Virus Evolution

Studies have shown that HCV subtype 3a had likely been circulating in South Asia before its global spread. However, the time and route of this dissemination remain unclear. For the first time, we generated host and virus genome-wide data for more than 500 patients infected with HCV subtype 3a from the UK, North America, Australia and New Zealand. We used the host genomic data to infer the ancestry of the patients and used this information to investigate the epidemic history of HCV subtype 3a. We observed that viruses from hosts of South Asian ancestry clustered together near the root of the tree, irrespective of the sampling country and that they were more diverse than viruses from other host ancestries. We also inferred that three independent transmission events resulted in the spread of the virus from South Asia to the UK, North America and the Australian continent. This initial spread happened during or soon after the end of the second world war. This was followed by an exponential growth in the effective population size of HCV subtype 3a worldwide and many independent transmissions between the UK, North America and Australian continent. Using both host and virus genomic information can be highly informative in studying the virus epidemic history especially in the context of chronic infections.

Abundant novel circular Rep-encoding ssDNA viruses (CRESS DNA viruses) have been discovered in the past decade, prompting a new appreciation for the ubiquity and genomic diversity of this group of viruses. Although highly divergent in the hosts they infect or are associated with, CRESS DNA viruses are united by the homologous replication-associated protein (Rep). An accurate genealogy of Rep can therefore provide insights into how these diverse families are related to each other. We used a dataset of eukaryote-associated CRESS DNA RefSeq genomes (n=926), which included representatives from all six established families and unclassified species. To assure an optimal Rep genealogy, we derived and tested a bespoke amino acid substitution model (named CRESS), which outperformed existing protein matrices in describing the evolution of Rep. The CRESS model-estimated Rep genealogy resolved the monophyly of Bacilladnaviridae and the reciprocal monophyly of Nanoviridae and the alpha-satellites when trees estimated with general matrices like LG did not. The most intriguing, previously unobserved result is a likely single origin of intron-containing Reps, which causes several geminivirus genera to group with Genomoviridae (bootstrap support 55%, aLRT SH-like support 0.997, 0.91-0.997 in trees estimated with established matrices). This grouping, which eliminates the monophyly of Geminiviridae, is supported by both domains of Rep, and appears to be related to our use of all RefSeq Reps instead of subsampling to get a smaller dataset. In addition to producing a trustworthy Rep genealogy, the derived CRESS matrix is proving useful for other analyses; it best fit alignments of capsid protein sequences from several CRESS DNA families and parvovirus NS1/Rep sequences.

Recombination plays an important role in the evolution of RNA viruses, as it allows the exchange of genetic material between viral lineages. Reassortment, a form of recombination specific to segmented genomes, involves the exchange of entire segments and has contributed significantly to the adaptation and spread of influenza viruses through novel genomic combinations, i.e., antigenic shifts. It is usually identified by phylogenetic discordance: differences in the topologies of trees reconstructed from different genomic segments. However, phylogenetic discordance can also result from error in reconstructing trees. To characterize the impact of reconstruction error, we curated a database of n = 11, 765 complete genomes of avian H5Nx influenza A viruses from avian hosts. We found evidence of widespread reassortment as measured by inferred subtree-prune-regraft (SPR) events, consistent with previous studies. Next, we ran replicate simulations of sequence evolution along the reference tree for the segment encoding hemagglutinin (HA), adjusting simulations for the lengths and clock rates of the other segments. These simulations provided a baseline for the expected amount of phylogenetic discordance in the absence of any reassortment. When sampling HA sequences at random from the database to build reference trees, we observed that simulating other segments without reassortment still yielded about 32% as many SPRs as the real segment data on average. The average proportion of SPRs without reassortment was greatly reduced (4%) if we selected an equivalent number of HA sequences retaining the most genetic diversity, which was consistent with the accuracy of phylogenetic reconstruction being the limiting factor. This implies that measuring reassortment by SPRs may have a high false positive rate, and that previous evidence of extensive reassortment in influenza viruses should be interpreted with caution. In addition, we observed that the SPRs reconstructed on simulated trees had significantly shorter distances between the prune and regraft locations than real trees. These results suggest that down-sampling sequences to maximize evolutionary divergence and filtering out the shortest SPRs may be effective measures against false positives.

RNA-dependent RNA polymerases (RdRps) are crucial for replication of RNA viruses and serve as key marker genes for defining deep taxonomic ranks and for understanding viral evolutionary history. Despite shared functions and conserved amino acid motifs, the high genetic diversity of RdRps complicates precise sequence comparisons across viral families, hindering accurate taxonomic classification of new species - especially important at the age of metagenomics. When available, three-dimensional (3D) RdRp structures can help address these challenges through structure-based alignments. However, such structures are scarce for understudied viruses infecting fungi and plants, preventing the investigation of their ecological and evolutionary links. In this study, we focused on the highy divergent order Sobelivirales. Using deep{-}learning structural modeling, we generated highly reliable 3D models on 20 representative viral species. Multiple structural alignment enabled the reconstruction of a robust phylogeny with improved quality and length. Based on this phylogeny, we proposed revisions of existing viral families and reclassified genera. Clade divergence dates were then estimated using the Prisoner of War model, which has previously revealed the ancient origin of the genus Sobemovirus. We provided here the first divergence time estimation between these plant and fungal viruses, dating back to 26.6 million years before present - significantly more recent than the divergence between their respective hosts. Our amino acid conservation analysis, validated on 99 other viral species, also identified molecular signatures of sobeliviral families and genera, which could help in future taxonomic assignment and diagnostic tools development. This interdisciplinary approach integrating structure modeling and date estimations offers new insights into the evolutionary divergence between fungus and plant viruses, with potential applications to other viral orders and families. Author summaryRNA-dependent RNA polymerases (RdRps) are essential for RNA virus replication and serve as important markers for classifying viruses and understanding their evolution. However, with the rising popularity of metagenomics and discovery of viruses with high genetic diversity in RdRps, it is difficult to compare viral families and accurately classify new species. When available, 3D structures of RdRps can help overcome this challenge through structural alignments. In this study, we focused on the highly divergent order of the Sobelivirales, using deep{-}learning models to generate reliable 3D structures for 20 representative viral species. These structural alignments allowed us to build a more accurate viral phylogeny. Based on this finding, we proposed updates to existing viral families and genera within the Sobelivirales order. We also estimated divergence dates using a model that previously uncovered the ancient origins of Sobemovirus. Notably, we provided the first estimate of when these plant and fungal viruses diverged - around 26.6 million years ago, which is much more recent than the separation of their hosts. We also identified molecular signatures that are useful for future virus classification and diagnosis, with potential applications to other viral groups.

The evolutionary dynamics of seasonal influenza A viruses (IAVs) have been well characterized at the population level, with antigenic drift known to be a major force in driving strain turnover. The evolution of IAV populations at the within-host level, however, is still less well characterized. Improving our understanding of within-host IAV evolution has the potential to shed light on the source of new strains, including new antigenic variants, at the population level. Existing studies have pointed towards the role that stochastic processes play in shaping within-host viral evolution in acute infections of both humans and pigs. Here, we apply a population genetic model called the Beta-with-Spikes approximation to longitudinal intrahost Single Nucleotide Variant (iSNV) frequency data to quantify the extent of genetic drift acting on IAV populations at the within-host scale. We estimate small effective population sizes in both human IAV infections (NE = 41, 95% confidence interval: [22-72]) and swine IAV infections (NE = 10, 95% confidence interval: [8-14]). Moreover, we evaluate the consistency of the observed iSNV dynamics with Wright-Fisher model simulations. For the human IAV dataset that we analyze, we find that observed within-host IAV evolutionary dynamics are consistent with this classic model at the estimated low effective population size. However, for the swine IAV dataset, we find statistical evidence for rejecting the classic Wright-Fisher model as the only process governing within-host iSNV frequency dynamics. Our results contribute to the growing number of studies that point towards the important role of genetic drift in shaping patterns of genetic diversity in IAV populations within acutely infected hosts. It further raises questions about whether and what other processes, such as spatial compartmentalization, viral progeny production dynamics with strong skew, or selection, may be needed to explain patterns of within-host IAV evolution.

A multitude of demographic, health, and genetic factors are associated with the risk of developing severe COVID-19 following infection by the SARS-CoV-2. There is a need to perform studies across human societies and to investigate the full spectrum of genetic variation of the virus. Using data from 869 COVID-19 patients in Bahrain between March 2020 and March 2021, we analyzed paired viral sequencing and non-genetic host data to understand host and viral determinants of severe COVID-19. We estimated the effects of demographic variables specific to the Bahrain population and found that the impact of health factors are largely consistent with other populations. To extend beyond the common variants of concern in the Spike protein analyzed by previous studies, we used a viral burden approach and detected a protective effect of low-frequency missense viral mutations in the RNA-dependent RNA polymerase (Pol) gene on disease severity. Our results contribute to the survey of severe COVID-19 in diverse populations and highlight the benefits of studying rare viral mutations.

Drosophila melanogaster is an important model for antiviral immunity in arthropods, but very few DNA viruses have been described from the family Drosophilidae. This deficiency limits our opportunity to use natural host-pathogen combinations in experimental studies, and may bias our understanding of the Drosophila virome. Here we report fourteen DNA viruses detected in a metagenomic analysis of approximately 6500 pool-sequenced Drosophila, sampled from 47 European locations between 2014 and 2016. These include three new Nudiviruses, a new and divergent Entomopox virus, a virus related to Leptopilina boulardi filamentous virus, and a virus related to Musca domestica salivary gland hypertrophy virus. We also find an endogenous genomic copy of Galbut virus, a dsRNA Partitivirus, segregating at very low frequency. Remarkably, we find that Drosophila Vesanto virus, a small DNA virus previously described as a Bidnavirus, may be composed of up to 12 segments and represent a new lineage of segmented DNA viruses. Two of the DNA viruses, Drosophila Kallithea nudivirus and Drosophila Vesanto virus are relatively common, found in 2% or more of wild flies. The others are rare, with many likely to be represented by a single infected fly. We find that virus prevalence in Europe reflects the prevalence seen in publicly-available datasets, with Drosophila Kallithea nudivirus and Drosophila Vesanto virus the only ones commonly detectable in public data from wild-caught flies and large population cages, and the other viruses being rare or absent. These analyses suggest that DNA viruses are at lower prevalence than RNA viruses in D. melanogaster, and may be less likely to persist in laboratory cultures. Our findings go some way to redressing an earlier bias toward RNA virus studies in Drosophila, and lay the foundation needed to harness the power of Drosophila as a model system for the study of DNA viruses.

Phytoplankton viruses are key players in aquatic ecosystems, where they control algal populations and affect nutrient flow. The ecological impact and evolution of these viruses can be understood by studying their life history traits, but very little is known about the life history diversity of related viruses. We quantified the life cycle of 34 strains in the genus Chlorovirus, which infects freshwater green algae. All chloroviral life history traits varied 5-to 75-fold across strains, in some cases rivaling the known trait range for all phytoplankton viruses. The trait variation affected viral growth rates but was not detectably constrained by life history trade-offs. This study represents the most in-depth characterization of algal viruses to date and raises the question whether all phytoplankton virus genera are equally diverse.

AbstractThe evolutionary relationships between hosts and their symbionts offer valuable insights into the origins, maintenance, and consequences of biological interactions. While co-divergence and host-switching have been extensively explored in systems, such as Wolbachia-arthropod symbioses or viruses infecting vertebrates, similar investigations in protistan parasites remain scarce. The Leishmania-Leishmaniavirus (LRV) association offers a rare opportunity to study co-divergence in a medically relevant symbiotic system, in which the virus modulates the parasitic disease severity in humans. Here, we used total RNA sequencing to capture both the Leishmania transcriptome and the LRV genome simultaneously, enabling the first comprehensive investigation of the co-evolutionary history of Leishmania and LRV across different hierarchical levels (subgenus, species, and population). We found significant positive correlations between the parasite and viral genetic distances at both the subgenus (R2 = 0.89; F = 10,913.49; p < 0.001) and species level (focusing on L. (Viannia) spp.; R2 = 0.59; F = 1,254; p < 0.001). This was corroborated by additional co-phylogenetic methods (global-fit and event-based), indicating a strong pattern of phylogenetic congruence between Leishmania and LRV up to the species level. Only at the population level, focusing on the interactions of L. (V.) braziliensis and L. (V.) guyanensis with LRV1, we found weaker co-phylogenetic signals accompanied by more instances of intraspecific host switching. Overall and for the first time, our findings provide analytical evidence for the co-evolution between Leishmania and LRV, with co-speciation as the predominant process, while also shedding light on how such symbioses were maintained over long evolutionary time scales.

Efforts to describe the diversity of viruses have largely focused on classifying viruses at the species level. However, substantial ecological diversity, both in virulence level and host range, is known within virus species. Here we demonstrate a proof of concept for easily discovering ecological diversity within a virus species taxon. We have focused on the West Nile Virus to take advantage of its broad host range in nature. We produced a genome-based phylogeny of world diversity of WNV and then used Ecotype Simulation 2 to hypothesize demarcation of genomes into 69 putative ecotypes (ecologically distinct populations), based only on clustering of genome sequences. Then we looked for evidence of ecological divergence among ecotypes based on differences in host bird associations within the Connecticut-New York region. Our results indicated significant heterogeneity among ecotypes for their associations with different bird hosts. Ecological diversity within other zoonotic viruses could be easily discovered using this approach. Opportunities for extending this line of research to human associations of virus ecotypes are limited by missing geographic metadata on human samples.

As the SARS-Cov-2 virus spreads around the world afflicting millions of people, it has undergone divergent genetic mutations. Although most of these mutations are expected to be inconsequential, some mutations in the spike protein structure have been hypothesized to affect the critical stage at which the virus invades human cells, which could affect transmission probability and disease expression. If true, then we expect an increased growth rate of reported COVID-19 cases in regions dominated by viruses with these altered proteins. We modeled early global infection dynamics based on clade assignment along with other demographic and meteorological factors previously found to be important. Clade, but not variant D614G which has been associated with increased viral load, enhanced our ability to describe early COVID-19 growth dynamics. Including clade identity in models significantly improved predictions over earlier work based only on weather and demographic variables. In particular, higher proportions of clade 19A and 19B were negatively correlated with COVID-19 growth rate, whereas higher proportions of 20A and 20C were positively correlated with growth rate. A strong interaction between the prevalence of clade 20C and relative humidity suggests that the impact of clade identity might be more important when coupled with certain weather conditions. In particular, 20C an 20A generate the highest growth rates when coupled with low humidity. Projections based on data through April 2020 suggest that, without intervention, COVID-19 has the potential to grow more quickly in regions dominated by the 20A and 20C clades, including most of South and North America.

The 2022 outbreak of monkeypox virus (MPXV), a double-stranded DNA virus, is remarkable for an unusually high number of single-nucleotide substitutions compared to earlier strains, with a strong bias toward C[-&gt;]T and G[-&gt;]A transitions consistent with the APOBEC3 cytidine deaminase activity. While APOBEC3-induced mutagenesis is well documented at the DNA level, its potential impact on MPXV RNA transcripts remains unclear. To assess whether APOBEC3 enzymes act on MPXV RNA, we analyzed RNA-seq data from infected samples. The enrichment of APOBEC-signature substitutions among high-frequency mismatched positions led us to consider two possibilities: RNA editing at hotspots or fixed DNA mutations. Multiple lines of evidence support the conclusion that these substitutions arise from DNA-level mutagenesis rather than RNA editing. These include a substantial number of G[-&gt;]A substitutions remaining after normalization by gene strand direction, a largely neutral impact of substitutions on protein-coding sequences, the lack of positional correlation with transcriptional features or RNA secondary structure typically associated with APOBEC action hotspots, and an overlap with known genomic mutations in MPXV strains. Analysis of the nucleotide context of observed substitutions indicated that APOBEC3A or APOBEC3B were likely drivers of DNA-level mutagenesis. ImportanceThe 2022 monkeypox virus (MPXV) outbreak showed an unusually high number of mutations thought to result from human antiviral enzymes of the APOBEC3 family. While such mutations have been clearly documented in the viral DNA, whether APOBEC3 also edits viral messenger RNA molecules remained unclear. In this study, we analyzed multiple publicly available MPXV RNA sequencing datasets to address this question. We found that the apparent APOBEC-like changes in RNA are best explained by fixed DNA mutations rather than active RNA editing. This finding helps clarify how MPXV evolves and adapts, suggesting that APOBEC3s role in shaping the virus likely operates at the DNA level. Understanding where and how these mutations occur provides insight into the viruss interaction with the human immune system and informs future studies on viral evolution and antiviral defenses.

Modern phylogeography aims at reconstructing the geographic diffusion of organisms based on their genomic sequences and spatial information. Phylogeographic approaches usually ignore the possibility of recombination, which decouples the evolutionary and geographic histories of different parts of the genome. Genomic regions of recombining or reassorting pathogens often originate and evolve at different times and locations, which characterised their unique spatial histories. Measuring the extent of these differences requires new methods to compare geographic information on phylogenetic trees reconstructed from different parts of the genome. Here we develop for the first time a set of measures of phylogeographic incompatibility aimed at detecting differences between geographical histories in terms of distances between phylogeographies. We study the effect of varying demography and recombination on phylogeographic incompatibilities using coalescent simulations. We further apply these measures to the evolutionary history of human and livestock pathogens, either reassorting or recombining, such as the Victoria and Yamagata lineages of influenza B and the O/Ind-2001 foot-and-mouth disease virus strain. Our results reveal diverse geographical paths of diffusion that characterise the origins and evolutionary histories of different viral genes and genomic segments. phylogeography, recombination, viral evolution

Cellular coinfection between multiple virions is a common feature of viral infections. The collective virus-virus interactions enabled by these coinfections can influence the fitness of viral populations and give rise to novel infection phenotypes. Multi-strain coinfections allow viral resources to be shared between multiple individuals, and enable genetic combination and recombination between genotypes, potentially giving rise to hybrid progeny with enhanced fitness. However, coinfection can also impose fitness costs in certain situations. For example, resource sharing among viruses can lead to the persistence of low-fitness genotypes, and reassortment between different strains can lead to negative inter-segment epistasis when genes are poorly matched to one another. Thus, the fitness implications of cellular coinfection are poorly defined and likely context dependent. To investigate the specific conditions that lead to positive or negative fitness consequences for multi-strain coinfections, we formulated a model in which different genotypes of a three-segment virus replicate under varying degrees of inter-strain mixing. We observed that increased mixing had negative fitness consequences under a variety of scenarios, and that this effect was exacerbated with increasing genetic divergence between strains. Inter-strain mixing only enhanced viral fitness (a) when positive genetic dominance interactions were at play, and (b) under very specific conditions of selective pressure. We also observed that reassortment arising from mixing could generate hybrid genotypes with higher fitness than either parental virus, but that these outcomes were relatively rare. Overall, using a model segmented virus, we found that the heterologous coinfection was deleterious under most conditions, suggesting that it may be beneficial for many viruses to limit the extent of cellular coinfection.

Zika virus (ZIKV) is a mosquito-borne flavivirus primarily transmitted among humans by Aedes aegypti. Over the past two decades, it has caused significant outbreaks associated with birth defects and neurological disorders. Phylogenetically, ZIKV consists of two main genotypes referred to as the African and Asian lineages, each exhibiting distinct biological properties. African lineage strains are transmitted more efficiently by mosquitoes, but pinpointing the genetic basis of this difference has remained challenging. Here, we address this question by comparing recent African and Asian strains using chimeric viruses, in which segments of the parental genomes are swapped. Our results show that the structural genes from the African strain enhance viral internalization, while the non-structural genes improve genome replication and infectious particle production in mosquito cells. In vivo mosquito transmission is most significantly influenced by the structural genes, although no single viral gene alone determines this effect. Additionally, we develop a stochastic model of in vivo viral dynamics in mosquitoes that mirrors the observed patterns, suggesting that the primary difference between the African and Asian strains lies in their ability to traverse the mosquito salivary glands. Overall, our findings suggest that the polygenic nature of ZIKV transmissibility has prevented Asian lineage strains from achieving the same epidemic potential as African lineage strains, underscoring the importance of lineage-specific adaptive landscapes in shaping ZIKV evolution and emergence.

Some viruses including the widespread human cytomegalovirus (HCMV) show a high level of genetic diversity within hosts and across the whole virus population even in relatively conserved genomic regions. To investigate how this diversity is formed, we earlier introduced and analyzed a population model for neutrally evolving viruses that persist in their hosts and are capable of reinfection, mutation and recombination. Based on these results, we fit our model to observed genotype frequencies from Austrian HCMV patients. Despite simply assuming neutral evolution, the model captures the data closely. The inferred parameters are discussed and may give insights about the roles of viral replication, reinfection, mutation and recombination for the evolution of HCMV.

Japanese encephalitis virus (JEV) is the dominant cause of viral encephalitis in the Asian region with 100,000 cases and 25,000 deaths reported annually. The genome is comprised of a single polyprotein that encodes three structural and seven non-structural proteins. We collated a dataset of 347 complete genomes from a number of public databases, and analysed the data for recombination, evolutionary selection and phylogenetic structure. There are low rates of recombination in JEV, subsequently recombination is not a major evolutionary force shaping JEV. We found a strong overall signal of purifying selection in the genome, which is the main force affecting the evolutionary dynamics in JEV. There are also a small number of genomic sites under episodic diversifying selection, especially in the envelope protein and non-structural proteins 3 and 5. Overall, these results support previous analyses of JEV evolutionary genomics and provide additional insight into the evolutionary processes shaping the distribution and adaptation of this important pathogenic arbovirus. Author SummaryThis comparative study of Japanese Encephalitis Virus is the largest genomic analysis of the virus to date. We undertake a suite of analyses to investigate phylogenetic relationships, rates of recombination and patterns of genomic selection. We show that recombination is not a significant driver of evolution in JEV, demonstrate support for previous phylogenetic reconstructions of the virus, and find a number of sites across the genome under episodic diversifying selection. These adaptive hotspots of evolution serve as key genomic points for the adaptive evolution of this important vector borne pathogen.

Since 2014, Brazil has experienced an unprecedented epidemic caused by chikungunya virus (CHIKV), with several waves of East-Central-South-African (ECSA) lineage transmission reported across the country. In 2018, Rio de Janeiro state, the third most populous state in Brazil, reported 41% of all chikungunya cases in the country. Here we use evolutionary and epidemiological analysis to estimate the timescale of CHIKV-ECSA-American lineage and its epidemiological patterns in Rio de Janeiro. We show that the CHIKV-ECSA outbreak in Rio de Janeiro derived from two distinct clades introduced from the Northeast region in mid-2015 (clade RJ1, n = 63/67 genomes from Rio de Janeiro) and mid-2017 (clade RJ2, n = 4/67). We detected evidence for positive selection in non-structural proteins linked with viral replication in the RJ1 clade (clade-defining: nsP4-A481D) and the RJ2 clade (nsP1-D351G). Finally, we estimate the CHIKV-ECSAs basic reproduction number (R0) to be between 1.2 to 1.6 and show that its instantaneous reproduction number (Rt) displays a strong seasonal pattern with peaks in transmission coinciding with periods of high Aedes aegypti transmission potential. Our results highlight the need for continued genomic and epidemiological surveillance of CHIKV in Brazil, particularly during periods of high ecological suitability, and show that selective pressures underline the emergence and evolution of the large urban CHIKV-ECSA outbreak in Rio de Janeiro.

Our knowledge of the diversity and evolution of the virosphere will likely increase dramatically with the study of microbial eukaryotes, including the microalgae in few RNA viruses have been documented to date. By combining meta-transcriptomic approaches with sequence and structural-based homology detection, followed by PCR confirmation, we identified 18 novel RNA viruses in two major groups of microbial algae - the chlorophytes and the chlorarachniophytes. Most of the RNA viruses identified in the green algae class Ulvophyceae were related to those from the families Tombusviridae and Amalgaviridae that have previously been associated with plants, suggesting that these viruses have an evolutionary history that extends to when their host groups shared a common ancestor. In contrast, seven ulvophyte associated viruses exhibited clear similarity with the mitoviruses that are most commonly found in fungi. This is compatible with horizontal virus transfer between algae and fungi, although mitoviruses have recently been documented in plants. We also document, for the first time, RNA viruses in the chlorarachniophytes, including the first observation of a negative-sense (bunya-like) RNA virus in microalgae. The other virus-like sequence detected in chlorarachniophytes is distantly related to those from the plant virus family Virgaviridae, suggesting that they may have been inherited from the secondary chloroplast endosymbiosis event that marked the origin of the chlorarachniophytes. More broadly, this work suggests that the scarcity of RNA viruses in algae most likely results from limited investigation rather than their absence. Greater effort is needed to characterize the RNA viromes of unicellular eukaryotes, including through structure-based methods that are able to detect distant homologies, and with the inclusion of a wider range of eukaryotic microorganisms. Author summaryRNA viruses are expected to infect all living organisms on Earth. Despite recent developments in and the deployment of large-scale sequencing technologies, our understanding of the RNA virosphere remains anthropocentric and largely restricted to human, livestock, cultivated plants and vectors for viral disease. However, a broader investigation of the diversity of RNA viruses, especially in protists, is expected to answer fundamental questions about their origin and long-term evolution. This study first investigates the RNA virus diversity in unicellular algae taxa from the phylogenetically distinct ulvophytes and chlorarachniophytes taxa. Despite very high levels of sequence divergence, we were able to identify 18 new RNA viruses, largely related to plant and fungi viruses, and likely illustrating a past history of horizontal transfer events that have occurred during RNA virus evolution. We also hypothesise that the sequence similarity between a chlorarachniophyte-associated virga-like virus and members of Virgaviridae associated with plants may represent inheritance from a secondary endosymbiosis event. A promising approach to detect the signals of distant virus homologies through the analysis of protein structures was also utilised, enabling us to identify potential highly divergent algal RNA viruses.

Cospeciation has been suggested to be the main force driving the evolution of herpesviruses, with viral species co-diverging with their hosts along more than 400 million years of evolutionary history. Recent studies, however, have been challenging this assumption, showing that other co-phylogenetic events, such as intrahost speciations and host switches play a central role on their evolution. Most of these studies, however, were performed with undated phylogenies, which may underestimate or overestimate the frequency of certain events. In this study we performed co-phylogenetic analyses using time-calibrated trees of herpesviruses and their hosts. This approach allowed us to (i) infer co-phylogenetic events over time, and (ii) integrate crucial information about continental drift and host biogeography to better understand virus-host evolution. We observed that cospeciations were in fact relatively rare events, taking place mostly after the Late Cretaceous (~100 Millions of years ago). Host switches were particularly common among alphaherpesviruses, where at least 10 transfers were detected. Among beta- and gammaherpesviruses, transfers were less frequent, with intrahost speciations followed by losses playing more prominent roles, especially from the Early Jurassic to the Early Cretaceous, when those viral lineages underwent several intrahost speciations. Our study reinforces the understanding that cospeciations are uncommon events in herpesvirus evolution. More than topological incongruences, mismatches in divergence times were the main disagreements between host and viral phylogenies. In most cases, host switches could not explain such disparities, highlighting the important role of losses and intrahost speciations in the evolution of herpesviruses.