Biology subjects

Dufloo, J.

Publications and source records attributed to Dufloo, J..

12 recordsLinked to original sources

bioRxiv2026

Anticipatory discovery of entry inhibitors against emerging viruses guided by viral phylogeny

Emerging viruses pose a major threat to global health, underscoring the need to improve antiviral preparedness. Here, we developed a phylogenetically-informed framework to identify entry inhibitors with potential activity against yet-to-emerge viruses. First, using pseudotypes displaying the receptor-binding proteins (RBPs) of 8 enveloped RNA viruses from different families, we screened a library of 2,320 FDA-approved compounds to identify candidate entry inhibitors. Hits were tested for potency, selectivity, consistency across cell types and pseudotyping vectors and, in some cases, were validated using authentic viruses. Then, to define the breadth of antiviral activity, 25 selected drugs were assayed against an expanded panel of 68 RBPs from 13 families, as well as against pairs of closely related RBPs. This revealed both narrow and broad-range inhibitors, including selective estrogen receptor modulators, alkaloids, aminoquinolines, and anidulafungin, which showed particularly broad activity. Importantly, RBPs from the same phylogenetic cluster frequently displayed correlated drug-sensitivity profiles, indicating that antiviral effects are predictable across closely related viruses. Our findings provide a proof of concept for anticipatory antiviral discovery, showing that phylogenetic relationships can guide the identification of entry inhibitors against potential future zoonotic threats.

microbiology↗

bioRxiv2026

Sialic acids are a barrier to the entry of non-influenza orthomyxoviruses

Sialic acids (SAs) are abundantly expressed on vertebrate cell surfaces and are widely recognized as key viral attachment factors, particularly for influenza viruses. However, their role remains understudied in other orthomyxoviruses, such as thogoto and quaranja viruses, which are tick-borne viruses sporadically infecting humans. Enzymatic removal of SAs increased the infectivity of Thogoto and Dhori viruses, as well as pseudotypes carrying the glycoproteins of Oz, Sinu, and Wellfleet Bay viruses. A similar effect on pseudotype infectivity was observed following the binding of specific lectins to SAs. These findings indicate that, in contrast to influenza viruses, SAs act as a barrier to the entry of these orthomyxoviruses. Experimental evolution of the Sinu and Wellfleet Bay virus glycoproteins revealed point mutations that partially overcame this barrier. Given the abundance of sialic acids in mucosal tissues, we speculate that SAs may contribute to the inability of thogoto and quaranjaviruses to transmit directly between vertebrate hosts. Our results also underscore the importance of monitoring the circulation of these viruses for potential changes in their transmission routes. Author summaryMany viruses, including influenza viruses, use sugar molecules called sialic acids (SAs) on the surface of host cells to promote viral entry. Here, we show that SAs can instead restrict infection for several lesser-known relatives of influenza virus that are primarily transmitted by ticks and can occasionally infect humans. Removal of SAs from human cells strongly increased entry of these non-influenza viruses, when using both non-replicative pseudotypes and authentic viruses. In addition, masking SAs also enhanced entry of viral pseudotypes. We further found that viral surface proteins can acquire mutations that partially reduce this restriction, indicating an ability by these viruses to adapt to SA-rich environments. Because SAs are abundant in vertebrate mucosal tissues such as the respiratory tract, our findings suggest a possible explanation for why these viruses do not transmit directly between vertebrate hosts and rely on tick bites for infection. More broadly, this work identifies a previously unrecognized host cell surface feature that limits viral entry and highlights the importance of monitoring viral evolution that could alter transmission potential.

microbiology↗

bioRxiv2025

Dipeptidase 1 is a functional receptor for coronavirus PHEV

Coronaviruses of the subgenus Embecovirus include several relevant pathogens such as the human seasonal coronaviruses HKU1 and OC43, bovine coronavirus, and porcine hemagglutinating encephalomyelitis virus (PHEV), among others. While sialic acid is thought to be required for embecovirus entry, protein receptors are unknown in most cases. Here we show that PHEV does not require sialic acid for entry and uses dipeptidase 1 (DPEP1) as a receptor. Cryo-electron microscopy revealed that PHEV, unlike other embecoviruses, samples open and closed conformations of its spike trimer at steady state. We found that the receptor binding domain (RBD) of the PHEV spike shares no detectable sequence homology or receptor usage with those of closely related viruses. In contrast, the X-ray structure of the RBD/DPEP1 complex showed that the elements involved in receptor binding are conserved across embecoviruses, revealing a striking versatility of the RBD to accommodate highly variable sequences that confer novel receptor specificities.

microbiology↗

bioRxiv2025

Identification and characterization of novel bat coronaviruses in Spain

The zoonotic transmission of bat coronaviruses poses a threat to human health. However, the diversity of bat-borne coronaviruses remains poorly characterized in many geographical areas. Here, we recovered six complete coronavirus genomes by performing a metagenomic analysis of fecal samples from hundreds of individual bats captured in Spain, a country with high bat species diversity. Three of these genomes corresponded to potentially novel coronavirus species belonging to the alphacoronavirus genus. Phylogenetic analyses revealed that some of these viruses are closely related to coronaviruses previously described in bats from other countries, suggesting the existence of a shared viral reservoir worldwide. Using viral pseudotypes, we investigated the receptor usage of the identified viruses and found that one of them can use human and bat ACE2, highlighting its zoonotic potential. However, the receptor usage of the other viruses remains unknown. This study broadens our understanding of coronavirus diversity and identifies research priorities for the prevention of zoonotic viral outbreaks. Author summaryBats carry many viruses, some of which can cross the species barrier and infect humans, a process known as zoonosis. In particular, bat-borne coronaviruses pose a significant threat to human health. To improve our pandemic preparedness, it is essential to characterize the diversity and zoonotic potential of bat coronaviruses. However, such research efforts have historically suffered from a strong geographical bias. For example, despite the rich bat diversity in Spain, few studies have searched for coronaviruses in Iberian bats. Here, we used viral metagenomics to test for the presence of coronaviruses in more than 200 bat samples collected across Spain. We detected six complete coronaviruses, three of which were proposed to be new viral species. We characterized their relationship to previously identified viruses and their ability to use known coronavirus receptors to enter cells, demonstrating that one virus could use human ACE2 as a receptor. Our results highlight the diversity of bat-borne coronaviruses in Spain, their zoonotic potential, and the need to better characterize coronavirus diversity worldwide.

microbiology↗

bioRxiv2024

Human cell adaptation of the swine acute diarrhea syndrome coronavirus spike protein

Swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently identified highly pathogenic swine coronavirus. In vitro, SADS-CoV can infect cell lines from many different species, including humans, highlighting its high zoonotic potential. Coronavirus spike glycoproteins play a critical role in viral entry and are involved in determining viral host range and cellular tropism. Here, we used experimental evolution to investigate how the SADS-CoV spike protein adapts to human cells and to identify potential variants with increased infectivity. We evolved a recombinant vesicular stomatitis virus expressing the SADS-CoV spike (rVSV-SADS) in three human cell lines. After ten passages, increased viral replication was observed, and spike mutations were identified by sequencing. Mutations were functionally characterized in terms of viral fitness, spike processing and fusogenicity. Our results thus identify potential human-adaptive mutations in the SADS-CoV spike that may further enhance its zoonotic potential. ImportanceCoronavirus transmission from animals represents a serious threat to humans. Pigs are of particular concern because of their proximity to humans and the several coronaviruses they harbor. In particular, the swine acute diarrhea syndrome coronavirus (SADS-CoV) is a recently identified highly pathogenic porcine coronavirus that has a very broad tropism in vitro, highlighting its high zoonotic risk. The coronavirus spike protein is a strong determinant of species tropism, and spike mutations may facilitate cross-species transmission. Here, to identify potential variants with increased ability to enter human cells, we used an experimental evolution approach to study how the SADS-CoV spike adapts to different human cell lines. These mutations, should they occur in nature, could potentially increase the zoonotic potential of SADS-CoV.

microbiology↗

bioRxiv2024

Viral entry is a weak barrier to zoonosis

Recent advances in viral metagenomics have led to the discovery of many mammalian viruses, but experimental tests to determine whether they pose a threat to humans are largely lacking. A first step for a virus to cross the species barrier is to penetrate host cells. Here, we use gene synthesis and viral pseudotyping to experimentally test the ability of viral receptor-binding proteins (RBPs) from >100 enveloped RNA viruses to mediate entry into human cells. Analysis of thousands of RBP-cell pairs demonstrated such ability for most viruses, with significant variation among the 14 viral families studied. Comparison of RBP-mediated infectivity with cellular gene expression data showed that viral entry is often not limited by the presence of a receptor and revealed the contribution of additional host factors. Our results prove the weakness of interspecies barriers at the early stages of infection and identify molecular interactions that enable viral zoonosis.

microbiology↗

bioRxiv2023

Temperature impacts SARS-CoV-2 spike fusogenicity and evolution

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33{degrees}C and 37{degrees}C, respectively). In addition, fever is a common COVID-19 symptom. SARS-CoV-2 has been shown to replicate more efficiently at low temperatures but the effect of temperature on different viral proteins remains poorly understood. Here, we investigate how temperature affects the SARS-CoV-2 spike function and evolution. We first observed that rising temperature from 33{degrees}C to 37{degrees}C or 39{degrees}C increased spike-mediated cell-cell fusion. We then experimentally evolved a recombinant vesicular stomatitis virus expressing the SARS-CoV-2 spike at these different temperatures. We found that spike-mediated cell-cell fusion was maintained during evolution at 39{degrees}C, but was lost in a high proportion of viruses evolved at 33{degrees}C or 37{degrees}C. Consistently, sequencing of the spikes evolved at 33{degrees}C or 37{degrees}C revealed the accumulation of mutations around the furin cleavage site, a region that determines cell-cell fusion, whereas this did not occur in spikes evolved at 39{degrees}C. Finally, using site-directed mutagenesis, we found that disruption of the furin cleavage site had a temperature-dependent effect on spike-induced cell-cell fusion and viral fitness. Our results suggest that variations in body temperature may affect the activity and diversification of the SARS-CoV-2 spike. ImportanceWhen it infects humans, SARS-CoV-2 is exposed to different temperaures (e.g. replication site, fever...). Temperature has been shown to strongly impact SARS-CoV-2 replication but how it affects the activity and evolution of the spike protein remains poorly understood. Here, we first show that high temperatures increase the SARS-CoV-2 spike fusogenicity. Then, we demonstrate that the evolution of the spike activity and variants depends on temperature. Finally, we show that the functional effect of specific spike mutations is temperature-dependent. Overall, our results suggest that temperature may be a factor influencing the activity and adapatation of the SARS-CoV-2 spike in vivo, which will help understanding viral tropism, pathogenesis, and evolution.

microbiology↗

bioRxiv2023

Cell type-specific adaptation of the SARS-CoV-2 spike

SARS-CoV-2 can infect various human tissues and cell types, principally via interaction with its cognate receptor ACE2. However, how the virus evolves in different cellular environments is poorly understood. Here, we used experimental evolution to study the adaptation of the SARS-CoV-2 spike to four human cell lines expressing different levels of key entry factors. After 20 passages, cell type-specific phenotypic changes were observed. Selected spike mutations were identified and functionally characterized in terms of entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, entry pathway and syncytia formation. We found that the effects of these mutations varied across cell types. Interestingly, two spike mutations (L48S and A372T) that emerged in cells expressing low ACE2 levels increased receptor affinity, syncytia induction, and entry efficiency under low-ACE2 conditions. Our results demonstrate specific adaptation of the SARS-CoV-2 spike to different cell types and have implications for understanding SARS-CoV-2 tissue tropism and evolution.

microbiology↗

bioRxiv2023

Sex and species associated differences in Complement-mediated immunity in Humans and Rhesus macaques

The complement system can be viewed as a moderator of innate immunity, instructor of humoral immunity, and regulator of adaptive immunity. While sex and aging are known to affect humoral and cellular immune systems, their impact on the complement pathway in humans and rhesus macaques, a commonly used non-human primate model system, have not been well-studied. To address this knowledge gap, we analyzed serum samples from 90 humans and 75 rhesus macaques for the abundance and activity of the complement system components. While sequences of cascade proteins were highly conserved, dramatically different levels were observed between species. Whereas the low levels detected in rhesus samples raised questions about the suitability of the test, differences in levels of complement proteins were observed in male and female humans. Levels of total and antibody-dependent deposition of C1q and C3b on a glycosylated antigen differed between human and rhesus, suggesting differential recognition of glycans. Functional differences in complement-mediated lysis of antibody-sensitized cells were observed in multiple assays and showed that human females frequently exhibited higher lytic activity than human males or rhesus macaques, which typically did not exhibit such sexual dimorphism. Other differences between species and sexes were observed in more narrow contexts--for only certain antibodies, antigens, or assays. Collectively, these results expand our knowledge of sexual dimorphism in the complement system in humans, identifying differences that appear to be absent from rhesus macaques.

immunology↗

bioRxiv2022

Resistance of Omicron subvariants BA.2.75.2, BA.4.6 and BQ.1.1 to neutralizing antibodies

Convergent evolution of SARS-CoV-2 Omicron BA.2, BA.4 and BA.5 lineages has led to the emergence of several new subvariants, including BA.2.75.2, BA.4.6. and BQ.1.1. The subvariants BA.2.75.2 and BQ.1.1 are expected to become predominant in many countries in November 2022. They carry an additional and often redundant set of mutations in the spike, likely responsible for increased transmissibility and immune evasion. Here, we established a viral amplification procedure to easily isolate Omicron strains. We examined their sensitivity to 6 therapeutic monoclonal antibodies (mAbs) and to 72 sera from Pfizer BNT162b2-vaccinated individuals, with or without BA.1/BA.2 or BA.5 breakthrough infection. Ronapreve (Casirivimab and Imdevimab) and Evusheld (Cilgavimab and Tixagevimab) lost any antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remained weakly active. BQ.1.1 was also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals were low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increased these titers, which remained about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increased more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitated their spread in immunized populations and raises concerns about the efficacy of most currently available mAbs.

microbiology↗

bioRxiv2022

Potent Human Broadly SARS-CoV-2 Neutralizing IgA and IgG Antibodies Effective Against Omicron BA.1 and BA.2

Memory B-cell and antibody responses to the SARS-CoV-2 spike protein contribute to long-term immune protection against severe COVID-19, which can also be prevented by antibody-based interventions. Here, wide SARS-CoV-2 immunoprofiling in COVID-19 convalescents combining serological, cellular and monoclonal antibody explorations, revealed humoral immunity coordination. Detailed characterization of a hundred SARS-CoV-2 spike memory B-cell monoclonal antibodies uncovered diversity in their repertoire and antiviral functions. The latter were influenced by the targeted spike region with strong Fc-dependent effectors to the S2 subunit and potent neutralizers to the receptor binding domain. Amongst those, Cv2.1169 and Cv2.3194 antibodies cross-neutralized SARS-CoV-2 variants of concern including Omicron BA.1 and BA.2. Cv2.1169, isolated from a mucosa-derived IgA memory B cell, demonstrated potency boost as IgA dimers and therapeutic efficacy as IgG antibodies in animal models. Structural data provided mechanistic clues to Cv2.1169 potency and breadth. Thus, potent broadly neutralizing IgA antibodies elicited in mucosal tissues can stem SARS-CoV-2 infection, and Cv2.1169 and Cv2.3194 are prime candidates for COVID-19 prevention and treatment.

immunology↗

bioRxiv2020

Syncytia formation by SARS-CoV-2 infected cells

Severe cases of COVID-19 are associated with extensive lung damage and the presence of infected multinucleated syncytial pneumocytes. The viral and cellular mechanisms regulating the formation of these syncytia are not well understood. Here, we show that SARS-CoV-2 infected cells express the viral Spike protein (S) at their surface and fuse with ACE2-positive neighbouring cells. Expression of S without any other viral proteins triggers syncytia formation. Type-I interferon (IFN)-induced transmembrane proteins (IFITMs), a family of restriction factors that block the entry of many viruses, inhibit S-mediated fusion, with IFITM1 being more active than IFITM2 and IFITM3. On the contrary, the TMPRSS2 serine protease, which is known to enhance infectivity of cell-free virions, processes both S and ACE2 and increases syncytia formation by accelerating the fusion process. TMPRSS2 thwarts the antiviral effect of IFITMs. Our results show that the pathological effects of SARS-CoV-2 are modulated by cellular proteins that either inhibit or facilitate syncytia formation. One Sentence SummarySyncytia produced by SARS-CoV-2 infected cells and regulation of their formation by IFITMs and TMPRSS2.

microbiology↗