Biology subjects

Bestebroer, T. M.

Publications and source records attributed to Bestebroer, T. M..

12 recordsLinked to original sources

bioRxiv2025

Mass mortality at penguin mega-colonies due to avian cholera confounds H5N1 HPAIV surveillance in Antarctica

In the austral summer 2023/2024, H5N1 high pathogenicity avian influenza virus (HPAIV) was reported for the first time in Antarctica. Concerns of HPAIV causing high mortality of seabirds and mammals prompted immediate efforts to track its spread and impact on endemic wildlife. In March 2024, we visited the Danger Islands archipelago, that hosts two mega-colonies of Adelie penguins, and observed an unusual mortality estimated in thousands of Adelie penguins and other species. Swabs and tissues were collected for molecular detection of infectious agents from 49 carcasses, and additional tissues for histology from a selection of 9 carcasses. We unexpectedly detected Pasteurella multocida DNA in 46 of 49 individuals, and diagnosed avian cholera, and not HPAI, as the cause of death of most of these animals. By metagenomics, we retrieved the genomic sequences of the Pasteurella multocida strain which caused the epizootic, and the phylogenetic analysis showed a close relation with strains previously reported in the Southern Ocean area. This study confirms avian cholera as a relevant cause of mortality in the Antarctic region, and overall highlights the importance of considering avian cholera in the differential diagnoses during mortality events in Antarctica, even with the concurrent circulation of HPAIV.

microbiology↗

bioRxiv2025

Enhanced human antigen-specific B cell responses using in vitro 3D tonsil cultures containing stromal cells

The structural complexity of secondary lymphoid organs (SLOs) and their role in shaping antigen-specific B cell responses, pose significant challenges in modeling human germinal center (GC) response in vitro. A human 3D lymphoid model incorporating lymphoid and stromal cell types recapitulates key immune and structural features, enabling the study of antigen-specific B and T cell interactions beyond current 2D culture limitations. In this study, human tonsil cells were cultured with and without tonsil-derived fibroblastic reticular cells (FRCs) either in 2D or within a 3D PEG-4MAL hydrogel culture. Antigen-specific B cell responses in co-cultures were studied by comparing unstimulated cultures to stimulation with antigen (SARS-CoV-2 spike (S) or Influenza hemagglutinin (HA), both with or without adjuvant R848), S-nanoparticles and influenza vaccines. Combination of FRCs with the 3D matrix significantly improved B and T cell survival and facilitated reaggregation into follicle-like structures. Antigen-specific responses were most pronounced in 3D FRC-supported co-cultures, with increasing S- or HA-specific B cell frequencies, antibody-secreting cell differentiation, and secretion of antigen-specific antibodies. Importantly, cell death and unspecific bystander activation was lowest in 3D FRC-supported cultures. Additionally, GC-associated chemokine receptors CXCR4 and CXCR5 showed distinct expression patterns on CD27CD38 B cells, reflecting GC-like dark and light zone organization typically observed in SLOs in vivo. Autologous and allogeneic FRC-supported cultures yielded comparable results, demonstrating the platforms potential for high-throughput applications. The 3D FRC-supported lymphoid cultures offer a physiologically relevant platform for studying human GC responses in vitro, supporting mechanistic research into adaptive immunity and enabling the screening of vaccine immunogens and adjuvants in a controlled setting.

immunology↗

bioRxiv2025

The expanding avian influenza panzootic: skua die-off in Antarctica

High pathogenicity avian influenza virus of subtype H5 (H5 HPAIV), clade 2.3.4.4b, invaded Antarctica in 2023. Here we show that H5 HPAIV caused high mortality in a breeding colony of skuas at one of ten sites we visited in March 2024. By combined virological and pathological analyses, we found that H5 HPAIV caused multi-organ necrosis and rapid death in skuas. Taken together with recent data, skuas in Antarctica are at risk of continued mortality from H5 HPAIV infection, threatening their already small populations. Conversely, because of their wide distribution and ecological relevance, skuas may play a substantial role in spread of the virus across Antarctica. Transdisciplinary surveillance is needed in coming years to monitor the impact of this poultry-origin disease on Antarcticas unique wildlife.

microbiology↗

bioRxiv2025

Mapping the complete influenza A virus infection cycle through single vRNP imaging

Cell-to-cell heterogeneity is a common feature of viral infection that can generate enormous complexity, complicating understanding of infection progression and interpretation of differences between viral variants. To overcome these challenges, we developed a technology to visualize the infection cycle of human influenza A virus (IAV) from start-to-finish in individual living cells with unprecedented spatial and temporal resolution, which identified numerous distinct pathways through which infection can progress. We show that heterogeneous viral gene expression drives infection cycle heterogeneity, and identified genome packaging, vRNP transcriptional activation and host cell division as major determinants of gene expression variability. Our work maps out the complete IAV infection cycle, identifies the origin of infection heterogeneity and provides a broadly-applicable technology for studying IAV and other viruses.

cell biology↗

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↗

bioRxiv2020

Glycan remodeled erythrocytes facilitate antigenic characterizationof recent A/H3N2 influenza viruses

During circulation in humans and natural selection to escape antibody recognition for decades, A/H3N2 influenza viruses emerged with altered receptor specificities. These viruses lost the ability to agglutinate erythrocytes critical for antigenic characterization and give low yields and acquire adaptive mutations when cultured in eggs and cells, contributing to recent vaccine challenges. We examined receptor specificities of A/H3N2 viruses, revealing that recent viruses compensated for decreased binding of the prototypic human receptor by recognizing 2,6-sialosides on extended LacNAc moieties. Erythrocyte glycomics showed an absence of extended glycans, providing a rationale for lack of agglutination by recent A/H3N2 viruses. A glycan remodeling approach installed functional receptors on erythrocytes, allowing antigenic characterization of recent A/H3N2 viruses and confirming the cocirculation of several antigenically different viruses in humans. Computational studies of HAs in complex with a sialoside having an extended LacNAc moiety revealed that mutations distal to the RBD reoriented the Y159 side chain, resulting in an extended receptor binding site.

biochemistry↗

bioRxiv2020

Comparison of three air samplers for the collection of four nebulized respiratory viruses

Viral respiratory tract infections are a leading cause of morbidity and mortality worldwide. Unfortunately, the transmission routes and shedding kinetics of respiratory viruses remain poorly understood. Air sampling techniques to quantify infectious viruses in the air are indispensable to improve intervention strategies to control and prevent spreading of respiratory viruses. Here, the collection of infectious virus with the six-stage Andersen cascade impactor was optimized with semi-solid gelatin as collection surface. Subsequently, the collection efficiency of the cascade impactor, the SKC BioSampler, and an in-house developed electrostatic precipitator was compared. In an in-vitro setup, influenza A virus, human metapneumovirus, parainfluenza virus type 3 and respiratory syncytial virus were nebulized and the amount of collected infectious virus and viral RNA was quantified with each air sampler. Whereas only low amounts of virus were collected using the electrostatic precipitator, high amounts were collected with the BioSampler and cascade impactor. The BioSampler allowed straight-forward sampling in liquid medium, whereas the more laborious cascade impactor allowed size fractionation of virus-containing particles. Depending on the research question, either the BioSampler or the cascade impactor can be applied in laboratory and field settings, such as hospitals to gain more insight into the transmission routes of respiratory viruses. Practical ImplicationsRespiratory viruses pose a continuous health threat, especially to vulnerable groups such as young children, immunocompromised individuals and the elderly. It is important to understand via which routes these viruses can transmit to and between individuals that are at risk. If we can determine the amount of a certain respiratory virus in the air, then this will help to predict the importance of transmission through the air for this virus. Most currently available air sampling devices have not been designed to collect infectious viruses from the air. Therefore, we here optimized and compared the performance of three air samplers for four different respiratory viruses.

microbiology↗

bioRxiv2020

SARS-CoV and SARS-CoV-2 are transmitted through the air between ferrets over more than one meter distance

SARS-CoV-2 emerged in late 2019 and caused a pandemic, whereas the closely related SARS-CoV was contained rapidly in 2003. Here, a newly developed experimental set-up was used to study transmission of SARS-CoV and SARS-CoV-2 through the air between ferrets over more than a meter distance. Both viruses caused a robust productive respiratory tract infection resulting in transmission of SARS-CoV-2 to two of four indirect recipient ferrets and SARS-CoV to all four. A control pandemic A/H1N1 influenza virus also transmitted efficiently. Serological assays confirmed all virus transmission events. Although the experiments did not discriminate between transmission via small aerosols, large droplets and fomites, these results demonstrate that SARS-CoV and SARS-CoV-2 can remain infectious while travelling through the air. Efficient virus transmission between ferrets is in agreement with frequent SARS-CoV-2 outbreaks in mink farms. Although the evidence for airborne virus transmission between humans under natural conditions is absent or weak for SARS-CoV and SARS-CoV-2, ferrets may represent a sensitive model to study interventions aimed at preventing virus transmission.

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↗