Biology subjects

Herfst, S.

Publications and source records attributed to Herfst, S..

15 recordsLinked to original sources

bioRxiv2026

Andes virus from the MV Hondius outbreak is highly pathogenic in hamsters and airborne shed

In May 2026, an outbreak of hantavirus pulmonary syndrome (HPS) caused by Andes virus (ANDV) was detected aboard the cruise ship MV Hondius, sparking public health measures in multiple countries. ANDV infections in humans are mostly sporadic, most often linked to exposure to infected rodents in South America, although small clusters of human cases, like the 2026 outbreak on the MV Hondius occur as well. It is not known whether virological factors may contribute to this distinct epidemiology, and no phenotypic characterisation of the newly emerged ANDV (ANDV2026) has been carried out to date. Here, using an ANDV isolate from a presymptomatic patient from the MV Hondius, we studied pathogenesis, identified sites of viral replication, characterized innate and adaptive immune responses, and quantified shedding in golden Syrian hamsters. ANDV2026 proved to be highly pathogenic in hamsters, causing severe pulmonary disease reminiscent of HPS. ANDV2026 triggered a delayed type-I IFN response and inflammatory response in the lungs consistent with the key pathological features of HPS, and we detected viral replication in both respiratory and extra-respiratory tissues, including the brain. Infected animals mounted neutralising antibodies and T cell responses, the latter directed predominantly against the L and N proteins. Importantly, ANDV2026-infected animals shed virus through multiple routes, and we detected infectious virus in air samplers connected to cages housing ANDV2026-infected hamsters. In contrast, control hamsters infected with a rodent-derived ANDV isolate (ANDV9717869) shed little virus and air samples were consistently negative. These findings consolidate established features of ANDV pathogenesis, while revealing properties not previously described. Phenotypic characterisation of patient-derived ANDV isolates in animal models can strengthen outbreak preparedness, identify markers of disease severity, and support the development of medical countermeasures.

microbiology↗

bioRxiv2024

Structural and immunological characterization of the H3 influenza hemagglutinin during antigenic drift

The quest for a universal influenza vaccine holds great promise for mitigating the global burden of influenza-related morbidity and mortality. However, challenges persist in identifying conserved epitopes capable of inducing protection. In this study, we explore the influence of glycan evolution on H3 hemagglutinin from 1968 to present day and its impacts on antigenicity and immunogenicity. We observe that the appearance of potential N-linked glycosylation sites in Sing/16 hemagglutinin head domain reduces the binding of broadly neutralizing antibodies and shifts the polyclonal immune response upon vaccination to target the stem. Furthermore, structural characterization of HK/68 and Sing/16 by cryo-electron microscopy shows that while HK/68 is resistant to enzymatic deglycosylation, removal of glycans destabilizes the hyperglycosylated head and membrane-proximal region in Sing/16. These insights expand our understanding of glycans beyond their role in protein folding and highlight the interplay among glycan integration and immune recognition to design a universal influenza vaccine.

immunology↗

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↗

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

Contemporary human H3N2 influenza A viruses require a low threshold of suitable glycan receptors for efficient infection

Recent human H3N2 influenza A viruses (IAV) have evolved to employ elongated glycans terminating in 2,6-linked sialic acid as their receptors. These glycans are displayed in low abundancies by cells commonly employed to propagate these viruses (MDCK and hCK), resulting in low or no viral propagation. Here, we examined whether the overexpression of the glycosyltransferases B3GNT2 and B4GALT1, which are responsible for the elongation of poly-N-acetyllactosamines (LacNAc), would result in improved A/H3N2 propagation. Stable overexpression of B3GNT2 and B4GALT1 in MDCK and hCK cells was achieved by lentiviral integration and subsequent antibiotic selection and confirmed by qPCR and protein mass spectrometry experiments. Flow cytometry and glycan mass spectrometry experiments using the B3GNT2 and/or B4GALT1 knock-in cells demonstrated increased binding of viral hemagglutinins and the presence of a larger number of LacNAc repeating units, especially on hCK-B3GNT2 cells. An increase in the number of glycan receptors did, however, not result in a greater infection efficiency of recent human H3N2 viruses. Based on these results, we propose that H3N2 IAVs require a low number of suitable glycan receptors to infect cells and that an increase in the glycan receptor display above this threshold does not result in improved infection efficiency.

microbiology↗

bioRxiv2022

Robustness of the ferret model for influenza risk assessment studies: a cross-laboratory exercise

Ferrets represent the preferred animal model for assessing the transmission potential of newly emerged zoonotic influenza viruses. However, heterogeneity among established experimental protocols and facilities across different laboratories may lead to variable results, complicating interpretation of transmission experimental data. Between 2018-2020, a global exercise was conducted by 11 participating laboratories to assess the range of variation in ferret transmission experiments using two common stock H1N1 influenza viruses that possess different transmission characteristics in ferrets. Inoculation route, dose, and volume were standardized, and all participating laboratories followed the same experimental conditions for respiratory droplet transmission, including a strict 1:1 donor:contact ratio. Additional host and environmental parameters likely to affect influenza transmission kinetics were monitored throughout. Overall transmission outcomes for both viruses across 11 laboratories were concordant, suggesting the robustness of the ferret model for zoonotic influenza risk assessment. To attain high confidence in identifying zoonotic influenza viruses with moderate-to-high or low transmissibility, our analyses support that as few as three but as many as five laboratories, respectively, would need to independently perform viral transmission experiments with concordant results. This exercise facilitates the development of a more homogenous protocol for ferret transmission experiments that are employed for the purposes of risk assessment.

microbiology↗

bioRxiv2021

Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D63N, D81N and A144G promoted virus replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

microbiology↗

bioRxiv2021

3D visualization of SARS-CoV-2 infection and receptor distribution in Syrian hamster lung lobes display distinct spatial arrangements

SARS-CoV-2 attaches to angiotensin-converting enzyme 2 (ACE2) to gain entry into cells after which the spike protein is cleaved by the transmembrane serine protease 2 (TMPRRS2) to facilitate viral-host membrane fusion. ACE2 and TMPRRS2 expression profiles have been analyzed at the genomic, transcriptomic, and single-cell RNAseq level, however, biologically relevant protein receptor organization in whole tissues is still poorly understood. To describe the organ-level architecture of receptor expression, related to the ability of ACE2 and TMPRRS2 to mediate infectivity, we performed a volumetric analysis of whole Syrian hamster lung lobes. Lung tissue of infected and control animals were stained using antibodies against ACE2 and TMPRRS2, combined with fluorescent spike protein and SARS-CoV-2 nucleoprotein staining. This was followed by light-sheet microscopy imaging to visualize expression patterns. The data demonstrates that infection is restricted to sites with both ACE2 and TMPRRS2, the latter is expressed in the primary and secondary bronchi whereas ACE2 is predominantly observed in the terminal bronchioles and alveoli. Conversely, infection completely overlaps at these sites where ACE2 and TMPRSS2 co-localize. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/435771v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@150fc65org.highwire.dtl.DTLVardef@1ea6a56org.highwire.dtl.DTLVardef@eb3c43org.highwire.dtl.DTLVardef@1c1877e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Multimerization- and glycosylation-dependent receptor binding of SARS-CoV-2 spike proteins

Receptor binding studies using recombinant SARS-CoV proteins have been hampered due to challenges in approaches creating spike protein or domains thereof, that recapitulate receptor binding properties of native viruses. We hypothesized that trimeric RBD proteins would be suitable candidates to study receptor binding properties of SARS-CoV-1 and -2. Here we created monomeric and trimeric fluorescent RBD proteins, derived from adherent HEK293T, as well as in GnTI mutant cells, to analyze the effect of complex vs high mannose glycosylation on receptor binding. The results demonstrate that trimeric fully glycosylated proteins are superior in receptor binding compared to monomeric and immaturely glycosylated variants. Although differences in binding to commonly used cell lines were minimal between the different RBD preparations, substantial differences were observed when respiratory tissues of experimental animals were stained. The RBD trimers demonstrated distinct ACE2 expression profiles in bronchiolar ducts and confirmed the higher binding affinity of SARS-CoV-2 over SARS-CoV-1. Our results show that fully glycosylated trimeric RBD proteins are attractive to analyze receptor binding and explore ACE2 expression profiles in tissues.

molecular biology↗

bioRxiv2020

SARS-CoV-2 neutralizing human antibodies protect against lower respiratory tract disease in a hamster model

Effective clinical intervention strategies for COVID-19 are urgently needed. Although several clinical trials have evaluated the use of convalescent plasma containing virus-neutralizing antibodies, the effectiveness has not been proven. We show that hamsters treated with a high dose of human convalescent plasma or a monoclonal antibody were protected against weight loss showing reduced pneumonia and pulmonary virus replication compared to control animals. However, a ten-fold lower dose of convalescent plasma showed no protective effect. Thus, variable and relatively low levels of virus neutralizing antibodies in convalescent plasma may limit their use for effective antiviral therapy, favouring concentrated, purified (monoclonal) antibodies.

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↗

bioRxiv2020

Comparative Pathogenesis Of COVID-19, MERS And SARS In A Non-Human Primate Model

A novel coronavirus, SARS-CoV-2, was recently identified in patients with an acute respiratory syndrome, COVID-19. To compare its pathogenesis with that of previously emerging coronaviruses, we inoculated cynomolgus macaques with SARS-CoV-2 or MERS-CoV and compared with historical SARS-CoV infections. In SARS-CoV-2-infected macaques, virus was excreted from nose and throat in absence of clinical signs, and detected in type I and II pneumocytes in foci of diffuse alveolar damage and mucous glands of the nasal cavity. In SARS-CoV-infection, lung lesions were typically more severe, while they were milder in MERS-CoV infection, where virus was detected mainly in type II pneumocytes. These data show that SARS-CoV-2 can cause a COVID-19-like disease, and suggest that the severity of SARS-CoV-2 infection is intermediate between that of SARS-CoV and MERS-CoV. One Sentence SummarySARS-CoV-2 infection in macaques results in COVID-19-like disease with prolonged virus excretion from nose and throat in absence of clinical signs.

microbiology↗