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Background Emerging 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. Methods We 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. Results We 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. Conclusions Our 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.

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°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 increasing temperature from 33°C to 37°C or 39°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°C but was lost in a high proportion of viruses that evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°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°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.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can infect various human tissues and cell types, principally via interaction with its cognate receptor angiotensin-converting enzyme-2 (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 twenty passages of a spike-expressing recombinant vesicular stomatitis virus (VSV), cell-type-specific phenotypic changes were observed and sequencing allowed the identification of sixteen adaptive spike mutations. We used VSV pseudotyping to measure the entry efficiency, ACE2 affinity, spike processing, TMPRSS2 usage, and entry pathway usage of all the mutants, alone or in combination. The fusogenicity of the mutant spikes was assessed with a cell-cell fusion assay. Finally, mutant recombinant VSVs were used to measure the fitness advantage associated with selected mutations. We found that the effects of these mutations varied across cell types, both in terms of viral entry and replicative fitness. 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.

S1 Fig. Workflow for the manual and automatic text-mining searches. A starting list of 6034 mammal viruses was used to obtain, which were then reviewed using both manual and PubmedKB-based automated strategies. The resulting virus-receptor pairs were combined with known databases and manually curated (see text for full description). M, manual strategy; PKB, PubmedKB strategy., S2 Fig. Roles of known receptors according to viral family. Only families with at least 10 known virus-host interactions are represented. Families of enveloped and non-enveloped viruses are shown, and within each group, families are sorted by the fraction of known receptors that are sufficient for viral entry (main plus alternative receptors)., S3 Fig. Research effort versus the known number of host proteins used as receptors for different viral families. Data points correspond to individual viruses. Those corresponding to the indicated family are shown in color (blue for non-enveloped viruses; yellow for enveloped viruses), and grey points correspond to all other viruses. The colored and grey dashed lines show the GLM prediction obtained specifically for the family and all viruses, respectively. Only families with at least 5 viral species in the dataset were considered., S1 Table. Database of virus receptors generated in this study. The following information is provided: the viral species, number of PubMed records and Genbank sequences available for each virus, viral family, presence of an envelope, number of host species, receptor symbol, receptor nature, functional role of the receptor, corresponding gene symbol, original publication PMID, year of discovery, and whether the virus-receptor interaction was reported in previous reviews and databases., S2 Table. Scores obtained from the GBM. The gene symbol, assigned score (probability of being a receptor), and whether the corresponding protein is a known receptor are indicated., S3 Table. Relevant features identified by the GBM. Gain represents the relative contribution of each variable to the model prediction. Cover indicates the relative number of observations that are related to a given variable.

The interaction of viral surface components with cellular receptors and other entry factors determines key features of viral infection such as host range, tropism and virulence. Despite intensive research, our understanding of these interactions remains limited. Here, we report a systematic analysis of published work on mammalian virus receptors and attachment factors. We build a dataset twice the size of those available to date and specify the role of each factor in virus entry. We identify cellular proteins that are preferentially used as virus receptors, which tend to be plasma membrane proteins with a high propensity to interact with other proteins. Using machine learning, we assign cell surface proteins a score that predicts their ability to function as virus receptors. Our results also reveal common patterns of receptor usage among viruses and suggest that enveloped viruses tend to use a broader repertoire of alternative receptors than non-enveloped viruses, a feature that might confer them with higher interspecies transmissibility.

SARS-CoV-2 infects both the upper and lower respiratory tracts, which are characterized by different temperatures (33°C and 37°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°C to 37°C or 39°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°C, but was lost in a high proportion of viruses evolved at 33°C or 37°C. Consistently, sequencing of the spikes evolved at 33°C or 37°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°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.

The transmission of viruses between different host species is a major source of emerging diseases and is of particular concern in the case of zoonotic transmission from mammals to humans. Several zoonosis risk factors have been identified, but it is currently unclear which viral traits primarily determine this process as previous work has focused on a few hundred viruses that are not representative of actual viral diversity. Here, we investigate fundamental virological traits that influence cross-species transmissibility and zoonotic propensity by interrogating a database of over 12,000 mammalian virus–host associations. Our analysis reveals that enveloped viruses tend to infect more host species and are more likely to be zoonotic than nonenveloped viruses, while other viral traits such as genome composition, structure, size, or the viral replication compartment play a less obvious role. This contrasts with the previous notion that viral envelopes did not significantly impact or even reduce zoonotic risk and should help better prioritize outbreak prevention efforts. We suggest several mechanisms by which viral envelopes could promote cross-species transmissibility, including structural flexibility of receptor-binding proteins and evasion of viral entry barriers.

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.

Anelloviruses represent the major and most diverse component of the healthy human virome, referred to as the anellome. In this study, we determined the anellome of 50 blood donors, forming two sex- and age-matched groups. Anelloviruses were detected in 86% of the donors. The number of detected anelloviruses increased with age and was approximately twice as high in men as in women. A total of 349 complete or nearly complete genomes were classified as belonging to torque teno virus (TTV), torque teno mini virus (TTMV), and torque teno midi virus (TTMDV) anellovirus genera (197, 88, and 64 sequences, respectively). Most donors had intergenus (69.8%) or intragenus (72.1%) coinfections. Despite the limited number of sequences, intradonor recombination analysis showed 6 intragenus recombination events in ORF1. As thousands of anellovirus sequences have been described recently, we finally analyzed the global diversity of human anelloviruses. Species richness and diversity were close to saturation in each anellovirus genus. Recombination was found to be the main factor promoting diversity, although its effect was significantly lower in TTV than in TTMV and TTMDV. Overall, our results suggest that differences in diversity between genera may be caused by variations in the relative contribution of recombination., [Importance] Anelloviruses are the most common human infectious viruses and are considered essentially harmless. Compared to other human viruses, they are characterized by enormous diversity, and recombination is suggested to play an important role in their diversification and evolution. Here, by analyzing the composition of the plasma anellome of 50 blood donors, we find that recombination is also a determinant of viral evolution at the intradonor level. On a larger scale, analysis of anellovirus sequences currently available in databases shows that their diversity is close to saturation and differs among the three human anellovirus genera and that recombination is the main factor explaining this intergenus variability. Global characterization of anellovirus diversity could provide clues about possible associations between certain virus variants and pathologies, as well as facilitate the implementation of unbiased PCR-based detection protocols, which may be relevant for using anelloviruses as endogenous markers of immune status.

Viral glycoproteins mediate entry into host cells, dictating host range and pathogenesis. In addition, they constitute the principal target of neutralizing antibody responses, making them important antigens in vaccine development. Recombinant vesicular stomatitis virus encoding foreign glycoproteins in lieu of its cognate glycoprotein can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of these proteins from viruses requiring high levels of biosafety containment or those lacking efficient infectious clone systems. However, the production of recombinant VSV can be technically challenging. In this work, we present an efficient and robust plasmid-based system for the production of recombinant vesicular stomatitis virus encoding foreign glycoproteins. We validate the system using the glycoproteins of different viral families, including numerous SARS-CoV-2 variants and arenaviruses. This described method can facilitate the study of both native vesicular stomatitis virus as well as vesicular stomatitis virus encoding foreign glycoproteins and can serve as the basis for the production of vesicular stomatitis virus-based vaccines.

The environmental impact of uncultured phages is shaped by their preferred life cycle (lytic or lysogenic). However, our ability to predict it is very limited. We aimed to discriminate between lytic and lysogenic phages by comparing the similarity of their genomic signatures to those of their hosts, reflecting their co-evolution. We tested two approaches: (1) similarities of tetramer relative frequencies, (2) alignment-free comparisons based on exact k = 14 oligonucleotide matches. First, we explored 5126 reference bacterial host strains and 284 associated phages and found an approximate threshold for distinguishing lysogenic and lytic phages using both oligonucleotide-based methods. The analysis of 6482 plasmids revealed the potential for horizontal gene transfer between different host genera and, in some cases, distant bacterial taxa. Subsequently, we experimentally analyzed combinations of 138 Klebsiella pneumoniae strains and their 41 phages and found that the phages with the largest number of interactions with these strains in the laboratory had the shortest genomic distances to K. pneumoniae. We then applied our methods to 24 single-cells from a hot spring biofilm containing 41 uncultured phage–host pairs, and the results were compatible with the lysogenic life cycle of phages detected in this environment. In conclusion, oligonucleotide-based genome analysis methods can be used for predictions of (1) life cycles of environmental phages, (2) phages with the broadest host range in culture collections, and (3) potential horizontal gene transfer by plasmids.

Supplemental information: Document S1. Figures S1–S7 and Table S5. Table S1. General features of the 46 isolated bacteriophages. Phage ID, phylogenetic classification in 13 groups, genome size and circularity, sequencing depth, GC %, CDS, putative lysogeny genes, taxonomic family, subfamily, and genus according to IGS values with the closest phage are provided. Additionally, concentration of phages for the spot assay, formation of plaque haloes, susceptible CLTs, RBP system and number of depolymerase domains (Dpos) are also shown. REP: transcription repressor/regulator, PAR: parAB genes, REC: recombinases. TSP: Tail spike protein. CBM: carbohydrate-binding module. Taxonomy ranks marked with a star can be reclassified based on relationships showed in this study. CLTs marked with a star indicate that bacteria were acapsular. Dpos marked with a star indicate putative soluble Dpos. Table S2. General features of the 138 Klebsiella clinical strains. The strain, sequencing details, virulence and antibiotic resistance scores, wzi allele, CLT and OLT inference, number of prophages, CRISPR loci, and presence of defense systems and secondary receptors are provided. The presence of a capsule was determined by visual inspection of colonies using light microscopy. Table S3. Phage-bacteria positive combinations. The strain used for phage isolation is indicated. Spotting: positive by the spot-assay. Virulent: positive by the killing planktonic or progeny assay. Table S4. Clustering of phage RBDs with putative depolymerase activity from this work and the literature. RBDs are identified by the phage name followed by the phage protein in which the domain was detected, and the positions considered after the removal of the anchor domain. Document S2. Article plus supplemental information.

Bacteriophages play key roles in bacterial ecology and evolution and are potential antimicrobials. However, the determinants of phage-host specificity remain elusive. Here, we isolate 46 phages to challenge 138 representative clinical isolates of Klebsiella pneumoniae, a widespread opportunistic pathogen. Spot tests show a narrow host range for most phages, with <2% of 6,319 phage-host combinations tested yielding detectable interactions. Bacterial capsule diversity is the main factor restricting phage host range. Consequently, phage-encoded depolymerases are key determinants of host tropism, and depolymerase sequence types are associated with the ability to infect specific capsular types across phage families. However, all phages with a broader host range found do not encode canonical depolymerases, suggesting alternative modes of entry. These findings expand our knowledge of the complex interactions between bacteria and their viruses and point out the feasibility of predicting the first steps of phage infection using bacterial and phage genome sequences.

The ability of natural selection to optimize traits depends on the topology of the genotype-fitness map (fitness landscape). Epistatic interactions produce rugged fitness landscapes, where adaptation is constrained by the presence of low-fitness intermediates. Here, we used simulations to explore how evolvability in rugged fitness landscapes is influenced by genetic complementation, a process whereby different sequence variants mutually compensate for their deleterious mutations. We designed our model inspired by viral populations, in which genetic variants are known to interact frequently through coinfection. Our simulations indicate that genetic complementation enables a more efficient exploration of rugged fitness landscapes. Although this benefit may be undermined by genetic parasites, its overall effect on evolvability remains positive in populations that exhibit strong relatedness between interacting sequences. Similar processes could operate in contexts other than viral coinfection, such as in the evolution of ploidy.

Figure S1: PCA of genomic signatures based on hexamer frequencies of the reference bacteria; Figure S2: k4freq-based distances of the bacterial genera to the reference phages; Figure S3: ROC curves for k4freq and k14exact; Figure S4: Statistical error analysis for different k-mer options; Figure S5: k14exact-based distances of the bacterial genera to the reference phages; Figure S6: Distances of 75 Klebsiella strains to 41 Klebsiella phages; Table S1: Bacterial genomes in the reference set; Table S2: Phage genomes in the reference set; Table S3: Plasmids in the reference set; Table S4: Summary of statistical comparisons of genomic distances; Table S5: Lysogenic activity of K. pneumoniae phages; Table S6: Bacterial single-cells and phages detected in their assemblies.

The emergence of cellular resistances to oncolytic viruses is an underexplored process that could compromise the efficacy of cancer virotherapy. Here, we isolated and characterized B16 mouse melanoma cells that evolved resistance to an oncolytic vesicular stomatitis virus (VSV-D51). RNA-seq revealed that resistance was associated to broad changes in gene expression, which typically involved chronic upregulation of interferon-stimulated genes. Innate immunity activation was maintained in the absence of the virus or other infection signals, and conferred cross-resistance to wild-type VSV and the unrelated Sindbis virus. Furthermore, we identified differentially expressed genes with no obvious role in antiviral immunity, such as Mnda, Psmb8 and Btn2a2, suggesting novel functions for these genes. Transcriptomic changes associated to VSV resistance were similar among B16 clones and in some clones derived from the mouse colon carcinoma cell line CT26, suggesting that oncolytic virus resistance involves certain conserved mechanisms and is therefore a potentially predictable process.

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 subvariant BQ.1.1 became predominant in many countries in December 2022. The subvariants 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) lose antiviral efficacy against BA.2.75.2 and BQ.1.1, whereas Xevudy (Sotrovimab) remaine weakly active. BQ.1.1 is also resistant to Bebtelovimab. Neutralizing titers in triply vaccinated individuals are low to undetectable against BQ.1.1 and BA.2.75.2, 4 months after boosting. A BA.1/BA.2 breakthrough infection increases these titers, which remains about 18-fold lower against BA.2.75.2 and BQ.1.1, than against BA.1. Reciprocally, a BA.5 breakthrough infection increases more efficiently neutralization against BA.5 and BQ.1.1 than against BA.2.75.2. Thus, the evolution trajectory of novel Omicron subvariants facilitates their spread in immunized populations and raises concerns about the efficacy of most available mAbs.