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.

Interspecies Recombination Has Driven the Macroevolution of Cassava Mosaic Begomoviruses.

Begomoviruses (family Geminiviridae, genus Begomovirus) significantly hamper crop production and threaten food security around the world. The frequent emergence of new begomovirus genotypes is facilitated by high mutation frequencies and the propensity to recombine and reassort. Homologous recombination has been especially implicated in the emergence of novel cassava mosaic begomovirus (CMB) genotypes, which cause cassava mosaic disease (CMD). Cassava (Manihot esculenta) is a staple food crop throughout Africa and an important industrial crop in Asia, two continents where production is severely constrained by CMD. The CMD species complex is comprised of 11 bipartite begomovirus species with ample distribution throughout Africa and the Indian subcontinent. While recombination is regarded as a frequent occurrence for CMBs, a revised, systematic assessment of recombination and its impact on CMB phylogeny is currently lacking. We assembled data sets of all publicly available, full-length DNA-A (n = 880) and DNA-B (n = 369) nucleotide sequences from the 11 recognized CMB species. Phylogenetic networks and complementary recombination detection methods revealed extensive recombination among the CMB sequences. Six out of the 11 species descended from unique interspecies recombination events. Estimates of recombination and mutation rates revealed that all species experience mutation more frequently than recombination, but measures of population divergence indicate that recombination is largely responsible for the genetic differences between species. Our results support that recombination has significantly impacted the CMB phylogeny and has driven speciation in the CMD species complex. IMPORTANCE Cassava mosaic disease (CMD) is a significant threat to cassava production throughout Africa and Asia. CMD is caused by a complex comprised of 11 recognized virus species exhibiting accelerated rates of evolution, driven by high frequencies of mutation and genetic exchange. Here, we present a systematic analysis of the contribution of genetic exchange to cassava mosaic virus species-level diversity. Most of these species emerged as a result of genetic exchange. This is the first study to report the significant impact of genetic exchange on speciation in a group of viruses.

Population diversity of cassava mosaic begomoviruses increases over the course of serial vegetative propagation.

Cassava mosaic disease (CMD) represents a serious threat to cassava, a major root crop for more than 300 million Africans. CMD is caused by single-stranded DNA begomoviruses that evolve rapidly, making it challenging to develop durable disease resistance. In addition to the evolutionary forces of mutation, recombination and reassortment, factors such as climate, agriculture practices and the presence of DNA satellites may impact viral diversity. To gain insight into the factors that alter and shape viral diversity in planta, we used high-throughput sequencing to characterize the accumulation of nucleotide diversity after inoculation of infectious clones corresponding to African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in the susceptible cassava landrace Kibandameno. We found that vegetative propagation had a significant effect on viral nucleotide diversity, while temperature and a satellite DNA did not have measurable impacts in our study. EACMCV diversity increased linearly with the number of vegetative propagation passages, while ACMV diversity increased for a time and then decreased in later passages. We observed a substitution bias toward C→T and G→A for mutations in the viral genomes consistent with field isolates. Non-coding regions excluding the promoter regions of genes showed the highest levels of nucleotide diversity for each genome component. Changes in the 5' intergenic region of DNA-A resembled the sequence of the cognate DNA-B sequence. The majority of nucleotide changes in coding regions were non-synonymous, most with predicted deleterious effects on protein structure, indicative of relaxed selection pressure over six vegetative passages. Overall, these results underscore the importance of knowing how cropping practices affect viral evolution and disease progression.

Truly ubiquitous CRESS DNA viruses scattered across the eukaryotic tree of life.

Until recently, most viruses detected and characterized were of economic significance, associated with agricultural and medical diseases. This was certainly true for the eukaryote-infecting circular Rep (replication-associated protein)-encoding single-stranded DNA (CRESS DNA) viruses, which were thought to be a relatively small group of viruses. With the explosion of metagenomic sequencing over the past decade and increasing use of rolling-circle replication for sequence amplification, scientists have identified and annotated copious numbers of novel CRESS DNA viruses - many without known hosts but which have been found in association with eukaryotes. Similar advances in cellular genomics have revealed that many eukaryotes have endogenous sequences homologous to viral Reps, which not only provide 'fossil records' to reconstruct the evolutionary history of CRESS DNA viruses but also reveal potential host species for viruses known by their sequences alone. The Rep protein is a conserved protein that all CRESS DNA viruses use to assist rolling-circle replication that is known to be endogenized in a few eukaryotic species (notably tobacco and water yam). A systematic search for endogenous Rep-like sequences in GenBank's non-redundant eukaryotic database was performed using tBLASTn. We utilized relaxed search criteria for the capture of integrated Rep sequence within eukaryotic genomes, identifying 93 unique species with an endogenized fragment of Rep in their nuclear, plasmid (one species), mitochondrial (six species) or chloroplast (eight species) genomes. These species come from 19 different phyla, scattered across the eukaryotic tree of life. Exogenous and endogenous CRESS DNA viral Rep tree topology suggested potential hosts for one family of uncharacterized viruses and supports a primarily fungal host range for genomoviruses.

Why are RNA virus mutation rates so damn high?

The high mutation rate of RNA viruses is credited with their evolvability and virulence. This Primer, however, discusses recent evidence that this is, in part, a byproduct of selection for faster genomic replication.

Existing Host Range Mutations Constrain Further Emergence of RNA Viruses.

RNA viruses are capable of rapid host shifting, typically due to a point mutation that confers expanded host range. As additional point mutations are necessary for further expansions, epistasis among host range mutations can potentially affect the mutational neighborhood and frequency of niche expansion. We mapped the mutational neighborhood of host range expansion using three genotypes of the double-stranded RNA (dsRNA) bacteriophage φ6 (wild type and two isogenic host range mutants) on the novel host Pseudomonas syringae pv. atrofaciens. Both Sanger sequencing of 50 P. syringae pv. atrofaciens mutant clones for each genotype and population Illumina sequencing revealed the same high-frequency mutations allowing infection of P. syringae pv. atrofaciens. Wild-type φ6 had at least nine different ways of mutating to enter the novel host, eight of which are in p3 (host attachment protein gene), and 13/50 clones had unchanged p3 genes. However, the two isogenic mutants had dramatically restricted neighborhoods: only one or two mutations, all in p3. Deep sequencing revealed that wild-type clones without mutations in p3 likely had changes in p12 (morphogenic protein), a region that was not polymorphic for the two isogenic host range mutants. Sanger sequencing confirmed that 10/13 of the wild-type φ6 clones had nonsynonymous mutations in p12, and 2 others had point mutations in p9 and p5. None of these genes had previously been associated with host range expansion in φ6. We demonstrate, for the first time, epistatic constraint in an RNA virus due to host range mutations themselves, which has implications for models of serial host range expansion.IMPORTANCE RNA viruses mutate rapidly and frequently expand their host ranges to infect novel hosts, leading to serial host shifts. Using an RNA bacteriophage model system (Pseudomonas phage φ6), we studied the impact of preexisting host range mutations on another host range expansion. Results from both clonal Sanger and Illumina sequencing show that extant host range mutations dramatically narrow the neighborhood of potential host range mutations compared to that of wild-type φ6. This research suggests that serial host-shifting viruses may follow a small number of molecular paths to enter additional novel hosts. We also identified new genes involved in φ6 host range expansion, expanding our knowledge of this important model system in experimental evolution.

Taxonomy of prokaryotic viruses: 2018-2019 update from the ICTV Bacterial and Archaeal Viruses Subcommittee.

This article is a summary of the activities of the ICTV's Bacterial and Archaeal Viruses Subcommittee for the years 2018 and 2019. Highlights include the creation of a new order, 10 families, 22 subfamilies, 424 genera and 964 species. Some of our concerns about the ICTV's ability to adjust to and incorporate new DNA- and protein-based taxonomic tools are discussed.

Overlooking the smallest matter: viruses impact biological invasions.

Parasites and pathogens have recently received considerable attention for their ability to affect biological invasions, however, researchers have largely overlooked the distinct role of viruses afforded by their unique ability to rapidly mutate and adapt to new hosts. With high mutation and genomic substitution rates, RNA and single-stranded DNA (ssDNA) viruses may be important constituents of invaded ecosystems, and could potentially behave quite differently from other pathogens. We review evidence suggesting that rapidly evolving viruses impact invasion dynamics in three key ways: (1) Rapidly evolving viruses may prevent exotic species from establishing self-sustaining populations. (2) Viruses can cause population collapses of exotic species in the introduced range. (3) Viruses can alter the consequences of biological invasions by causing population collapses and extinctions of native species. The ubiquity and frequent host shifting of viruses make their ability to influence invasion events likely. Eludicating the viral ecology of biological invasions will lead to an improved understanding of the causes and consequences of invasions, particularly as regards establishment success and changes to community structure that cannot be explained by direct interspecific interactions among native and exotic species.

Variability in P1 gene redefines phylogenetic relationships among cassava brown streak viruses.

BACKGROUND: Cassava brown streak disease is emerging as the most important viral disease of cassava in Africa, and is consequently a threat to food security. Two distinct species of the genus Ipomovirus (family Potyviridae) cause the disease: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). To understand the evolutionary relationships among the viruses, 64 nucleotide sequences from the variable P1 gene from major cassava producing areas of east and central-southern Africa were determined. METHODS: We sequenced an amplicon of the P1 region of 31 isolates from Malawi and Tanzania. In addition to these, 33 previously reported sequences of virus isolates from Uganda, Kenya, Tanzania, Malawi and Mozambique were added to the analysis. RESULTS: Phylogenetic analyses revealed three major P1 clades of Cassava brown streak viruses (CBSVs): in addition to a clade of most CBSV and a clade containing all UCBSV, a novel, intermediate clade of CBSV isolates which has been tentatively called CBSV-Tanzania (CBSV-TZ). Virus isolates of the distinctive CBSV-TZ had nucleotide identities as low as 63.2 and 63.7% with other members of CBSV and UCBSV respectively. CONCLUSIONS: Grouping of P1 gene sequences indicated for distinct sub-populations of CBSV, but not UCBSV. Representatives of all three clades were found in both Tanzania and Malawi.

Cell tropism predicts long-term nucleotide substitution rates of mammalian RNA viruses.

The high rates of RNA virus evolution are generally attributed to replication with error-prone RNA-dependent RNA polymerases. However, these long-term nucleotide substitution rates span three orders of magnitude and do not correlate well with mutation rates or selection pressures. This substitution rate variation may be explained by differences in virus ecology or intrinsic genomic properties. We generated nucleotide substitution rate estimates for mammalian RNA viruses and compiled comparable published rates, yielding a dataset of 118 substitution rates of structural genes from 51 different species, as well as 40 rates of non-structural genes from 28 species. Through ANCOVA analyses, we evaluated the relationships between these rates and four ecological factors: target cell, transmission route, host range, infection duration; and three genomic properties: genome length, genome sense, genome segmentation. Of these seven factors, we found target cells to be the only significant predictors of viral substitution rates, with tropisms for epithelial cells or neurons (P<0.0001) as the most significant predictors. Further, one-tailed t-tests showed that viruses primarily infecting epithelial cells evolve significantly faster than neurotropic viruses (P<0.0001 and P<0.001 for the structural genes and non-structural genes, respectively). These results provide strong evidence that the fastest evolving mammalian RNA viruses infect cells with the highest turnover rates: the highly proliferative epithelial cells. Estimated viral generation times suggest that epithelial-infecting viruses replicate more quickly than viruses with different cell tropisms. Our results indicate that cell tropism is a key factor in viral evolvability.

Contrasting genetic structure between two begomoviruses infecting the same leguminous hosts.

Begomoviruses are whitefly-transmitted, ssDNA plant viruses and are among the most damaging pathogens causing epidemics in economically important crops worldwide. Wild/non-cultivated plants play a crucial epidemiological role, acting as begomovirus reservoirs and as 'mixing vessels' where recombination can occur. Previous work suggests a higher degree of genetic variability in begomovirus populations from non-cultivated hosts compared with cultivated hosts. To assess this supposed host effect on the genetic variability of begomovirus populations, cultivated (common bean, Phaseolus vulgaris, and lima bean, Phaseolus lunatus) and non-cultivated (Macroptilium lathyroides) legume hosts were sampled from two regions of Brazil. A total of 212 full-length DNA-A genome segments were sequenced from samples collected between 2005 and 2012, and populations of the begomoviruses Bean golden mosaic virus (BGMV) and Macroptilium yellow spot virus (MaYSV) were obtained. We found, for each begomovirus species, similar genetic variation between populations infecting cultivated and non-cultivated hosts, indicating that the presumed genetic variability of the host did not a priori affect viral variability. We observed a higher degree of genetic variation in isolates from MaYSV populations than BGMV populations, which was explained by numerous recombination events in MaYSV. MaYSV and BGMV showed distinct distributions of genetic variation, with the BGMV population (but not MaYSV) being structured by both host and geography.

Frequent migration of introduced cucurbit-infecting begomoviruses among Middle Eastern countries.

BACKGROUND: In the early 2000s, two cucurbit-infecting begomoviruses were introduced into the eastern Mediterranean basin: the Old World Squash leaf curl virus (SLCV) and the New World Watermelon chlorotic stunt virus (WmCSV). These viruses have been emerging in parallel over the last decade in Egypt, Israel, Jordan, Lebanon and Palestine. METHODS: We explored this unique situation by assessing the diversity and biogeography of the DNA-A component of SLCV and WmCSV in these five countries. RESULTS: There was fairly low sequence variation in both begomovirus species (SLCV π = 0.0077; WmCSV π = 0.0066). Both viruses may have been introduced only once into the eastern Mediterranean basin, but once established, these viruses readily moved across country boundaries. SLCV has been introduced at least twice into each of all five countries based on the absence of monophyletic clades. Similarly, WmCSV has been introduced multiple times into Jordan, Israel and Palestine. CONCLUSIONS: We predict that uncontrolled movement of whiteflies among countries in this region will continue to cause SLCV and WmCSV migration, preventing strong genetic differentiation of these viruses among these countries.

Rates of evolutionary change in viruses: patterns and determinants.

Understanding the factors that determine the rate at which genomes generate and fix mutations provides important insights into key evolutionary mechanisms. We review our current knowledge of the rates of mutation and substitution, as well as their determinants, in RNA viruses, DNA viruses and retroviruses. We show that the high rate of nucleotide substitution in RNA viruses is matched by some DNA viruses, suggesting that evolutionary rates in viruses are explained by diverse aspects of viral biology, such as genomic architecture and replication speed, and not simply by polymerase fidelity.

Cassava brown streak virus evolves with a nucleotide-substitution rate that is typical for the family Potyviridae.

The ipomoviruses (family Potyviridae) that cause cassava brown streak disease (cassava brown streak virus [CBSV] and Uganda cassava brown streak virus [UCBSV]) are damaging plant pathogens that affect the sustainability of cassava production in East and Central Africa. However, little is known about the rate at which the viruses evolve and when they emerged in Africa - which inform how easily these viruses can host shift and resist RNAi approaches for control. We present here the rates of evolution determined from the coat protein gene (CP) of CBSV (Temporal signal in a UCBSV dataset was not sufficient for comparable analysis). Our BEAST analysis estimated the CBSV CP evolves at a mean rate of 1.43 × 10-3 nucleotide substitutions per site per year, with the most recent common ancestor of sampled CBSV isolates existing in 1944 (95% HPD, between years 1922 - 1963). We compared the published measured and estimated rates of evolution of CPs from ten families of plant viruses and showed that CBSV is an average-evolving potyvirid, but that members of Potyviridae evolve more quickly than members of Virgaviridae and the single representatives of Betaflexiviridae, Bunyaviridae, Caulimoviridae and Closteroviridae.

A multispecies polyadenylation site model.

BACKGROUND: Polyadenylation is present in all three domains of life, making it the most conserved post-transcriptional process compared with splicing and 5'-capping. Even though most mammalian poly(A) sites contain a highly conserved hexanucleotide in the upstream region and a far less conserved U/GU-rich sequence in the downstream region, there are many exceptions. Furthermore, poly(A) sites in other species, such as plants and invertebrates, exhibit high deviation from this genomic structure, making the construction of a general poly(A) site recognition model challenging. We surveyed nine poly(A) site prediction methods published between 1999 and 2011. All methods exploit the skewed nucleotide profile across the poly(A) sites, and the highly conserved poly(A) signal as the primary features for recognition. These methods typically use a large number of features, which increases the dimensionality of the models to crippling degrees, and typically are not validated against many kinds of genomes. RESULTS: We propose a poly(A) site model that employs minimal features to capture the essence of poly(A) sites, and yet, produces better prediction accuracy across diverse species. Our model consists of three dior-trinucleotide profiles identified through principle component analysis, and the predicted nucleosome occupancy flanking the poly(A) sites. We validated our model using two machine learning methods: logistic regression and linear discriminant analysis. Results show that models achieve 85-92% sensitivity and 85-96% specificity in seven animals and plants. When we applied one model from one species to predict poly(A) sites from other species, the sensitivity scores correlate with phylogenetic distances. CONCLUSIONS: A four-feature model geared towards small motifs was sufficient to accurately learn and predict poly(A) sites across eukaryotes.

Synonymous site variation due to recombination explains higher genetic variability in begomovirus populations infecting non-cultivated hosts.

Begomoviruses are ssDNA plant viruses that cause serious epidemics in economically important crops worldwide. Non-cultivated plants also harbour many begomoviruses, and it is believed that these hosts may act as reservoirs and as mixing vessels where recombination may occur. Begomoviruses are notoriously recombination-prone, and also display nucleotide substitution rates equivalent to those of RNA viruses. In Brazil, several indigenous begomoviruses have been described infecting tomatoes following the introduction of a novel biotype of the whitefly vector in the mid-1990s. More recently, a number of viruses from non-cultivated hosts have also been described. Previous work has suggested that viruses infecting non-cultivated hosts have a higher degree of genetic variability compared with crop-infecting viruses. We intensively sampled cultivated and non-cultivated plants in similarly sized geographical areas known to harbour either the weed-infecting Macroptilium yellow spot virus (MaYSV) or the crop-infecting Tomato severe rugose virus (ToSRV), and compared the molecular evolution and population genetics of these two distantly related begomoviruses. The results reinforce the assertion that infection of non-cultivated plant species leads to higher levels of standing genetic variability, and indicate that recombination, not adaptive selection, explains the higher begomovirus variability in non-cultivated hosts.

Frequent coinfection reduces RNA virus population genetic diversity.

The masking of deleterious mutations by complementation and the reassortment of virus segments (virus sex) are expected to increase population genetic diversity among coinfecting viruses. Conversely, clonally reproducing or noncoinfecting virus populations may experience clonal interference where viral clones compete with one another, preventing selective sweeps. This dynamic reduces the efficiency of selection and increases the genetic diversity. To determine the relative influences of these forces on population genetic diversity, we evolved 6 populations of bacteriophage φ6 under conditions promoting or preventing coinfection. Following 300 generations, we isolated and partially sequenced 10 clones from each population. We found greater diversity among asexual populations than sexual populations. Moreover, sexual populations did not show greater relative fitnesses than asexual populations, implying that reduced genetic variation did not result from purifying selection. However, sexual populations were less genetically robust than asexual populations and likely more prone to the deleterious epistatic effects of mutations. As such, a neutral mutation on the asexually evolved (robust) background could be profoundly deleterious on the sexually evolved (brittle) background. This could facilitate sexual populations undergoing greater purifying selection to remove deleterious mutations, but this selection is not reflected by increases in average population fitness. Our results bolster a growing literature suggesting that RNA virus segmentation is probably not a mechanism that evolved because it provides a generalized benefit of sex.

The how of counter-defense: viral evolution to combat host immunity.

Viruses are locked in an evolutionary arms race with their hosts. What ultimately determines viral evolvability, or capacity for adaptive evolution, is their ability to efficiently explore and expand sequence space while under the selective regime imposed by their ecology, which includes innate and adaptive host defenses. Viral genomes have significantly higher evolutionary rates than their host counterparts and should have advantages relative to their slower-evolving hosts. However, functional constraints on virus evolutionary landscapes along with the modularity and mutational tolerance of host defense proteins may help offset the advantage conferred to viruses by high evolutionary rates. Additionally, cellular life forms from all domains of life possess many highly complex defense mechanisms that act as hurdles to viral replication. Consequently, viruses constantly probe sequence space through mutation and genetic exchange and are under pressure to optimize diverse counter-defense strategies.

Limited role of recombination in the global diversification of begomovirus DNA-B proteins.

Approximately half of the characterized begomoviruses have bipartite genomes, but the second genomic segment, the DNA-B, is understudied relative to the DNA-A, which is homologous to the entire genome of monopartite begomoviruses. We examined the evolutionary history of the two proteins encoded by the DNA-B, the genes of which make up ∼60% of the DNA-B segment, from all bipartite begomovirus species. Our dataset of 131 movement protein (MP) and nuclear shuttle protein (NSP) sequences confirmed the deep split between Old World (OW) and New World (NW) species, and showed strong support for deep, congruent branches among the OW sequences of the MP and NSP. NW sequences were much less diverse and had poor phylogenetic resolution; over half of nodes in both the NSP and MP NW clades were supported by <50% bootstrap support. This poor resolution hampered our ability to detect incongruent phylogenies between the MP and NSP datasets, and we found no statistical evidence for recombination within our MP and NSP datasets. Finally, we quantified the sequence diversity between the NW and OW proteins, showing that the NW MP has particularly low diversity, suggesting it has been subject to different evolutionary pressures than the NW NSP.