Biology subjects

Fouchier, R. A. M.

Publications and source records attributed to Fouchier, R. A. M..

16 recordsLinked to original sources

bioRxiv2026

NGS-RFA/FRA: A High Throughput Experimental and Computational Pipeline for Selective Mutational Scanning in Parallel

Influenza viruses evade vaccine and infection mediated immunity by accumulating mutations in their hemagglutinin (HA) protein. Predicting this evolution might be possible via selective mutational scanning (SMS) - the generation of many specific mutants of interest from currently circulating viruses and characterizing their escape potential and fitness with high accuracy (Mogling 2016). However, this task is challenging, even when focusing on a reduced set of key HA positions (Koel et al. 2013). Here we describe a high-throughput SMS method to address this challenge. Our approach consists of a three-stage pipeline: (1) a parallel optimized virus rescue process that generates balanced target mutant virus libraries (2) an assay to assess replicative fitness and neutralisation of these variants as a mixture, and (3) a bespoke statistical model to quantify statistically significant differences between these observables. We tested the pipeline on libraries of up to 134 variants finding excellent correlation to classical hemagglutination inhibition (HI) and plaque growth assays used to assess antigenic phenotype and replicative fitness respectively, as well as remarkable repeatability overall. Notably, the method reduces the timeline required to carry out such assessments with classical methods from about a year to several weeks. By enabling rapid and efficient characterization of influenza virus variants, this approach has the potential to greatly enhance surveillance efforts, transforming reactive monitoring into proactive forecasting.

evolutionary biology↗

bioRxiv2026

Loss of hemagglutination ability by H3N2 influenza A virus, subclade K.

Seasonal human H3N2 influenza viruses, subclade K (J.2.4.1), have been the predominant influenza A viruses in the Northern hemisphere influenza season of 2025/2026. Since 2024, the vaccine virus A/Darwin/6/21 has emerged in different antigenic variants. Antigenic changes are frequently caused by amino acid substitutions near the hemagglutinin (HA) receptor-binding pocket, which can also affect receptor binding properties, such as hemagglutination. Hemagglutination is crucial for assessing antigenicity using the hemagglutination inhibition (HAI) assay, and a loss of binding to turkey erythrocytes could significantly hamper this process. In this study, we explored how substitutions in or around the HA receptor-binding site affect binding to glycans at the molecular level. We employed ELISA, glycan array, flow cytometry, hemagglutination assays, and tissue staining. Substitutions at positions 140, 192, and 223 establish clade J viruses that emerged in 2024. Computational analysis of HA in complex with an elongated glycan reveals that mutation F192 forms a CH-Pi interaction to stabilize the binding. Based on this background, substitutions in antigenic sites A and B within subclade K viruses exhibit a binding preference for elongated glycans, which are not displayed on turkey erythrocytes. Conversely, our previously established glyco-remodeled erythrocytes are efficiently bound by these subclade K H3N2 viruses and could support influenza surveillance and vaccine development.

microbiology↗

bioRxiv2026

Predicting the antigenic evolution of seasonal influenza viruses using phylogenetic convergence

The antigenic evolution of human seasonal influenza viruses is primarily driven by single amino acid substitutions immediately adjacent to the receptor binding site in the hemagglutinin (HA) protein. The ability to predict these substitutions would allow vaccine strains to be selected with an understanding of likely future antigenic variation. Here, we estimate the effect of HA substitutions on viral fitness using measurements of convergent evolution in a large phylogeny. We show that the substitutions which have historically caused major antigenic changes in H3N2 influenza viruses were nearly always one of few substitutions near the HA receptor binding site estimated to be under positive selection in sequences collected before the antigenic transition, based on convergent acquisition of the substitution in multiple independent lineages. Furthermore, this signal predates the establishment of the major clade containing the antigenic substitution by more than one year, so is highly informative for prospective prediction.

evolutionary biology↗

bioRxiv2026

Development of a multi-species luciferase-based double antigen ELISA for the detection of antibodies against Influenza A virus H5 clade 2.3.4.4b

The highly pathogenic avian influenza viruses (HPAIV) of subtype H5N1 represent a major threat to animal and public health. The current panzootic with H5 clade 2.3.4.4b has caused numerous, widespread outbreaks in various domestic and wild avian species with high mortalities, massive losses and a high frequency of spillover events to unexpected novel mammalian hosts such as dairy cows. The global H5N1 situation raises serious concerns about zoonotic risks due to effective mammal-to-mammal transmission. Therefore, it is critical to increase surveillance intensity of a broadened species range, particularly at the human-animal interface. For this purpose, reliable and cost-effective serological tools that are easy to perform and suitable for high-throughput screenings are critically needed. The newly developed double antigen ELISA format employing a luminescence-based detection technology has demonstrated to comply with such prerequisites. The assay allowed the detection of H5-specific antibodies even early after infection or vaccination in a wide range of birds and mammals including humans. It further demonstrated superior analytical sensitivity and high specificity for antibodies directed against H5 hemagglutinin of clade 2.3.4.4b as no cross-reactivity with other hemagglutinin subtypes was observed. Thus, the assay represents a valuable contribution to existing serological diagnostic tests for a clade-optimized detection of influenza A virus antibodies in a broad range of species. ImportanceThe ongoing HPAIV H5N1 panzootic has caused numerous outbreaks in domestic and wild animals with frequent spillover events to unexpected host species, which underscores the importance of an intensified surveillance. However, sensitive and specific multi-species serological assays represent a major gap. For this purpose, we developed a novel double antigen ELISA which employs an innovative luminescence-based read-out strategy. The test allowed a highly sensitive and specific detection of H5-specific antibodies even early after infection or vaccination in a wide range of avian and mammalian species including humans. It therefore represents a significant contribution to improving species-independent serological diagnostic tools for the detection of influenza A virus antibodies.

microbiology↗

bioRxiv2025

Language models learn to represent antigenic properties of human influenza A(H3) virus

Given that influenza vaccine effectiveness depends on a good antigenic match between the vaccine and circulating viruses, it is important to assess the antigenic properties of newly emerging variants continuously. With the increasing application of real-time pathogen genomic surveillance, a key question is if antigenic properties can reliably be predicted from influenza virus genomic information. Based on validated linked datasets of influenza virus genomic and wet lab experimental results, in silico models may be of use to learn to predict immune escape of variants of interest starting from the protein sequence only. In this study, we compared several machine-learning methods to reconstruct antigenic map coordinates for HA1 protein sequences of influenza A(H3N2) virus, to rank substitutions responsible for major antigenic changes, and to recognize variants with novel antigenic properties that may warrant future vaccine updates. Methods based on deep learning language models (BiLSTM and ProtBERT) and more classical approaches based solely on genetic distances and physicochemical properties of amino acid sequences had comparable performances over the coarser features of the map, but the first two performed better over fine-grained features like single amino acid-driven antigenic change and in silico deep mutational scanning experiments to rank the substitutions with the largest impact on antigenic properties. Given that the best performing model that produces protein embeddings is agnostic to the specific pathogen, the presented approach may be applicable to other pathogens.

genomics↗

bioRxiv2024

Emergence and Dynamics of Usutu and West Nile Viruses in the Netherlands, 2016-2022

BackgroundMosquito-borne arboviruses, including Usutu virus (USUV) and West Nile virus (WNV), are emerging threats in Europe, with changes in climate, land use, and increased travel and trade influencing their dynamics. Understanding the emergence and establishment of these viruses in new regions is critical for informing targeted mitigation of drivers of emergence and enhancing public and wildlife health preparedness. MethodsSeven years (2016-2022) of interconnected studies were conducted in the Netherlands. Live birds were sampled by volunteer bird ringers, dead birds were referred for sampling by citizen-scientists and zoological institutions, and mosquitoes were trapped. Samples were tested for USUV and WNV using RT-PCR and screened for Orthoflavivirus antibodies using protein microarray and neutralization assays. Sequencing and phylogenetic analyses were performed. FindingsUSUV was first detected in the Netherlands in 2016, caused large outbreaks in birds until 2018, and resurged in 2022. One dominant, enzootic lineage, co-circulated alongside several limited introductions of a second lineage. A localized WNV lineage 2 outbreak occurred in live birds and mosquitoes in 2020, with another positive bird in 2022 and serological evidence of continued circulation. InterpretationWe provide the first comprehensive, multi-year documentation of two emerging arboviruses in the Netherlands. USUV was enzootically maintained over seven years and was associated with substantial bird mortality. WNV is in an early stage of establishment with no bird mortality observed. Our integrated wildlife sampling was crucial in detecting a human WNV outbreak, bringing this emerging threat to public health attention. FundingZonMw, Dutch Research Council (NWO), European Union, Government of the Netherlands

microbiology↗

bioRxiv2024

A vaccine antigen central in influenza A(H5) virus antigenic space confers subtype-wide immunity

Highly pathogenic avian influenza A(H5) viruses globally impact wild and domestic birds, and mammals, including humans, underscoring their pandemic potential. The antigenic evolution of the A(H5) hemagglutinin (HA) poses challenges for pandemic preparedness and vaccine design. Here, the global antigenic evolution of the A(H5) HA was captured in a high-resolution antigenic map. The map was used to engineer immunogenic and antigenically central vaccine HA antigens, eliciting antibody responses that broadly cover the A(H5) antigenic space. In ferrets, a central antigen protected as well as homologous vaccines against heterologous infection with two antigenically distinct viruses. This work showcases the rational design of subtype-wide influenza A(H5) pre-pandemic vaccines and demonstrates the value of antigenic maps for the evaluation of vaccine-induced immune responses through antibody profiles.

microbiology↗

bioRxiv2024

Transient RNA structures underlie highly pathogenic avian influenza virus genesis

Highly pathogenic avian influenza viruses (HPAIVs) cause severe disease and high fatality in poultry1. They emerge exclusively from H5 and H7 low pathogenic avian influenza viruses (LPAIVs)2. Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for LPAIV-to-HPAIV transition3,4, the exact mechanisms underlying said insertion have remained unknown. Here we used an innovative combination of bioinformatic models to predict RNA structures forming around the influenza virus RNA polymerase during replication, and circular sequencing5 to reliably detect nucleotide insertions. We show that transient H5 hemagglutinin RNA structures predicted to trap the polymerase on purine-rich sequences drive nucleotide insertions characteristic of MBCSs, providing the first strong empirical evidence of RNA structure involvement in MBCS acquisition. Insertion frequencies at the H5 cleavage site were strongly affected by substitutions in flanking genomic regions altering predicted transient RNA structures. Introduction of H5-like cleavage site sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions never observed before in H6 viruses. Our results demonstrate that nucleotide insertions that underlie H5 HPAIV emergence result from a previously unknown RNA-structure-driven diversity-generating mechanism, which could be shared with other RNA viruses.

microbiology↗

bioRxiv2023

Asymmetrical Bi-antennary Glycans Prepared by a Stop-and-Go Strategy Reveal Receptor Binding Evolution of Human Influenza A Viruses

Glycan binding properties of respiratory viruses have been difficult to probe due to a lack of biological relevant glycans for binding studies. Here, a stop-and-go chemoenzymatic methodology is presented that gave access to a panel of 32 asymmetrical bi-antennary N-glycans having various numbers of N-acetyl lactosamine (LacNAc) repeating units capped by 2,3- or 2,6-sialosides resembling structures found in airway tissues. It exploits that the branching enzymes MGAT1 and MGAT2 can utilize unnatural UDP-2-deoxy-2-trifluoro-N-acetamido-glucose (UDP-GlcNTFA) as donor. The TFA moiety of the resulting glycans can be hydrolyzed to give GlcNH2 at one of the antennae that temporarily blocks extension by glycosyl transferases. The N-glycans were printed as a microarray that was probed for receptor binding specificities of evolutionary distinct human A(H3N2) and A(H1N1)pdm09 viruses. It was found that not only the sialoside type but also the length of the LacNAc chain and presentation at the 1,3-antenna of N-glycans is critical for binding. Early A(H3N2) viruses bound to 2,6-sialosides at a single LacNAc moiety at the 1,3-antenna whereas later viruses required the sialoside to be presented at a tri-LacNAc moiety. Surprisingly, most of the A(H3N2) viruses that appeared after 2021 regained binding capacity to sialosides presented at a di-LacNAc moiety. As a result, these viruses agglutinate erythrocytes again, commonly employed for antigenic characterization of influenza viruses. Human A(H1N1)pdm09 viruses have similar receptor binding properties as recent A(H3N2) viruses. The data indicates that an asymmetric N-glycan having 2,6-sialoside at a di-LacNAc moiety is a commonly employed receptor by human influenza A viruses.

biochemistry↗

bioRxiv2022

Strong phylogenetic and ecological effects on host competency for avian influenza in Australian wild birds

Host susceptibility to parasites is mediated by intrinsic and external factors such as genetics, age or season. While key features have been revealed for avian influenza A virus (AIV) in waterfowl of the Northern Hemisphere, the role of host phylogeny has received limited attention. Herein, we analysed 12339 oropharyngeal and cloacal swabs and 10826 serum samples collected over 11 years from wild birds in Australia. As well as describing species-level differences in prevalence and seroprevalence, we reveal that host phylogeny is a key driver in susceptibility. We confirm the role of age in AIV seroprevalence and viral prevalence. Seasonality effects appear less pronounced than in the Northern Hemisphere, while annual variations are potentially linked to El Nino- Southern Oscillation. Taken together, our study provides new insights into evolutionary ecology of AIV in its avian hosts, defining distinctive processes on the continent of Australia and expanding our understanding of AIV globally.

microbiology↗

bioRxiv2022

Mapping SARS-CoV-2 antigenic relationships and serological responses

During the SARS-CoV-2 pandemic, multiple variants escaping pre-existing immunity emerged, causing concerns about continued protection. Here, we use antigenic cartography to analyze patterns of cross-reactivity among a panel of 21 variants and 15 groups of human sera obtained following primary infection with 10 different variants or after mRNA-1273 or mRNA-1273.351 vaccination. We find antigenic differences among pre-Omicron variants caused by substitutions at spike protein positions 417, 452, 484, and 501. Quantifying changes in response breadth over time and with additional vaccine doses, our results show the largest increase between 4 weeks and >3 months post-2nd dose. We find changes in immunodominance of different spike regions depending on the variant an individual was first exposed to, with implications for variant risk assessment and vaccine strain selection. One sentence summaryAntigenic Cartography of SARS-CoV-2 variants reveals amino acid substitutions governing immune escape and immunodominance patterns.

immunology↗

bioRxiv2021

Evolutionary features of a prolific subtype of avian influenza A virus in European waterfowl

Avian influenza A virus (AIV) is ubiquitous in waterfowl, and detected annually at high prevalence in waterfowl during the Northern Hemipshere autumn. Some AIV subtypes are globally common in waterfowl, such as H3N8, H4N6, and H6N2, and are detected in the same populations at high frequency, annually. In order to investigate genetic features associated to the long-term maintenance of common subtypes in migratory ducks, we sequenced 248 H4 viruses isolated across 8 years (2002-2009) from Mallards (Anas platyrhynchos) sampled in southeast Sweden. Phylogenetic analyses showed that both H4 and N6 sequences fell into in three distinct lineages, structured by year of isolation. Specifically, across the eight years of the study, we observed lineage replacement, whereby a different HA lineage circulated in the population each year. Analysis of deduced amino acid sequences of the HA lineages illustrated key differences in regions of the globular head of hemagglutinin that overlap with established antigentic sites in homologous hemagglutinin H3, suggesting the possibility of antigenic differences among these HA lineages. Beyond HA, lineage replacement was common to all segments, such that novel genome constellations were detected across years. A dominant genome constellation would rapidly amplify in the duck population, followed by unlinking of gene segments as a result of reassortment within 2-3 weeks following introduction. These data help reveal the evolutionary dynamics exhibited by AIV on both annual and decadal scales in an important reservoir host.

evolutionary biology↗

bioRxiv2020

Insertions of codons encoding basic amino acids in H7 hemagglutinins of influenza A viruses occur by recombination with RNA at hotspots near snoRNA binding sites

The presence of multiple basic amino acids in the protease cleavage site of the hemagglutinin (HA) protein is the main molecular determinant of virulence of highly pathogenic avian influenza (HPAI) viruses. Recombination of HA RNA with other RNA molecules of host or virus origin is a dominant mechanism of multi basic cleavage site (MBCS) acquisition for H7 subtype HA. Using alignments of HA RNA sequences from documented cases of MBCS insertion due to recombination, we show that such recombination with host RNAs is most likely to occur at particular hotspots in ribosomal RNAs (rRNAs), transfer RNAs (tRNAs) and viral RNAs. The locations of these hotspots in highly abundant RNAs indicate that RNA recombination is facilitated by the binding of small nucleolar RNA (snoRNA) near the recombination points.

microbiology↗

bioRxiv2020

SARS-CoV-2 is transmitted via contact and via the air between ferrets.

SARS-CoV-2, a coronavirus that newly emerged in China in late 2019 1,2 and spread rapidly worldwide, caused the first witnessed pandemic sparked by a coronavirus. As the pandemic progresses, information about the modes of transmission of SARS-CoV-2 among humans is critical to apply appropriate infection control measures and to slow its spread. Here we show that SARS-CoV-2 is transmitted efficiently via direct contact and via the air (via respiratory droplets and/or aerosols) between ferrets. Intranasal inoculation of donor ferrets resulted in a productive upper respiratory tract infection and long-term shedding, up to 11 to 19 days post-inoculation. SARS-CoV-2 transmitted to four out of four direct contact ferrets between 1 and 3 days after exposure and via the air to three out of four independent indirect recipient ferrets between 3 and 7 days after exposure. The pattern of virus shedding in the direct contact and indirect recipient ferrets was similar to that of the inoculated ferrets and infectious virus was isolated from all positive animals, showing that ferrets were productively infected via either route. This study provides experimental evidence of robust transmission of SARS-CoV-2 via the air, supporting the implementation of community-level social distancing measures currently applied in many countries in the world and informing decisions on infection control measures in healthcare settings 3.

microbiology↗

bioRxiv2018

Epistatic interactions can moderate the antigenic effect of substitutions in hemagglutinin of influenza H3N2 virus

We previously showed that single amino acid substitutions at seven positions in hemagglutinin determined major antigenic change of influenza H3N2 virus. Here, the impact of two such substitutions was tested in eleven representative H3 hemagglutinins to investigate context-dependence effects. The antigenic effect of substitutions introduced at hemagglutinin position 145 was fully independent of the amino acid context of the representative hemagglutinins. Antigenic change caused by substitutions introduced at hemagglutinin position 155 was variable and context-dependent. Our results suggest that epistatic interactions with contextual amino acids in the hemagglutinin can moderate the magnitude of antigenic change.

microbiology↗

bioRxiv2018

Avian influenza viruses in wild birds: virus evolution in a multi-host ecosystem

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter or stage. The Republic of Georgia is located at the intersection of three migratory flyways: Central Asian Flyway, East Asian/East African Flyway and Black Sea/Mediterranean Flyway. For six consecutive years (2010-2016), we collected AIV samples from various duck and gull species that breed, migrate and overwinter in Georgia. We found substantial subtype diversity of viruses that varied in prevalence from year to year. Low pathogenic (LP)AIV subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, H16N3, plus two H5N5 and H5N8 highly pathogenic (HP)AIVs belonging to clade 2.3.4.4. Whole genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences for LPAIVs among different host species in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide diversity. Hemagglutinin clade 2.3.4.4 H5N8 viruses, circulated in Eurasia during 2014-2015 did not reassort, but analysis after its subsequent dissemination during 2016-2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions with maintenance of local AIV viruses in Georgia, whereas other lineages showed considerable genetic inter-relationship with viruses circulating in other parts of Eurasia and Africa, despite relative under-sampling in the area.\n\nImportanceWaterbirds (e.g., gulls/ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate dispersal of AIV at inter-continental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or re-assort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates of AIV evolutionary dynamics within a multi-host ecosystem at a stop-over site where three major migratory flyways intersect. Our analysis of this ecosystem over a six-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to both mitigating the risk of incursion into domestic poultry and potential risk to mammalian hosts including humans.

microbiology↗