An efficient plasmid-based system for the recovery of recombinant vesicular stomatitis virus encoding foreign glycoproteins.

Viral glycoproteins mediate entry into host cells, thereby 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 (VSV) encoding foreign glycoproteins can provide a convenient and safe surrogate system to interrogate the function, evolution, and antigenicity of viral glycoproteins from viruses that are difficult to manipulate or those requiring high biosafety level containment. 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 VSV encoding foreign glycoproteins. We validate the system using glycoproteins from different viral families, including arenaviruses, coronaviruses, and hantaviruses, as well as highlight their utility for studying the effects of mutations on viral fitness. Overall, the methods described herein can facilitate the study of both native and recombinant VSV encoding foreign glycoproteins and can serve as the basis for the production of VSV-based vaccines.

Experimental evolution of an RNA virus in Caenorhabditis elegans.

The discovery of Orsay virus (OrV), the first virus infecting wild populations of Caenorhabditis elegans, has boosted studies of viral immunity pathways in this nematode. Considering the many advantages that C. elegans offers for fundamental research in host-pathogen interactions, this pathosystem has high potential to become a model system for experimental virus evolution studies. However, the evolutionary constraints - i.e, the balance between genetic variation, selection, drift and historical contingency- operating in this pathosystem have barely been explored. Here we describe for the first time an evolution experiment of two different OrV strains in C. elegans. Comparison of the two ancestral strains showed differences in infectivity and sequence, and highlighted the importance of consistently normalize viral inocula for meaningful comparisons among strains. After 10 serial passages of evolution, we report slight changes in infectivity and non-synonymous mutations fixed in the evolved viral populations. In addition, we observed numerous minor variants emerging in the viral population. These minor variants were not randomly distributed along the genome but concentrated in polymorphic genomic regions. Overall, our work established the grounds for future experimental virus evolution studies using Caenorhabditis nematodes.

Cell type-specific adaptation 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.

Accumulation Dynamics of Defective Genomes during Experimental Evolution of Two Betacoronaviruses.

Virus-encoded replicases often generate aberrant RNA genomes, known as defective viral genomes (DVGs). When co-infected with a helper virus providing necessary proteins, DVGs can multiply and spread. While DVGs depend on the helper virus for propagation, they can in some cases disrupt infectious virus replication, impact immune responses, and affect viral persistence or evolution. Understanding the dynamics of DVGs alongside standard viral genomes during infection remains unclear. To address this, we conducted a long-term experimental evolution of two betacoronaviruses, the human coronavirus OC43 (HCoV-OC43) and the murine hepatitis virus (MHV), in cell culture at both high and low multiplicities of infection (MOI). We then performed RNA-seq at regular time intervals, reconstructed DVGs, and analyzed their accumulation dynamics. Our findings indicate that DVGs evolved to exhibit greater diversity and abundance, with deletions and insertions being the most common types. Notably, some high MOI deletions showed very limited temporary existence, while others became prevalent over time. We observed differences in DVG abundance between high and low MOI conditions in HCoV-OC43 samples. The size distribution of HCoV-OC43 genomes with deletions differed between high and low MOI passages. In low MOI lineages, short and long DVGs were the most common, with an additional cluster in high MOI lineages which became more prevalent along evolutionary time. MHV also showed variations in DVG size distribution at different MOI conditions, though they were less pronounced compared to HCoV-OC43, suggesting a more random distribution of DVG sizes. We identified hotspot regions for deletions that evolved at a high MOI, primarily within cistrons encoding structural and accessory proteins. In conclusion, our study illustrates the widespread formation of DVGs during betacoronavirus evolution, influenced by MOI and cell- and virus-specific factors.

Unveiling the hidden viromes across the animal tree of life: insights from a taxonomic classification pipeline applied to invertebrates of 31 metazoan phyla.

Invertebrates constitute the majority of animal species on Earth, including most disease-causing agents or vectors, with more diverse viromes when compared to vertebrates. Recent advancements in high-throughput sequencing have significantly expanded our understanding of invertebrate viruses, yet this knowledge remains biased toward a few well-studied animal lineages. In this study, we analyze invertebrate DNA and RNA viromes for 31 phyla using 417 publicly available RNA-Seq data sets from diverse environments in the marine-terrestrial and marine-freshwater gradients. This study aims to (i) estimate virome compositions at the family level for the first time across the animal tree of life, including the first exploration of the virome in several phyla, (ii) quantify the diversity of invertebrate viromes and characterize the structure of invertebrate-virus infection networks, and (iii) investigate host phylum and habitat influence on virome differences. Results showed that a set of few viral families of eukaryotes, comprising Retroviridae, Flaviviridae, and several families of giant DNA viruses, were ubiquitous and highly abundant. Nevertheless, some differences emerged between phyla, revealing for instance a less diverse virome in Ctenophora compared to the other animal phyla. Compositional analysis of the viromes showed that the host phylum explained over five times more variance in composition than its habitat. Moreover, significant similarities were observed between the viromes of some phylogenetically related phyla, which could highlight the influence of co-evolution in shaping invertebrate viromes.IMPORTANCEThis study significantly enhances our understanding of the global animal virome by characterizing the viromes of previously unexamined invertebrate lineages from a large number of animal phyla. It showcases the great diversity of viromes within each phylum and investigates the role of habitat shaping animal viral communities. Furthermore, our research identifies dominant virus families in invertebrates and distinguishes phyla with analogous viromes. This study sets the road toward a deeper understanding of the virome across the animal tree of life.

Decay of HCoV-OC43 infectivity is lower in cell debris-containing media than in fresh culture media.

In the quantitative description of viral dynamics within cell cultures and, more broadly, in modeling within-host viral infections, a question that commonly arises is whether the degradation of a fraction of the virus could be disregarded in comparison with the massive synthesis of new viral particles. Surprisingly, quantitative data on the synthesis and degradation rates of RNA viruses in cell cultures are scarce. In this study, we investigated the decay of the human betacoronavirus OC43 (HCoV-OC43) infectivity in cell culture lysates and in fresh media. Our findings revealed a significantly slower viral decay rate in the medium containing lysate cells compared to the fresh medium. This observation suggests that the presence of cellular debris from lysed cells may offer protection or stabilize virions, slowing down their degradation. Moreover, the growth rate of HCoV-OC43 infectivity is significantly higher than degradation as long as there are productive cells in the medium, suggesting that, as a first approximation, degradation can be neglected during early infection.

Identification of epigenetically regulated genes involved in plant-virus interaction and their role in virus-triggered induced resistance.

BACKGROUND: Plant responses to a wide range of stresses are known to be regulated by epigenetic mechanisms. Pathogen-related investigations, particularly against RNA viruses, are however scarce. It has been demonstrated that Arabidopsis thaliana plants defective in some members of the RNA-directed DNA methylation (RdDM) or histone modification pathways presented differential susceptibility to the turnip mosaic virus. In order to identify genes directly targeted by the RdDM-related RNA Polymerase V (POLV) complex and the histone demethylase protein JUMONJI14 (JMJ14) during infection, the transcriptomes of infected mutant and control plants were obtained and integrated with available chromatin occupancy data for various epigenetic proteins and marks. RESULTS: A comprehensive list of virus-responsive gene candidates to be regulated by the two proteins was obtained. Twelve genes were selected for further characterization, confirming their dynamic regulation during the course of infection. Several epigenetic marks on their promoter sequences were found using in silico data, raising confidence that the identified genes are actually regulated by epigenetic mechanisms. The altered expression of six of these genes in mutants of the methyltransferase gene CURLY LEAF and the histone deacetylase gene HISTONE DEACETYLASE 19 suggests that some virus-responsive genes may be regulated by multiple coordinated epigenetic complexes. A temporally separated multiple plant virus infection experiment in which plants were transiently infected with one virus and then infected by a second one was designed to investigate the possible roles of the identified POLV- and JMJ14-regulated genes in wild-type (WT) plants. Plants that had previously been stimulated with viruses were found to be more resistant to subsequent virus challenge than control plants. Several POLV- and JMJ14-regulated genes were found to be regulated in virus induced resistance in WT plants, with some of them poisoned to be expressed in early infection stages. CONCLUSIONS: A set of confident candidate genes directly regulated by the POLV and JMJ14 proteins during virus infection was identified, with indications that some of them may be regulated by multiple epigenetic modules. A subset of these genes may also play a role in the tolerance of WT plants to repeated, intermittent virus infections.

Emergence of two distinct spatial folds in a pair of plant virus proteins encoded by nested genes.

Virus genomes may encode overlapping or nested open reading frames that increase their coding capacity. It is not known whether the constraints on spatial structures of the two encoded proteins limit the evolvability of nested genes. We examine the evolution of a pair of proteins, p22 and p19, encoded by nested genes in plant viruses from the genus Tombusvirus. The known structure of p19, a suppressor of RNA silencing, belongs to the RAGNYA fold from the alpha+beta class. The structure of p22, the cell-to-cell movement protein from the 30K family widespread in plant viruses, is predicted with the AlphaFold approach, suggesting a single jelly-roll fold core from the all-beta class, structurally similar to capsid proteins from plant and animal viruses. The nucleotide and codon preferences impose modest constraints on the types of secondary structures encoded in the alternative reading frames, nonetheless allowing for compact, well-ordered folds from different structural classes in two similarly-sized nested proteins. Tombusvirus p22 emerged through radiation of the widespread 30K family, which evolved by duplication of a virus capsid protein early in the evolution of plant viruses, whereas lineage-specific p19 may have emerged by a stepwise increase in the length of the overprinted gene and incremental acquisition of functionally active secondary structure elements by the protein product. This evolution of p19 toward the RAGNYA fold represents one of the first documented examples of protein structure convergence in naturally occurring proteins.

Genetic basis of Arabidopsis thaliana responses to infection by naïve and adapted isolates of turnip mosaic virus.

Plant viruses account for enormous agricultural losses worldwide, and the most effective way to combat them is to identify genetic material conferring plant resistance to these pathogens. Aiming to identify genetic associations with responses to infection, we screened a large panel of Arabidopsis thaliana natural inbred lines for four disease-related traits caused by infection by A. thaliana-naïve and -adapted isolates of the natural pathogen turnip mosaic virus (TuMV). We detected a strong, replicable association in a 1.5 Mb region on chromosome 2 with a 10-fold increase in relative risk of systemic necrosis. The region contains several plausible causal genes as well as abundant structural variation, including an insertion of a Copia transposon into a Toll/interleukin receptor (TIR-NBS-LRR) coding for a gene involved in defense, that could be either a driver or a consequence of the disease-resistance locus. When inoculated with TuMV, loss-of-function mutant plants of this gene exhibited different symptoms than wild-type plants. The direction and severity of symptom differences depended on the adaptation history of the virus. This increase in symptom severity was specific for infections with the adapted isolate. Necrosis-associated alleles are found worldwide, and their distribution is consistent with a trade-off between resistance during viral outbreaks and a cost of resistance otherwise, leading to negative frequency-dependent selection.

Phenotypic and genomic changes during Turnip mosaic virus adaptation to Arabidopsis thaliana mutants lacking epigenetic regulatory factors.

In this study, we investigated how an emerging RNA virus evolves, interacts, and adapts to populations of a novel host species with defects in epigenetically controlled plant defense mechanisms. Mutations in epigenetic regulatory pathways would exert different effects on defense-response genes but also induce large-scale alterations in cellular physiology and homeostasis. To test whether these effects condition the emergence and subsequent adaptation of a viral pathogen, we have evolved five independent lineages of a naive turnip mosaic virus (TuMV) strain in a set of Arabidopsis thaliana genotypes carrying mutations that influence important elements of two main epigenetic pathways and compare the results with those obtained for viral lineages evolved in wild-type plants. All evolved lineages showed adaptation to the lack of epigenetically regulated responses through significant increases in infectivity, virulence, and viral load although the magnitude of the improvements strongly depended on the plant genotype. In early passages, these traits evolved more rapidly, but the rate of evolution flattened out in later ones. Viral load was positively correlated with different measures of virulence, though the strength of the associations changed from the ancestral to the evolved viruses. High-throughput sequencing was used to evaluate the viral diversity of each lineage, as well as characterizing the nature of fixed mutations, evolutionary convergences, and potential targets of TuMV adaptation. Within each lineage, we observed a net increase in genome-wide genetic diversity, with some instances where nonsynonymous alleles experienced a transient rise in abundance before being displaced by the ancestral allele. In agreement with previous studies, viral VPg protein has been shown as a key player in the adaptation process, even though no obvious association between fixed alleles and host genotype was found.

Viral strain-dependent impact of plant developmental stages on the nestedness and modularity of plant-virus interaction matrices.

This study examines the specificity of adaptation of lineages of turnip mosaic virus that were experimentally evolved from naïve and preadapted strains to Arabidopsis thaliana plants at various plant developmental stages. We conducted a cross-infection experiment involving three plant developmental stages and assessed the progression of disease and symptoms. We found a significative interaction between the host developmental stage where the virus evolved and the host developmental stage in which the virus was tested. The analysis of the resulting interaction matrices revealed significant nestedness for viruses evolved from the naïve strain, but not for those originating from the preadapted one. Furthermore, there was an absence of modularity across all matrices. Our findings suggest that the past adaptation history of the ancestral strain influences its future evolution, and each plant developmental stage imposes unique selective constraints. The study highlights the complexity of host-parasite interactions and the potential influence of the host's developmental stage on viral adaptation.

The International Virus Bioinformatics Meeting 2023.

The 2023 International Virus Bioinformatics Meeting was held in Valencia, Spain, from 24-26 May 2023, attracting approximately 180 participants worldwide. The primary objective of the conference was to establish a dynamic scientific environment conducive to discussion, collaboration, and the generation of novel research ideas. As the first in-person event following the SARS-CoV-2 pandemic, the meeting facilitated highly interactive exchanges among attendees. It served as a pivotal gathering for gaining insights into the current status of virus bioinformatics research and engaging with leading researchers and emerging scientists. The event comprised eight invited talks, 19 contributed talks, and 74 poster presentations across eleven sessions spanning three days. Topics covered included machine learning, bacteriophages, virus discovery, virus classification, virus visualization, viral infection, viromics, molecular epidemiology, phylodynamic analysis, RNA viruses, viral sequence analysis, viral surveillance, and metagenomics. This report provides rewritten abstracts of the presentations, a summary of the key research findings, and highlights shared during the meeting.

Host developmental stages shape the evolution of a plant RNA virus.

Viruses are obligate pathogens that entirely rely on their hosts to complete their infectious cycle. The outcome of viral infections depends on the status of the host. Host developmental stage is an important but sometimes overlooked factor impacting host-virus interactions. This impact is especially relevant in a context where climate change and human activities are altering plant development. To better understand how different host developmental stages shape virus evolution, we experimentally evolved turnip mosaic virus (TuMV) on Arabidopsis thaliana at three different developmental stages: vegetative (juvenile), bolting (transition) and reproductive (mature). After infecting plants with an Arabidopsis-naive or an Arabidopsis-well-adapted TuMV isolate, we observed that hosts in later developmental stages were prone to faster and more severe infections. This observation was extended to viruses belonging to different genera. Thereafter, we experimentally evolved lineages of the naive and the well-adapted TuMV isolates in plants from each of the three developmental stages. All evolved viruses enhanced their infection traits, but this increase was more intense in viruses evolved in younger hosts. The genomic changes of the evolved viral lineages revealed mutation patterns that strongly depended on the founder viral isolate as well as on the developmental stage of the host wherein the lineages were evolved. This article is part of the theme issue 'Infectious disease ecology and evolution in a changing world'.

Four principles to establish a universal virus taxonomy.

A universal taxonomy of viruses is essential for a comprehensive view of the virus world and for communicating the complicated evolutionary relationships among viruses. However, there are major differences in the conceptualisation and approaches to virus classification and nomenclature among virologists, clinicians, agronomists, and other interested parties. Here, we provide recommendations to guide the construction of a coherent and comprehensive virus taxonomy, based on expert scientific consensus. Firstly, assignments of viruses should be congruent with the best attainable reconstruction of their evolutionary histories, i.e., taxa should be monophyletic. This fundamental principle for classification of viruses is currently included in the International Committee on Taxonomy of Viruses (ICTV) code only for the rank of species. Secondly, phenotypic and ecological properties of viruses may inform, but not override, evolutionary relatedness in the placement of ranks. Thirdly, alternative classifications that consider phenotypic attributes, such as being vector-borne (e.g., "arboviruses"), infecting a certain type of host (e.g., "mycoviruses," "bacteriophages") or displaying specific pathogenicity (e.g., "human immunodeficiency viruses"), may serve important clinical and regulatory purposes but often create polyphyletic categories that do not reflect evolutionary relationships. Nevertheless, such classifications ought to be maintained if they serve the needs of specific communities or play a practical clinical or regulatory role. However, they should not be considered or called taxonomies. Finally, while an evolution-based framework enables viruses discovered by metagenomics to be incorporated into the ICTV taxonomy, there are essential requirements for quality control of the sequence data used for these assignments. Combined, these four principles will enable future development and expansion of virus taxonomy as the true evolutionary diversity of viruses becomes apparent.

Plant Virus Adaptation to New Hosts: A Multi-scale Approach.

Viruses are studied at each level of biological complexity: from within-cells to ecosystems. The same basic evolutionary forces and principles operate at each level: mutation and recombination, selection, genetic drift, migration, and adaptive trade-offs. Great efforts have been put into understanding each level in great detail, hoping to predict the dynamics of viral population, prevent virus emergence, and manage their spread and virulence. Unfortunately, we are still far from this. To achieve these ambitious goals, we advocate for an integrative perspective of virus evolution. Focusing in plant viruses, we illustrate the pervasiveness of the above-mentioned principles. Beginning at the within-cell level, we describe replication modes, infection bottlenecks, and cellular contagion rates. Next, we move up to the colonization of distal tissues, discussing the fundamental role of random events. Then, we jump beyond the individual host and discuss the link between transmission mode and virulence. Finally, at the community level, we discuss properties of virus-plant infection networks. To close this review we propose the multilayer network theory, in which elements at different layers are connected and submit to their own dynamics that feed across layers, resulting in new emerging properties, as a way to integrate information from the different levels.

A binary interaction map between turnip mosaic virus and Arabidopsis thaliana proteomes.

Viruses are obligate intracellular parasites that have co-evolved with their hosts to establish an intricate network of protein-protein interactions. Here, we followed a high-throughput yeast two-hybrid screening to identify 378 novel protein-protein interactions between turnip mosaic virus (TuMV) and its natural host Arabidopsis thaliana. We identified the RNA-dependent RNA polymerase NIb as the viral protein with the largest number of contacts, including key salicylic acid-dependent transcription regulators. We verified a subset of 25 interactions in planta by bimolecular fluorescence complementation assays. We then constructed and analyzed a network comprising 399 TuMV-A. thaliana interactions together with intravirus and intrahost connections. In particular, we found that the host proteins targeted by TuMV are enriched in different aspects of plant responses to infections, are more connected and have an increased capacity to spread information throughout the cell proteome, display higher expression levels, and have been subject to stronger purifying selection than expected by chance. The proviral or antiviral role of ten host proteins was validated by characterizing the infection dynamics in the corresponding mutant plants, supporting a proviral role for the transcriptional regulator TGA1. Comparison with similar studies with animal viruses, highlights shared fundamental features in their mode of action.

Single-cell RNA-sequencing data analysis reveals a highly correlated triphasic transcriptional response to SARS-CoV-2 infection.

Single-cell RNA sequencing (scRNA-seq) is currently one of the most powerful techniques available to study the transcriptional response of thousands of cells to an external perturbation. Here, we perform a pseudotime analysis of SARS-CoV-2 infection using publicly available scRNA-seq data from human bronchial epithelial cells and colon and ileum organoids. Our results reveal that, for most genes, the transcriptional response to SARS-CoV-2 infection follows a non-linear pattern characterized by an initial and a final down-regulatory phase separated by an intermediate up-regulatory stage. A correlation analysis of transcriptional profiles suggests a common mechanism regulating the mRNA levels of most genes. Interestingly, genes encoded in the mitochondria or involved in translation exhibited distinct pseudotime profiles. To explain our results, we propose a simple model where nuclear export inhibition of nsp1-sensitive transcripts will be sufficient to explain the transcriptional shutdown of SARS-CoV-2 infected cells.

Virus classification based on in-depth sequence analyses and development of demarcation criteria using the Betaflexiviridae as a case study.

Currently, many viruses are classified based on their genome organization and nucleotide/amino acid sequence identities of their capsid and replication-associated proteins. Although biological traits such as vector specificities and host range are also considered, this later information is scarce for the majority of recently identified viruses, characterized only from genomic sequences. Accordingly, genomic sequences and derived information are being frequently used as the major, if not only, criteria for virus classification and this calls for a full review of the process. Herein, we critically addressed current issues concerning classification of viruses in the family Betaflexiviridae in the era of high-throughput sequencing and propose an updated set of demarcation criteria based on a process involving pairwise identity analyses and phylogenetics. The proposed framework has been designed to solve the majority of current conundrums in taxonomy and to facilitate future virus classification. Finally, the analyses performed herein, alongside the proposed approaches, could be used as a blueprint for virus classification at-large.