness (VE) against outpatient, laboratory-confirmed influenza was calculated among participants of the US Influenza VE Network using a test-negative design. RESULTS: Of 2561 participants enrolled from December 12, 2022 to April 30, 2023, 91 laboratory-confirmed influenza cases primarily had A(H1N1)pdm09 (6B.1A.5a.2a.1) or A(H3N2) (3C.2a1b.2a.2b). Overall, VE was 30% (95% confidence interval -9%, 54%); low late-season activity precluded estimation for most subgroups. CONCLUSIONS: 2022-23 late-season outpatient influenza VE was not statistically significant. Genomic characterization may improve the identification of influenza viruses that circulate postinfluenza peak.

CRISPR-Based Assays for Point-of-Need Detection and Subtyping of Influenza.

The high disease burden of influenza virus poses a significant threat to human health. Optimized diagnostic technologies that combine speed, sensitivity, and specificity with minimal equipment requirements are urgently needed to detect the many circulating species, subtypes, and variants of influenza at the point of need. Here, we introduce such a method using Streamlined Highlighting of Infections to Navigate Epidemics (SHINE), a clustered regularly interspaced short palindromic repeats (CRISPR)-based RNA detection platform. Four SHINE assays were designed and validated for the detection and differentiation of clinically relevant influenza species (A and B) and subtypes (H1N1 and H3N2). When tested on clinical samples, these optimized assays achieved 100% concordance with quantitative RT-PCR. Duplex Cas12a/Cas13a SHINE assays were also developed to detect two targets simultaneously. This study demonstrates the utility of this duplex assay in discriminating two alleles of an oseltamivir resistance (H275Y) mutation as well as in simultaneously detecting influenza A and human RNAse P in patient samples. These assays have the potential to expand influenza detection outside of clinical laboratories for enhanced influenza diagnosis and surveillance.

Cross-protective efficacy and safety of an adenovirus-based universal influenza vaccine expressing nucleoprotein, hemagglutinin, and the ectodomain of matrix protein 2.

It is necessary to develop universal vaccines that act broadly and continuously to combat regular seasonal epidemics of influenza and rare pandemics. The aim of this study was to find the optimal dose regimen for the efficacy and safety of a mixture of previously developed recombinant adenovirus-based vaccines that expressed influenza nucleoprotein, hemagglutinin, and ectodomain of matrix protein 2 (rAd/NP and rAd/HA-M2e). The vaccine efficacy and safety were measured in the immunized mice with the mixture of rAd/NP and rAd/HA-M2e intranasally or intramuscularly. The minimum dose that would be efficacious in a single intranasal administration of the vaccine mixture and cross-protective efficacy against various influenza strains were examined. In addition, the immune responses that may affect the cross-protective efficacy were measured. We found that intranasal administration is an optimal route for 107 pfu of vaccine mixture, which is effective against pre-existing immunity against adenovirus. In a study to find the minimum dose with vaccine efficacy, the 106 pfu of vaccine mixture showed higher antibody titers to the nucleoprotein than did the same dose of rAd/NP alone in the serum of immunized mice. The 106 pfu of vaccine mixture overcame the morbidity and mortality of mice against the lethal dose of pH1N1, H3N2, and H5N1 influenza infections. No noticeable side effects were observed in single and repeated toxicity studies. We found that the mucosal administration of adenovirus-based universal influenza vaccine has both efficacy and safety, and can provide cross-protection against various influenza infections even at doses lower than those previously known to be effective.

Seasonal antigenic prediction of influenza A H3N2 using machine learning.

Antigenic characterization of circulating influenza A virus (IAV) isolates is routinely assessed by using the hemagglutination inhibition (HI) assays for surveillance purposes. It is also used to determine the need for annual influenza vaccine updates as well as for pandemic preparedness. Performing antigenic characterization of IAV on a global scale is confronted with high costs, animal availability, and other practical challenges. Here we present a machine learning model that accurately predicts (normalized) outputs of HI assays involving circulating human IAV H3N2 viruses, using their hemagglutinin subunit 1 (HA1) sequences and associated metadata. Each season, the model learns an updated nonlinear mapping of genetic to antigenic changes using data from past seasons only. The model accurately distinguishes antigenic variants from non-variants and adaptively characterizes seasonal dynamics of HA1 sites having the strongest influence on antigenic change. Antigenic predictions produced by the model can aid influenza surveillance, public health management, and vaccine strain selection activities.

Molecular characterization of influenza virus circulating in Nepal in the year 2019.

Influenza (sometimes referred to as "flu") is a contagious viral infection of the airways in the lungs that affects a significant portion of the world's population. Clinical symptoms of influenza virus infections can range widely, from severe pneumonia to moderate or even asymptomatic sickness. If left untreated, influenza can have more severe effects on the heart, brain, and lungs than on the respiratory tract and can necessitate hospitalization. This study was aimed to investigate and characterize all types of influenza cases prevailing in Nepal and to analyze seasonal occurrence of Influenza in Nepal in the year 2019. A cross sectional, retrospective and descriptive study was carried out at National Influenza Center (NIC), National Public Health Laboratory Kathmandu Nepal for the period of one year (Jan-Dec 2019). A total of 3606 throat swab samples from various age groups and sexes were processed at the NIC. The specimens were primarily stored at 4 °C and processed using ABI 7500 RT PCR system for the identification of Influenza virus types and subtypes. Data accessed for research purpose were retrieved from National Influenza Centre (NIC) on 1st Jan 2020. Of the total 3606 patients suspected of having influenza infection, influenza viruses were isolated from 1213 (33.6%) patients with male predominance. The highest number of infection was caused by Influenza A/Pdm09 strain 739 (60.9%) followed by Influenza B 304 (25.1%) and Influenza A/H3 169 (13.9%) and most remarkable finding of this study was the detection of H5N1 in human which is the first ever case of such infection in human from Nepal. Similar to other tropical nations, influenza viruses were detected year-round in various geographical locations of Nepal. The influenza virus type and subtypes that were in circulation in Nepal were comparable to vaccine candidate viruses, which the currently available influenza vaccine may prevent.

Interim 2023/2024 Season Influenza Vaccine Effectiveness in Primary and Secondary Care in the United Kingdom.

BACKGROUND: We report 2023/2024 season interim influenza vaccine effectiveness for three studies, namely, primary care in Great Britain, hospital settings in Scotland and hospital settings in England. METHODS: A test negative design was used to estimate vaccine effectiveness. RESULTS: Estimated vaccine effectiveness against all influenzas ranged from 63% (95% confidence interval 46 to 75%) to 65% (41 to 79%) among children aged 2-17, from 36% (20 to 49%) to 55% (43 to 65%) among adults 18-64 and from 40% (29 to 50%) to 55% (32 to 70%) among adults aged 65 and over. CONCLUSIONS: During a period of co-circulation of influenza A(H1N1)pdm09 and A(H3N2) in the United Kingdom, evidence for effectiveness of the influenza vaccine in both children and adults was found.

The antigenic landscape of human influenza N2 neuraminidases from 2009 until 2017.

Human H3N2 influenza viruses are subject to rapid antigenic evolution which translates into frequent updates of the composition of seasonal influenza vaccines. Despite these updates, the effectiveness of influenza vaccines against H3N2-associated disease is suboptimal. Seasonal influenza vaccines primarily induce hemagglutinin-specific antibody responses. However, antibodies directed against influenza neuraminidase (NA) also contribute to protection. Here, we analysed the antigenic diversity of a panel of N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. The antigenic breadth of these NAs was determined based on the NA inhibition (NAI) of a broad panel of ferret and mouse immune sera that were raised by infection and recombinant N2 NA immunisation. This assessment allowed us to distinguish at least four antigenic groups in the N2 NAs derived from human H3N2 viruses that circulated between 2009 and 2017. Computational analysis further revealed that the amino acid residues in N2 NA that have a major impact on susceptibility to NAI by immune sera are in proximity of the catalytic site. Finally, a machine learning method was developed that allowed to accurately predict the impact of mutations that are present in our N2 NA panel on NAI. These findings have important implications for the renewed interest to develop improved influenza vaccines based on the inclusion of a protective NA antigen formulation.

Two proteins, the hemagglutinin and the neuraminidase, protrude from the surface of the influenza virus. Their detection by the immune system allows the host organism to mount defences against the viral threat. The virus evolves in response to this pressure, which manifests as changes in the appearance of its hemagglutinin and neuraminidase. This process, known as antigenic drift, leads to the proteins evading detection. It is also why flu vaccines require frequent updates, as they rely on 'training' the immune system to recognise the most important strains in circulation ­ primarily by exposing it to appropriate versions of hemagglutinin. While the antigenic drift of hemagglutinin has been extensively studied, much less is known about how the neuraminidase accumulates mutations, and how these affect the immune response. To investigate this question, Catani et al. selected 43 genetically distant neuraminidases from human viral samples isolated between 2009 and 2017. Statistical analyses were applied to define their relatedness, revealing that a group of closely related neuraminidases predominated from 2009 to 2015, before they were being taken over by a second group. A third group, which was identified in viruses isolated in 2013, was remarkably close to the neuraminidase of strains that circulated in the late 1990s. The fourth and final group of neuraminidases was derived from influenza viruses that normally circulate in pigs but can also occasionally infect humans. Next, Catani et al. examined the immune response that these 43 neuraminidases could elicit in mice, as well as in ferrets ­ the animal most traditionally used in influenza research. This allowed them to pinpoint which changes in the neuraminidase sequences were important to escape recognition by the host. Data obtained from the two model species were comparable, suggesting that these experiments could be conducted on mice going forward, which are easier to work with than ferrets. Finally, Catani et al. used machine learning to build a computational model that could predict how strongly the immune system would respond to a specific neuraminidase variant. These findings could help guide the development of new vaccines that include neuraminidases tailored to best prime and train the immune system against a larger variety of strains. This may aid the development of 'supra-seasonal' vaccines that protect against a broad range of influenza viruses, reducing the need for yearly updates.

Low antiviral resistance in Influenza A and B viruses isolated in Mexico from 2010 to 2023.

The most widely used class of antivirals available for Influenza treatment are the neuraminidase inhibitors (NAI) Oseltamivir and Zanamivir. However, amino acid (AA) substitutions in the neuraminidase may cause reduced inhibition or high antiviral resistance. In Mexico, the current state of knowledge about NAI susceptibility is scarce, in this study we report the results of 14 years of Influenza surveillance by phenotypic and genotypic methods. A total of 255 isolates were assessed with the NAI assay, including Influenza A(H1N1)pdm09, A(H3N2) and Influenza B (IBV). Furthermore, 827 sequences contained in the GISAID platform were analyzed in search of relevant mutations.Overall, five isolates showed highly reduced inhibition or reduced inhibition to Oseltamivir, and two showed reduced inhibition to Zanamivir in the NAI assays. Additionally, five A(H1N1)pdm09 sequences from the GISAID possessed AA substitutions associated to reduced inhibition to Oseltamivir and none to Zanamivir. Oseltamivir resistant A(H1N1)pdm09 harbored the H275Y mutation. No genetic mutations were identified in Influenza A(H3N2) and IBV. Overall, these results show that in Mexico the rate of NAI resistance is low (0.6%), but it is essential to continue the Influenza surveillance in order to understand the drug susceptibility of circulating strains.

Nanopore sequencing of influenza A and B in Oxfordshire and the United Kingdom, 2022-23.

OBJECTIVES: We evaluated Nanopore sequencing for influenza surveillance. METHODS: Influenza A and B PCR-positive samples from hospital patients in Oxfordshire, UK, and a UK-wide population survey from winter 2022-23 underwent Nanopore sequencing following targeted rt-PCR amplification. RESULTS: From 941 infections, successful sequencing was achieved in 292/388 (75 %) available Oxfordshire samples: 231 (79 %) A/H3N2, 53 (18 %) A/H1N1, and 8 (3 %) B/Victoria and in 53/113 (47 %) UK-wide samples. Sequencing was more successful at lower Ct values. Most same-sample replicate sequences had identical haemagglutinin segments (124/141, 88 %); 36/39 (92 %) Illumina vs. Nanopore comparisons were identical, and 3 (8 %) differed by 1 variant. Comparison of Oxfordshire and UK-wide sequences showed frequent inter-regional transmission. Infections were closely-related to 2022-23 vaccine strains. Only one sample had a neuraminidase inhibitor resistance mutation. 849/941 (90 %) Oxfordshire infections were community-acquired. 63/88 (72 %) potentially healthcare-associated cases shared a hospital ward with ≥ 1 known infectious case. 33 epidemiologically-plausible transmission links had sequencing data for both source and recipient: 8 were within ≤ 5 SNPs, of these, 5 (63 %) involved potential sources that were also hospital-acquired. CONCLUSIONS: Nanopore influenza sequencing was reproducible and antiviral resistance rare. Inter-regional transmission was common; most infections were genomically similar. Hospital-acquired infections are likely an important source of nosocomial transmission and should be prioritised for infection prevention and control.

Exploration of influenza A virus PA protein-associated cellular proteins discloses its impact on mitochondrial function.

Influenza A virus can infect respiratory tracts and may cause severe illness in humans. Proteins encoded by influenza A virus can interact with cellular factors and dysregulate host biological processes to support viral replication and cause pathogenicity. The influenza viral PA protein is not only a subunit of influenza viral polymerase but also a virulence factor involved in pathogenicity during infection. To explore the role of the influenza virus PA protein in regulating host biological processes, we performed immunoprecipitation and LC‒MS/MS to globally identify cellular factors that interact with the PA proteins of the influenza A H1N1, 2009 pandemic H1N1, and H3N2 viruses. The results demonstrated that proteins located in the mitochondrion, proteasome, and nucleus are associated with the PA protein. We further discovered that the PA protein is partly located in mitochondria by immunofluorescence and mitochondrial fractionation and that overexpression of the PA protein reduces mitochondrial respiration. In addition, our results revealed the interaction between PA and the mitochondrial matrix protein PYCR2 and the antiviral role of PYCR2 during influenza A virus replication. Moreover, we found that the PA protein could also trigger autophagy and disrupt mitochondrial homeostasis. Overall, our research revealed the impacts of the influenza A virus PA protein on mitochondrial function and autophagy.

A combination influenza mRNA vaccine candidate provided broad protection against diverse influenza virus challenge.

Influenza viruses present a significant threat to global health. The production of a universal vaccine is considered essential due to the ineffectiveness of current seasonal influenza vaccines against mutant strains. mRNA technology offers new prospects in vaccinology, with various candidates for different infectious diseases currently in development and testing phases. In this study, we encapsulated a universal influenza mRNA vaccine. The vaccine encoded influenza hemagglutinin (HA), nucleoprotein (NP), and three tandem repeats of matrix protein 2 (3M2e). Twice-vaccinated mice exhibited strong humoral and cell-mediated immune responses in vivo. Notably, these immune responses led to a significant reduction in viral load of the lungs in challenged mice, and also conferred protection against future wild-type H1N1, H3N2, or H5N1 influenza virus challenges. Our findings suggest that this mRNA-universal vaccine strategy for influenza virus may be instrumental in mitigating the impact of future influenza pandemics.

Probing altered receptor specificities of antigenically drifting human H3N2 viruses by chemoenzymatic synthesis, NMR, and modeling.

Prototypic receptors for human influenza viruses are N-glycans carrying α2,6-linked sialosides. Due to immune pressure, A/H3N2 influenza viruses have emerged with altered receptor specificities that bind α2,6-linked sialosides presented on extended N-acetyl-lactosamine (LacNAc) chains. Here, binding modes of such drifted hemagglutinin's (HAs) are examined by chemoenzymatic synthesis of N-glycans having 13C-labeled monosaccharides at strategic positions. The labeled glycans are employed in 2D STD-1H by 13C-HSQC NMR experiments to pinpoint which monosaccharides of the extended LacNAc chain engage with evolutionarily distinct HAs. The NMR data in combination with computation and mutagenesis demonstrate that mutations distal to the receptor binding domain of recent HAs create an extended binding site that accommodates with the extended LacNAc chain. A fluorine containing sialoside is used as NMR probe to derive relative binding affinities and confirms the contribution of the extended LacNAc chain for binding.