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Sanjuan, R.

Publications and source records attributed to Sanjuan, R..

At least 19 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

Phenotypic Heterogeneity Shapes Phage Resistance and Cocktail Efficacy in Klebsiella pneumoniae

The emergence of phage-resistant bacteria poses a significant challenge to the success of phage therapy. Although phage cocktails can delay resistance, their efficacy relies on the ability to target the full spectrum of resistant variants, which are often more diverse than that represented by clonal isolates. In this study, we investigated how phenotypic diversity within phage-resistant Klebsiella pneumoniae influences susceptibility to newly isolated phages and the effectiveness of phage cocktails. We isolated phages from three cultures resistant to a capsule-dependent phage: a heterogeneous acapsular population, an acapsular mutant with a stable phenotype, and a capsule-reverted isolate that regained capsule expression upon removal of phage pressure. These phages differed in host tropism and in their capacity to delay resistance emergence when combined with the capsule-dependent phage. Phages targeting acapsular variants, particularly those isolated from the heterogeneous population, were the most effective, exhibiting strong synergy. Single-cell analyses further revealed that sustained selective pressure from the capsule-dependent phage prevents capsule reversion and maintains cocktail efficacy. Overall, our results highlight the importance of accounting for phenotypic heterogeneity when designing phage therapies and support population-level approaches for optimizing phage cocktail composition. ImportancePhage therapy is a promising alternative to antibiotics, but its success is often limited by the rapid emergence of phage-resistant bacteria. These resistant populations can be highly heterogeneous, comprising both stable mutants and variants with reversible, non-genetic resistance. In this study, we explore how this phenotypic diversity influences the effectiveness of phage cocktails. By isolating new phages and testing them in combination, we demonstrate that the selective pressure exerted by specific phages can prevent the reversion in transiently resistant variants, thereby sustaining treatment efficacy. Our findings highlight the need to consider not only the range of bacterial targets but also how phage pressure shapes bacterial population dynamics. This work offers a more refined strategy for designing phage cocktails with improved clinical potential.

microbiology↗
bioRxiv2025

Atypical Replication Error Pattern and Limited Repair Efficiency Contribute to Elevated Mutation Rate in Phage lambda

Understanding the rate and nature of spontaneous mutations is crucial for understanding and modulating the pace and trajectory of evolution. Yet, both remain poorly characterized in double-stranded DNA (dsDNA) phages, despite their relevance for phage-based therapies. Here, we address this gap for the dsDNA phage lambda, using four complementary approaches: mutation accumulation assay combined with whole-genome sequencing, mutation visualization assay, duplex sequencing and fluctuation assay. We find that the mutation rate of wild-type phage lambda is 4.9 {+/-} 1.8 x10-9 per base per replication, approximately 15 times lower than previously estimated and about 20 times higher than that of its host, Escherichia coli (E. coli). Inactivation of Mismatch Repair (MMR), a major conserved cellular system for mutation avoidance, increases the lambda mutation rate by only 2-to 10-fold, in contrast to the approximately 150-fold increase in E. coli, however lambda does not exhibit the characteristic mutational bias associated with MMR deficiency. Interestingly replication of the lambda genome generates an error spectrum distinct from that of E. coli, characterized by a marked increase in transversions, poorly repaired by MMR. Together, these results reveal that lambda exhibits a replication error profile that is less amenable to repair, likely contributing to its elevated mutation rate.

microbiology↗
bioRxiv2025

Phenotypic resistance to phage infection through capsule expression shutdown in Klebsiella pneumoniae

Capsule loss is a major mechanism by which bacteria evade phage infection. This process has traditionally been attributed to mutations in capsule biosynthesis genes. Here, we investigated phage resistance in Klebsiella pneumoniae, a medically relevant encapsulated bacterium. Phage infection rapidly selected for resistant acapsular cells. As expected, transcriptomic analysis revealed a marked downregulation of capsule biosynthesis genes. However, full genome sequencing showed that capsule loss occurred without evidence of mutations, and acapsular phage-resistant cells were able to rapidly restore their capsule once phage pressure was removed. These findings highlight that phage-driven selective pressure can act on non-heritable variation in gene expression, providing a faster and more flexible resistance mechanism compared to the traditional mutation-selection process. IMPORTANCEBacteriophages are increasingly considered as alternatives or complements to antibiotics, particularly against multidrug-resistant pathogens like Klebsiella pneumoniae. A key barrier to effective phage therapy is the rapid emergence of bacterial resistance. Capsule loss is a common resistance mechanism, traditionally linked to genetic mutations. Here, we show that K. pneumoniae can evade phage infection through reversible, non-mutational downregulation of capsule biosynthesis. This phenotypic adaptation allows rapid resistance development without compromising long-term fitness. Our findings reveal a flexible, non-genetic resistance strategy that may limit the durability of phage therapy and should be considered in future phage treatment designs.

microbiology↗
bioRxiv2025

Rodents as Potential Reservoirs for Toroviruses

Emerging zoonotic viruses pose a significant threat to global health. The order Nidovirales includes diverse viruses, such as coronaviruses, which are well known for their zoonotic potential. Toroviruses are a less-studied genus within Nidovirales primarily associated with gastrointestinal diseases in ungulates, although some evidence suggests their presence in humans. In this study, we report the discovery of a novel torovirus from a fecal sample of a dormouse (Eliomys quercinus) in Spain, which we named Dormouse torovirus (DToV). This represents the first complete genome of a rodent-associated torovirus. The 28,555-nucleotide genome encodes the six characteristic torovirus open reading frames, but these exhibit low amino acid sequence identity (44.3-86.3%) compared to other toroviruses, indicating that DToV likely represents a new viral species. Bayesian analysis of the ORF1b suggests that DToV diverged from known toroviruses approximately 1300 years ago. Moreover, the basal phylogenetic position of DToV suggests that rodents may represent a reservoir for this viral genus. Our findings expand the known torovirus host range, underscore their potential for cross-species transmission, and highlight the importance of continued surveillance of wildlife viruses. ImportanceCurrently, there is concern about the potential emergence of new viruses from natural reservoirs. This has led to an increasing search taking advantage of new massive sequencing methodologies. In our study, we have identified a complete genome of a torovirus in a rodent species in Spain. Toroviruses are a group related to coronaviruses and cause gastrointestinal diseases in ungulates, such as cows or pigs. However, they have not been studied in depth, as there is no clear evidence of their ability to infect humans. Our results suggest that rodents may be the natural reservoir of toroviruses and underline their potential for cross-species transmission. Based on the results obtained from monitoring wildlife for viruses with zoonotic potential, the next step will be to implement new approaches in the laboratory to characterise the ability of these viruses to infect humans.

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

Complete genomes of DNA viruses in faecal samples from small terrestrial mammals in Spain

Viromics studies are allowing us to understand not only the enormous diversity of the virosphere, but also the potential threat posed by emerging viruses. Regarding the latter, the main concern lies in monitoring the presence of RNA viruses, but the zoonotic potential of some DNA viruses, on which we have focused in the present study, should also be highlighted. For this purpose, we analysed 160 faecal samples from 14 species belonging to three orders of small terrestrial mammals (i.e. Rodentia, Lagomorpha and Eulypotyphla). This allowed us to identify a total of 25 complete or near-complete genomes belonging to the families Papillomaviridae, Polyomaviridae, Adenoviridae, Circoviridae and Genomoviridae, 18 of which could be considered new species or types. Our results provide a significant increase in the number of complete genomes of DNA viruses of European origin with zoonotic potential in databases, which are at present clearly under-represented compared to RNA viruses.

microbiology↗
bioRxiv2024

A new clade of pararetroviruses distantly related to hepadnaviruses and nackednaviruses

Group VII of the Baltimore classification comprises reverse-transcribing, non-integrated DNA viruses, also known as pararetroviruses. These include the hepadnaviruses, a family of small enveloped DNA viruses that infect vertebrates, but also a sister family of non-enveloped fish viruses, the nackednaviruses. Here we describe the complete sequence of a new pararetrovirus found in the feces of an insectivorous bat. This virus encodes a core protein and a reverse transcriptase but no envelope protein. A database search identified a viral sequence from a permafrost sample as its closest relative. The two viruses form a cluster that occupies a basal phylogenetic position relative to hepadnaviruses and nackednaviruses, with an estimated divergence time of 500 million years. These findings may lead to the definition of a new viral family and support the hypothesis that ancestral animal pararetroviruses were non-enveloped.

evolutionary biology↗
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

Unlocking data in Klebsiella lysogens to predict capsular type-specificity of phage depolymerases

Viral entry is a critical step in the infection process. Klebsiella spp. and other clinically relevant bacteria often express a complex polysaccharide capsule that acts as a barrier to phage entry. In turn, most Klebsiella phages encode depolymerases for capsule removal. This virus-host arms race has led to extensive genetic diversity in both capsules and depolymerases, complicating our ability to understand their interaction. This study exploits the information encoded in Klebsiella prophages to model the interplay between the bacteria, the prophages, and their depolymerases, using a graph neural network and a sequence clustering-based method. Both approaches showed significant predictive ability for prophages capsular tropism and, importantly, were transferrable to lytic phages. In addition to creating a comprehensive database linking depolymerase sequences to their specific targets, this study demonstrates the predictability of phage-host interactions at the subspecies level, providing new insights for improving the therapeutic and industrial applicability of phages.

bioinformatics↗
bioRxiv2024

Genetic diversity and cross-species transmissibility of bat-associated picornaviruses from Spain

BackgroundEmerging zoonotic diseases arise from cross-species transmission events between wild or domesticated animals and humans, with bats being one of the major reservoirs of zoonotic viruses. Viral metagenomics has led to the discovery of many viruses, but efforts have mainly been focused on some areas of the world and on certain viral families. MethodsWe set out to describe full-length genomes of new picorna-like viruses by collecting feces from hundreds of bats captured in different regions of Spain. Viral sequences were obtained by high-throughput Illumina sequencing and analyzed phylogenetically to classify them in the context of known viruses. Linear discriminant analysis (LDA) was performed to infer likely hosts based on genome composition. ResultsWe found five complete or nearly complete genomes belonging to the family Picornaviridae, including a new species of the subfamily Ensavirinae. LDA suggested that these were true vertebrate viruses, rather than viruses from the bat diet. Some of these viruses were related to picornaviruses previously found in other bat species from distant geographical regions. We also found a calhevirus genome that most likely belongs to a proposed new family within the order Picornavirales, and for which genome composition analysis suggested a plant host. ConclusionsOur findings describe new picorna-like viral species and variants circulating in the Iberian Peninsula, illustrate the wide geographical distribution and interspecies transmissibility of picornaviruses, and suggest new hosts for calheviruses.

microbiology↗
bioRxiv2024

Full genome sequencing of dozens of new DNA viruses found in Spanish bat faeces

Bats are natural hosts of multiple viruses, many of which have clear zoonotic potential. The search for emerging viruses has been aided by the implementation of metagenomic tools, which have also enabled the detection of unprecedented viral diversity. Currently, this search is mainly focused on RNA viruses, which are largely over-represented in databases. To compensate for this research bias, we analyzed fecal samples from 189 Spanish bats belonging to 22 different species using viral metagenomics. This allowed us to identify 50 complete or near-complete viral genomes belonging to the families Adenoviridae, Circoviridae, Genomoviridae, Papillomaviridae, Parvoviridae, Polyomaviridae and Smacoviridae. Of these, 28 could constitute new species, doubling the number of viruses currently described in Europe. These findings open the door to a more thorough analysis of bat DNA viruses and their zoonotic potential. IMPORTANCEMetagenomics has become a fundamental tool to characterize the global virosphere, allowing us to understand the existing viral diversity and its ecological implications, but also to identify new and emerging viruses. RNA viruses have a higher zoonotic potential, but this risk is also present for some DNA virus families. In our study, we have analyzed the DNA fraction of faecal samples from 22 Spanish bat species, identifying 50 complete or near-complete genomes of different viral families with zoonotic potential. This doubles the number of genomes currently described in Europe. Metagenomic data often produce partial genomes that can be difficult to analyse. Our work, however, has characterised a large number of complete genomes, thus facilitating their taxonomic classification and enabling different analyses to be carried out to evaluate their zoonotic potential. For example, recombination studies are relevant, since this phenomenon could play a major role in cross-species transmission.

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↗
bioRxiv2024

DepoScope: accurate phage depolymerase annotation and domain delineation using large language models

Bacteriophages (phages) are viruses that infect bacteria. Many of them produce specific enzymes called depolymerases to break down external polysaccharide structures. Accurate annotation and domain identification of these depolymerases are challenging due to their inherent sequence diversity. Hence, we present DepoScope, a machine learning tool that combines a fine-tuned ESM-2 model with a convolutional neural network to precisely identify depolymerase sequences and their enzymatic domains. To accomplish this, we curated a dataset from the INPHARED phage genome database, created a polysaccharide-degrading domain database, and applied sequential filters to construct a high-quality dataset, which are subsequently used to train DepoScope. Our work is the first approach that combines sequence-level predictions with amino-acid-level predictions for an accurate depolymerase detection and functional domain identification. In that way, we believe that DepoScope can enhance our understanding of phage-host interactions at the level of depolymerases. Summary with Key MessagesO_LIPhage depolymerases are proteins that play a crucial role in the first step of a phage replication cycle. As a result, they are both important from a biological perspective and a therapeutical perspective. C_LIO_LICurrent methods to accurately annotate phage depolymerases and their associated enzymatic domains remains challenging due to their inherent high sequence diversity. C_LIO_LIWe have developed DepoScope, a language-based artificial intelligence model that can accurately identify phage depolymerases and their specific enzymatic domains. C_LIO_LIWe provide full public access to the DepoScope code and database to give broad access to the research community and promote further research. C_LI

bioinformatics↗
bioRxiv2024

Targeted phage hunting to specific Klebsiella pneumoniae clinical isolates is an efficient antibiotic resistance and infection control strategy

Klebsiella pneumoniae is one of the most threatening multi-drug resistant pathogens today, with phage therapy being a promising alternative for personalized treatments. However, the intrinsic capsule diversity in Klebsiella spp. poses a substantial barrier to phage host range, complicating the development of broad-spectrum phage-based treatments. Here, we have isolated and genomically characterized phages capable of infecting each of the acquired 77 reference serotypes of Klebsiella spp., including capsular types widespread among high-risk K. pneumoniae clones causing nosocomial infections. We demonstrated the possibility of isolating phages for all capsular types in the collection, revealing high capsular specificity among taxonomically related phages, in contrast to a few phages that exhibited broad-spectrum infection capabilities. To decipher the determinants of the specificity of these phages, we focused on their receptor-binding proteins, with particular attention to depolymerase domains. We also explored the possibility of designing a broad-spectrum phage cocktail based on phages isolated in reference capsular type strains, and determining the ability to lysate relevant clinical isolates. Interestingly, a combination of 12 phages capable of infecting 60% of the reference Klebsiella spp. serotypes was tested on a panel of carbapenem-resistant K. pneumoniae clinical isolates. Our results suggest that in a highly variable encapsulated bacterial host, phage hunting must be directed to the specific Klebsiella isolates. This work is a step forward in the understanding of the complexity of phage-host interactions, and highlights the importance of implementing precise and phage-specific strategies to treat K. pneumoniae infections worldwide.

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↗