Background:Seasonal human influenza viruses can escape from antibody-mediated neutralization when amino acid changes occur in the hemagglutinin protein. Routine surveillance identified circulation of an A(H3N2) virus variant in the Netherlands with amino acid substitutions at hemagglutinin positions 158 and 189. These amino acid positions were previously responsible for antigenic change of influenza A(H3N2) viruses and potentially lead to escape of this variant from vaccine-mediated immunity. Aim:To characterize the emergence and antigenic properties of N158K and K189R double substitution virus variants. Methods:We analyzed the geographical and temporal dynamics of the double-substitution variant using a phylogeographic approach and used hemagglutination inhibition assays and antigenic cartography methods to map its antigenic properties. Results:A(H3N2) viruses carrying K189R were first detected in Guatemala in June 2024, before subsequently gaining the N158K substitution, which was intially detected in Colombia in November 2024, followed by detection in the Netherlands in December 2024. However, detections within Europe remained almost entirely confined to the Netherlands. The proportion of viruses carrying the N158K and K189R substitutions increased to 16% - 24% per collection week of sequenced Dutch viruses during the peak of the epidemic of the 2024-2025 respiratory season. Antigenic characterization of viruses with N158K and K189R substitutions indicated that these are antigenically distinct from the A(H3N2) components of 2025-2026 Northern Hemisphere vaccines, showing 8-192-fold reduction in hemagglutination inhibition titers with antisera against the vaccine strain compared to antisera against the homologous virus. Conclusions:Influenza A(H3N2) viruses with N158K and K189R escaped recognition by antibodies raised against the 2024-2025 and 2025/2026 Northern Hemipshere vaccine strains in hemagglutination inhibition assays. These variants circulated widely in the Netherlands during the 2024-2025 influenza season, raising concerns about reduced vaccine-mediated protection if such variants would spread more broadly during 2025-2026 Northern Hemipshere season.

Background Highly pathogenic avian influenza H5N1 viruses of the A/Goose/Guangdong/1/1996 lineage pose a global threat to wildlife, domestic animals, and humans. Cross-species transmission events to mammals, including humans, in the past 4 years highlight this threat. For influenza A viruses, crucial determinants of cross-species and intraspecies transmission to and among mammals include attachment to and replication in respiratory airway epithelial cells. Although these determinants have been studied for H5N1 viruses in the past, limited studies for clade 2.3.4.4b viruses exist. Therefore, the aim of this study was to determine the ability of recent clade 2.3.4.4b H5N1 viruses to attach to human respiratory tissues, to replicate in human airway epithelial cells and the associated immune response. Methods In this in-vitro study, we investigated three H5N1 clade 2.3.4.4b viruses (H5N1Gull2022, H5N1Polecat2022, and H5N1Bovine2024) in comparison with previously studied 2.1.3.2 H5N1 (H5N12005) and a seasonal H3N2 virus. First, we compared virus attachment patterns by virus histochemistry. Second, we investigated the infection and replication efficiency, and innate immune responses in infected human respiratory epithelium in vitro. Third, we measured polymerase complex activity using a minigenome assay. Findings Clade 2.3.4.4b viruses and H5N12005 virus differed by five amino acids located near the receptor binding site of the haemagglutinin. All clade 2.3.4.4b viruses attached more efficiently to cells of the human upper and lower respiratory tract compared with H5N12005 virus. All clade 2.3.4.4b viruses replicated in human nasal and tracheobronchial respiratory epithelium cultures. In the tracheobronchial respiratory epithelium cultures, H5N1Gull2022 virus replicated more efficiently than H5N12005 virus (p=0·0050) and reached titres similar to H3N22003 virus. Polymerase complex activity of H5N1Gull2022 virus was not significantly different from that of H5N12005 and was significantly lower compared with H3N22003 virus (p≤0·0001). Infection with H5N1Gull2022 virus induced a broader antiviral immune response than H5N12005 virus. Interpretation Clade 2.3.4.4b H5N1 viruses have phenotypic characteristics that are different from a clade 2.1.3.2 H5N12005 virus. The ability of clade 2.3.4.4b viruses to attach to and replicate in respiratory epithelium likely contributes to an increased risk for both human infection and virus adaptation to humans. Funding The EU, the Dutch Research Council, the Netherlands Organization for Health Research and Development, and the Dutch Ministries of Agriculture, Fisheries, Food Security and Nature, and Health, Welfare and Sport.

BackgroundZoonotic influenza, including infections with avian and swine influenza A viruses (swIAV), is a notifiable disease in the Netherlands. Human cases infected with swIAV have previously been rarely detected in the Netherlands.AimWe aimed to describe detection and characterisation of Eurasian avian-like swIAV infections in humans in the Netherlands 2020-2023.MethodsThe Dutch National Influenza Center coordinates different activities to monitor respiratory infections and circulating human influenza viruses. This monitoring includes sentinel surveillance in general practitioner practices, community participatory surveillance and characterisation of influenza viruses received from diagnostic laboratories. A subset of the specimens positive for influenza A virus from the monitoring activities are sent for further characterisation. We characterised swIAV from human patients using whole genome sequencing, tested the viruses for antiviral susceptibility and in haemagglutination inhibition assays for antigenic characterisation and compared them with previous detections from humans and pigs.ResultsAvian-like swine influenza virus was detected in three persons presenting with mild respiratory symptoms, and all recovered fully. Only one patient had close contact with pigs shortly before the start of symptoms. Sequence analyses of the viruses showed clustering with swAIV from pigs in a recently initiated surveillance system on pig farms.ConclusionsThese human cases show that swIAV viruses with zoonotic potential are enzootic in the Netherlands. Finding them by coincidence suggests human infections might occur more frequently than noticed.

ABSTRACT Avian A/H7 influenza viruses are a global threat to animal and human health. These viruses continue to cause outbreaks in poultry and have caused the highest number of reported zoonotic infections to date, highlighting their pandemic threat. Evidence for antigenic diversification of avian A/H7 influenza viruses exists; however, knowledge of the drivers and molecular basis of antigenic evolution of these viruses is limited. Here, antigenic cartography was used to analyze the global antigenic diversity of A/H7 influenza viruses and to determine the molecular basis of antigenic change in A/H7N9 viruses. A phylogenetic tree based on all available A/H7 HA sequences was generated, from which 52 representative, genetically diverse, antigens were selected for antigenic characterization using hemagglutination inhibition assays. The resulting data were used to compute an antigenic map using multidimensional scaling algorithms. High antigenic relatedness was observed between antigens and sera belonging to genetically divergent A/H7 (sub)lineages. The most striking antigenic change relative to the timespan of virus isolation was observed for the A/H7N9 viruses isolated between 2013 and 2019 in China. Amino acid changes at positions 116, 118, 125, 130, 151, and 217 in the hemagglutinin globular head were found to be the main determinants of antigenic evolution between A/H7N9 influenza virus prototypes. The A/H7 antigenic map and knowledge of the molecular determinants of their antigenic evolution will aid pandemic preparedness against A/H7 influenza viruses, specifically regarding the design of novel vaccines and vaccination strategies. IMPORTANCE A/H7 avian influenza viruses cause outbreaks in poultry globally, resulting in outbreaks with significant socio-economical impact and zoonotic risks. Occasionally, poultry vaccination programs have been implemented to reduce the burden of these viruses, which might result in an increased immune pressure accelerating antigenic evolution. In fact, evidence for antigenic diversification of A/H7 influenza viruses exists, posing challenges to pandemic preparedness and the design of vaccination strategies efficacious against drifted variants. Here, we performed a comprehensive analysis of the global antigenic diversity of A/H7 influenza viruses and identified the main substitutions in the hemagglutinin responsible for antigenic evolution in A/H7N9 viruses isolated between 2013 and 2019. The A/H7 antigenic map and knowledge of the molecular determinants of their antigenic evolution add value to A/H7 influenza virus surveillance programs, the design of vaccines and vaccination strategies, and pandemic preparedness.

Seasonal influenza vaccination takes into account primarily hemagglutinin (HA)-specific neutralizing antibody responses. However, the accumulation of substitutions in the antigenic regions of HA (i.e., antigenic drift) occasionally results in a mismatch between the vaccine and circulating strains. To prevent poor vaccine performance, we investigated whether an antigenically matched neuraminidase (NA) may compensate for reduced vaccine efficacy due to a mismatched HA. Ferrets were vaccinated twice with adjuvanted split inactivated influenza vaccines containing homologous HA and NA (vacH3N2), only homologous HA (vacH3N1), only homologous NA (vacH1N2), heterologous HA and NA (vacH1N1), or phosphate-buffered saline (vacPBS), followed by challenge with H3N2 virus (A/Netherlands/16190/1968). Ferrets vaccinated with homologous HA (vacH3N2 and vacH3N1) displayed minimum fever and weight loss compared to vacH1N1 and vacPBS ferrets, while ferrets vaccinated with NA-matched vacH1N2 displayed intermediate fever and weight loss. Vaccination with vacH1N2 further led to a reduction in virus shedding from the nose and undetectable virus titers in the lower respiratory tract, similarly to when the homologous vacH3N2 was used. Some protection was observed upon vacH1N1 vaccination, but this was not comparable to that observed for vacH1N2, again highlighting the important role of NA in vaccine-induced protection. These results illustrate that NA antibodies can prevent severe disease caused by influenza virus infection and that an antigenically matched NA in seasonal vaccines might prevent lower respiratory tract complications. This underlines the importance of considering NA during the yearly vaccine strain selection process, which may be particularly beneficial in seasons when the HA component of the vaccine is mismatched. IMPORTANCE Despite the availability of vaccines, influenza virus infections continue to cause substantial morbidity and mortality in humans. Currently available influenza vaccines take primarily the hemagglutinin (HA) into account, but the highly variable nature of this protein as a result of antigenic drift has led to a recurrent decline in vaccine effectiveness. While the protective effect of neuraminidase (NA) antibodies has been highlighted by several studies, there are no requirements with regard to quantity or quality of NA in licensed vaccines, and NA immunity remains largely unexploited. Since antigenic changes in HA and NA are thought to occur asynchronously, NA immunity could compensate for reduced vaccine efficacy when drift in HA occurs. By matching and mismatching the HA and NA components of monovalent split inactivated vaccines, we demonstrated the potential of NA immunity to protect against disease, virus replication in the lower respiratory tract, and virus shedding in the ferret model.

BACKGROUND:10 days after the first reported case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the Netherlands (on Feb 27, 2020), 55 (4%) of 1497 health-care workers in nine hospitals located in the south of the Netherlands had tested positive for SARS-CoV-2 RNA. We aimed to gain insight in possible sources of infection in health-care workers. METHODS:We did a cross-sectional study at three of the nine hospitals located in the south of the Netherlands. We screened health-care workers at the participating hospitals for SARS-CoV-2 infection, based on clinical symptoms (fever or mild respiratory symptoms) in the 10 days before screening. We obtained epidemiological data through structured interviews with health-care workers and combined this information with data from whole-genome sequencing of SARS-CoV-2 in clinical samples taken from health-care workers and patients. We did an in-depth analysis of sources and modes of transmission of SARS-CoV-2 in health-care workers and patients. FINDINGS:Between March 2 and March 12, 2020, 1796 (15%) of 12 022 health-care workers were screened, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete and near-complete genome sequences from 50 health-care workers and ten patients. Most sequences were grouped in three clusters, with two clusters showing local circulation within the region. The noted patterns were consistent with multiple introductions into the hospitals through community-acquired infections and local amplification in the community. INTERPRETATION:Although direct transmission in the hospitals cannot be ruled out, our data do not support widespread nosocomial transmission as the source of infection in patients or health-care workers. FUNDING:EU Horizon 2020 (RECoVer, VEO, and the European Joint Programme One Health METASTAVA), and the National Institute of Allergy and Infectious Diseases, National Institutes of Health.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Ten days after the first reported case of SARS-CoV-2 infection in the Netherlands, 3.9% of healthcare workers (HCWs) in nine hospitals located in the South of the Netherlands tested positive for SARS-CoV-2 RNA. The extent of nosocomial transmission that contributed to the HCW infections was unknown. We combined epidemiological data, collected by means of structured interviews of HCWs, with whole genome sequencing (WGS) of SARS-CoV-2 in clinical samples from HCWs and patients in three of nine hospitals that participated in the HCW screening, to perform an in-depth analysis of sources and modes of transmission of SARS -CoV-2 in HCWs and patients. A total of 1,796 out of 12,022 HCWs (15%) of the three participating hospitals were screened, based on clinical symptoms, of whom 96 (5%) tested positive for SARS-CoV-2. We obtained complete genome sequences of 50 HCWs and 18 patients. Most sequences grouped in 3 clusters, with 2 clusters displaying local circulation within the region. The observed patterns are most consistent with multiple introductions into the hospitals through community acquired infections, and local amplification in the community. Although direct transmission in the hospitals cannot be ruled out, the data does not support widespread nosocomial transmission as source of infection in patients or healthcare workers. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study has been partly funded by H2020 projects RECoVer (no. 101003589), VEO (No. 874735) and NIAID/NIH contract HHSN272201400008C ### Author Declarations All relevant ethical guidelines have been followed; any necessary IRB and/or ethics committee approvals have been obtained and details of the IRB/oversight body are included in the manuscript. Yes All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes All data is available upon request

SARS-CoV-2 is a novel coronavirus that has rapidly spread across the globe. In the Netherlands, the first case of SARS-CoV-2 has been notified on the 27 th of February. Here, we describe the first three weeks of the SARS-CoV-2 outbreak in the Netherlands, which started with several different introductory events from Italy, Austria, Germany and France followed by local amplification in, and later also, outside the South of the Netherlands. The timely generation of whole genome sequences combined with epidemiological investigations facilitated early decision making in an attempt to control local transmission of SARS-CoV-2 in the Netherlands.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Extra-epitopic amino acid residues affect recognition of human influenza A viruses (IAVs) by CD8+ T-lymphocytes (CTLs) specific for the highly conserved HLA-A*0201 restricted M158-66 epitope located in the matrix 1 (M1) protein. These residues are absent in the M1 protein of the 2009-pandemic IAV (H1N1pdm09). Consequently, stimulation with M1 protein of H1N1pdm09 IAV resulted in stronger activation and lytic activity of M158-66-specific CTLs than stimulation with seasonal H3N2 IAVs. During >6 years of circulation in the human population, descendants of the H1N1pdm09 virus had accumulated 4 other amino acid substitutions. However, these did not affect M158-66-specific CTL activation.

Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity.IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

Background. High-pathogenicity avian influenza viruses continue to circulate in poultry and wild birds and occasionally infect humans, sometimes with fatal outcomes. Development of vaccines is a priority to prepare for potential pandemics but is complicated by antigenic variation of the surface glycoprotein hemagglutinin. We report the immunological profile induced by human immunization with modified vaccinia virus Ankara (MVA) expressing the hemagglutinin gene of influenza A(H5N1) virus A/Vietnam/1194/04 (rMVA-H5). Methods. In a double-blinded phase 1/2a clinical trial, 79 individuals received 1 or 2 injections of rMVA-H5 or vector control. Twenty-seven study subjects received a booster immunization after 1 year. The breadth, magnitude, and properties of vaccine-induced antibody and T-cell responses were characterized. Results. rMVA-H5 induced broadly reactive antibody responses, demonstrated by protein microarray, hemagglutination inhibition, virus neutralization, and antibody-dependent cellular cytotoxicity assays. Antibodies cross-reacted with antigenically distinct H5 viruses, including the recently emerged subtypes H5N6 and H5N8 and the currently circulating subtype H5N1. In addition, the induction of T cells specific for H5 viruses of 2 different clades was demonstrated. Conclusions. rMVA-H5 induced immune responses that cross-reacted with H5 viruses of various clades. These findings validate rMVA-H5 as vaccine candidate against antigenically distinct H5 viruses.

Influenza viruses are responsible for substantial morbidity and mortality during seasonal epidemics. Vaccination is the most effective method to prevent infection, however due to antigenic drift of the viral surface protein hemagglutinin (HA), annual influenza virus vaccination is required. In addition to seasonal viruses, certain (avian) influenza A viruses of other subtypes, like H5N1 or H7N9, cause sporadic zoonotic infections. Therefore, the availability of game-changing novel vaccines that induce “universal” immune responses to a wide variety of influenza A virus subtypes is highly desirable. The quest for universal influenza vaccines has fueled the interest in broadly-reactive antibodies specific for the stalk of hemagglutinin (HA) and biological activities of antibodies other than direct virus neutralization, like antibody-dependent cellular cytotoxicity (ADCC). In the present study, we investigated the ADCC response upon influenza virus vaccination and infection in humans using a robust ADCC assay that is based on the use of recombinant HA and a continuous NK cell line that expresses FcγRIII (CD16). This assay offers advantages over existing methods, like ease to perform and possibilities to standardize. We showed that HA-specific ADCC mediating antibodies are induced by vaccination with adjuvanted trivalent seasonal and monovalent H1N1pdm09 inactivated vaccines, and by infection with H1N1pdm09 virus. In addition, the use of chimeric influenza HA with a H1 stem but antigenically irrelevant head domain derived from an avian virus allowed detection of H1-stalk-specific ADCC mediating antibodies. This assay will facilitate the assessment of ADCC mediating serum antibodies after (universal) influenza vaccination or infection and may define ADCC activity as a correlate of (cross-) protection in the future.

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. blast analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa.

To the Editor: Aquatic birds form a natural reservoir of avian influenza viruses from which new human and animal influenza viruses originate.After initial detection in 2010 in China, a new highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype reemerged in ducks in South Korea in 2014 (1,2).The hemagglutinin gene of this virus was distantly related to those of H5N1 subtypes that have caused infections in humans since 1997 (3).

Background: Hepatitis E virus (HEV) genotype 3 is recognised as an emerging pathogen in industrialised countries. The currently commercially available HEV-specific enzyme linked immunosorbent assays (ELISAs) are primarily designed for the detection of antibodies against genotypes 1 (Burma) and 2 (Mexico) and may not sensitively detect HEV genotypes 3 or 4.Objectives: This study aimed to evaluate the analytical and clinical performances of eight commercially available HEV serum antibody immunoglobulin M (IgM)- and immunoglobulin G (IgG)-specific ELISAs for genotype 1 and 3 HEV infections in a clinical setting and to study the antibody responses against HEV of immunocompromised versus immunocompetent patient groups.Study design: Analytical performance and diagnostic sensitivity and specificity were assessed using well-defined reference samples and samples from patients with polymerase chain reaction (PCR)-confirmed HEV infection (n=88) and a specificity panel (n=98).Results: Limiting dilutions indicated that the highest analytical sensitivity in head-to-head comparison was measured for the Mikrogen_new IgG assay. Taking the serum working dilutions of each assay into account, the Wantai IgG assay was the most sensitive assay. Receiver operator curve (ROC) analysis showed area under the curve (AUC) values of 0.943, 0.964, 0.969, 0.971, 0.974 and 0.994 for the DSI, Mikrogen_old, MP Diagnostics, Mikrogen_new, Wantai and DiaPro anti-HEV IgM assays, respectively. The highest specificity of currently available assays was found for the IgM Wantai assay (>99%). If anti-HEV IgM and IgG results from each supplier were combined, DSI and Wantai assays were able to detect the highest number of (passed) HEV infections.Conclusions: Our study showed that current commercial HEV ELISAs could be used to diagnose HEV genotype 3 infection adequately in a clinical setting. (C) 2013 Elsevier B.V. All rights reserved.