Showing papers in "Virus Evolution in 2015"

RDP4: Detection and analysis of recombination patterns in virus genomes.

Abstract: RDP4 is the latest version of recombination detection program (RDP), a Windows computer program that implements an extensive array of methods for detecting and visualising recombination in, and stripping evidence of recombination from, virus genome sequence alignments. RDP4 is capable of analysing twice as many sequences (up to 2,500) that are up to three times longer (up to 10 Mb) than those that could be analysed by older versions of the program. RDP4 is therefore also applicable to the analysis of bacterial full-genome sequence datasets. Other novelties in RDP4 include (1) the capacity to differentiate between recombination and genome segment reassortment, (2) the estimation of recombination breakpoint confidence intervals, (3) a variety of ‘recombination aware’ phylogenetic tree construction and comparison tools, (4) new matrix-based visualisation tools for examining both individual recombination events and the overall phylogenetic impacts of multiple recombination events and (5) new tests to detect the influences of gene arrangements, encoded protein structure, nucleic acid secondary structure, nucleotide composition, and nucleotide diversity on recombination breakpoint patterns. The key feature of RDP4 that differentiates it from other recombination detection tools is its flexibility. It can be run either in fully automated mode from the command line interface or with a graphically rich user interface that enables detailed exploration of both individual recombination events and overall recombination patterns.

The evolution, diversity and host associations of rhabdoviruses

Abstract: Metagenomic studies are leading to the discovery of a hidden diversity of RNA viruses. These new viruses are poorly characterized and new approaches are needed predict the host species these viruses pose a risk to. The rhabdoviruses are a diverse family of RNA viruses that includes important pathogens of humans, animals, and plants. We have discovered thirty-two new rhabdoviruses through a combination of our own RNA sequencing of insects and searching public sequence databases. Combining these with previously known sequences we reconstructed the phylogeny of 195 rhabdovirus sequences, and produced the most in depth analysis of the family to date. In most cases we know nothing about the biology of the viruses beyond the host they were identified from, but our dataset provides a powerful phylogenetic approach to predict which are vector-borne viruses and which are specific to vertebrates or arthropods. By reconstructing ancestral and present host states we found that switches between major groups of hosts have occurred rarely during rhabdovirus evolution. This allowed us to propose seventy-six new likely vector-borne vertebrate viruses among viruses identified from vertebrates or biting insects. Based on currently available data, our analysis suggests it is likely there was a single origin of the known plant viruses and arthropod-borne vertebrate viruses, while vertebrate- and arthropod-specific viruses arose at least twice. There are also few transitions between aquatic and terrestrial ecosystems. Viruses also cluster together at a finer scale, with closely related viruses tending to be found in closely related hosts. Our data therefore suggest that throughout their evolution, rhabdoviruses have occasionally jumped between distantly related host species before spreading through related hosts in the same environment. This approach offers a way to predict the most probable biology and key traits of newly discovered viruses.

Editorial: A home for virology, ecology, epidemiology, and evolutionary biology.

Abstract: Viruses make headline news on an almost daily basis. Sometimes the news is positive, a report on the development of new anti-viral drugs or a reduction in transmission, perhaps. However, often the story will relate to a gloomier theme, for example, the appearance of new viral epidemics, the evolution of drug resistance, or falling vaccine coverage. The appearance of Ebola virus in West Africa since 2014 represents just the latest of a long series of devastating viruses that have emerged or expanded in humans in recent years, including Middle East Respiratory Syndrome (MERS) and Severe Acute Respiratory Syndrome (SARS) coronaviruses, Chikungunya virus, highly pathogenic avian influenza viruses, West Nile virus, and various human enteroviruses, and bunyaviruses. This list is both selective and anthropocentric and excludes numerous new epidemics in livestock (e.g., Schmallenberg virus), crop (e.g., tomato torrado virus), and wild animal populations (e.g., phocine distemper virus). The impacts of viral epidemics may extend beyond death and illness to cause substantial economic losses and social instability. Such effects are not limited to new or exotic viruses, as established and well-characterized viral diseases persist despite tremendous efforts to control and eradicate them. Important pathogens in this category include HIV/AIDS, human influenza viruses, dengue viruses, hepatitis viruses, human papillomaviruses, and rabies virus. One reason that viruses are such potent adversaries is their great potential for genetic diversity and evolvability, a characteristic that they owe to a combination of short generation times, very large population sizes, and (in some but not all instances) error-prone replication mechanisms. Strains that escape host immune responses, are resistant to … [↵][1]*Corresponding author. E-mail: sfelena{at}ibmcp.upv.es; oliver.pybus{at}zoo.ox.ac.uk. [1]: #xref-corresp-1-1

The global distribution of Banana bunchy top virus reveals little evidence for frequent recent, human-mediated long distance dispersal events.

Abstract: Banana bunchy top virus (BBTV; family Nanoviridae, genus Babuvirus) is a multi-component single-stranded DNA virus, which infects banana plants in many regions of the world, often resulting in large-scale crop losses. Weanalyzed 171 banana leaf samples from fourteen countries and recovered, cloned, and sequenced 855 complete BBTV components including ninety-four full genomes. Importantly, full genomes were determined from eight countries, where previously no full genomes were available (Samoa, Burundi, Republic of Congo, Democratic Republic of Congo, Egypt, Indonesia, the Philippines, and the USA [HI]). Accounting for recombination and genome component reassortment, we examined the geographic structuring of global BBTV populations to reveal that BBTV likely originated in Southeast Asia, that the current global hotspots of BBTV diversity are Southeast Asia/Far East and India, and that BBTV populations circulating elsewhere in the world have all potentially originated from infrequent introductions. Most importantly, we find that rather than the current global BBTV distribution being due to increases in human-mediated movements of bananas over the past few decades, it is more consistent with a pattern of infrequent introductions of the virus to different parts of the world over the past 1,000 years.

Host ecology determines the dispersal patterns of a plant virus.

Abstract: Since its isolation in 1966 in Kenya, rice yellow mottle virus (RYMV) has been reported throughout Africa resulting in one of the economically most important tropical plant emerging diseases. A thorough understanding of RYMV evolution and dispersal is critical to manage viral spread in tropical areas that heavily rely on agriculture for subsistence. Phylogenetic analyses have suggested a relatively recent expansion, perhaps driven by the intensification of agricultural practices, but this has not yet been examined in a coherent statistical framework. To gain insight into the historical spread of RYMV within Africa rice cultivations, we analyse a dataset of 300 coat protein gene sequences, sampled from East to West Africa over a 46-year period, using Bayesian evolutionary inference. Spatiotemporal reconstructions date the origin of RMYV back to 1852 (1791-1903) and confirm Tanzania as the most likely geographic origin. Following a single long-distance transmission event from East to West Africa, separate viral populations have been maintained for about a century. To identify the factors that shaped the RYMV distribution, we apply a generalised linear model (GLM) extension of discrete phylogenetic diffusion and provide strong support for distances measured on a rice connectivity landscape as the major determinant of RYMV spread. Phylogeographic estimates in continuous space further complement this by demonstrating more pronounced expansion dynamics in West Africa that are consistent with agricultural intensification and extensification. Taken together, our principled phylogeographic inference approach shows for the first time that host ecology dynamics have shaped the historical spread of a plant virus.

Role of the host restriction factor APOBEC3 on papillomavirus evolution.

Abstract: More than 270 different types of papillomaviruses have been discovered in a wide array of animal species. Despite the great diversity of papillomaviruses, little is known about the evolutionary processes that drive host tropism and the emergence of oncogenic genotypes. Although host defense mechanisms have evolved to interfere with various aspects of a virus life cycle, viruses have also coevolved copious strategies to avoid host antiviral restriction. Our and other studies have shown that the cytidine deaminase APOBEC3 family members edit HPV genomes and restrict virus infectivity. Thus, we hypothesized that host restriction by APOBEC3 served as selective pressure during papillomavirus evolution. To test this hypothesis, we analyzed the relative abundance of all dinucleotide sequences in full-length genomes of 274 papillomavirus types documented in the Papillomavirus Episteme database (PaVE). Here, we report that TC dinucleotides, the preferred target sequence of several human APOBEC3 proteins (hA3A, hA3B, hA3F, and hA3H), are highly depleted in papillomavirus genomes. Given that HPV infection is highly tissue-specific, the expression levels of APOBEC3 family members were analyzed. The basal expression levels of all APOBEC3 isoforms, excluding hA3B, are significantly higher in mucosal skin compared with cutaneous skin. Interestingly, we reveal that Alphapapillomaviruses (alpha-PVs), a majority of which infects anogenital mucosa, display the most dramatic reduction in TC dinucleotide content. Computer modeling and reconstruction of ancestral alpha-PV genomes suggest that TC depletion occurred after the alpha-PVs diverged from their most recent common ancestor. In addition, we found that TC depletion in alpha-PVs is greatly affected by protein coding potential. Taken together, our results suggest that PVs replicating in tissues with high APOBEC3 levels may have evolved to evade restriction by selecting for variants that contain reduced APOBEC3 target sites in their genomes.

A diverse group of small circular ssDNA viral genomes in human and non-human primate stools.

Abstract: Viral metagenomics sequencing of fecal samples from outbreaks of acute gastroenteritis from the US revealed the presence of small circular ssDNA viral genomes encoding a replication initiator protein (Rep). Viral genomes were ∼2.5 kb in length, with bi-directionally oriented Rep and capsid (Cap) encoding genes and a stem loop structure downstream of Rep. Several genomes showed evidence of recombination. By digital screening of an in-house virome database (1.04 billion reads) using BLAST, we identified closely related sequences from cases of unexplained diarrhea in France. Deep sequencing and PCR detected such genomes in 7 of 25 US (28 percent) and 14 of 21 French outbreaks (67 percent). One of eighty-five sporadic diarrhea cases in the Gambia was positive by PCR. Twenty-two complete genomes were characterized showing that viruses from patients in the same outbreaks were closely related suggesting common origins. Similar genomes were also characterized from the stools of captive chimpanzees, a gorilla, a black howler monkey, and a lemur that were more diverse than the human stool-associated genomes. The name smacovirus is proposed for this monophyletic viral clade. Possible tropism include mammalian enteric cells or ingested food components such as infected plants. No evidence of viral amplification was found in immunodeficient mice orally inoculated with smacovirus-positive stool supernatants. A role for smacoviruses in diarrhea, if any, remains to be demonstrated.

Elucidating the phylodynamics of endemic rabies virus in eastern Africa using whole-genome sequencing

Abstract: Many of the pathogens perceived to pose the greatest risk to humans are viral zoonoses, responsible for a range of emerging and endemic infectious diseases. Phylogeography is a useful tool to understand the processes that give rise to spatial patterns and drive dynamics in virus populations. Increasingly, whole-genome information is being used to uncover these patterns, but the limits of phylogenetic resolution that can be achieved with this are unclear. Here, whole-genome variation was used to uncover fine-scale population structure in endemic canine rabies virus circulating in Tanzania. This is the first whole-genome population study of rabies virus and the first comprehensive phylogenetic analysis of rabies virus in East Africa, providing important insights into rabies transmission in an endemic system. In addition, sub-continental scale patterns of population structure were identified using partial gene data and used to determine population structure at larger spatial scales in Africa. While rabies virus has a defined spatial structure at large scales, increasingly frequent levels of admixture were observed at regional and local levels. Discrete phylogeographic analysis revealed long-distance dispersal within Tanzania, which could be attributed to human-mediated movement, and we found evidence of multiple persistent, co-circulating lineages at a very local scale in a single district, despite on-going mass dog vaccination campaigns. This may reflect the wider endemic circulation of these lineages over several decades alongside increased admixture due to human-mediated introductions. These data indicate that successful rabies control in Tanzania could be established at a national level, since most dispersal appears to be restricted within the confines of country borders but some coordination with neighbouring countries may be required to limit transboundary movements. Evidence of complex patterns of rabies circulation within Tanzania necessitates the use of whole-genome sequencing to delineate finer scale population structure that can that can guide interventions, such as the spatial scale and design of dog vaccination campaigns and dog movement controls to achieve and maintain freedom from disease.

Evolutionary reversion of live viral vaccines: Can genetic engineering subdue it?

Abstract: Attenuated, live viral vaccines have been extraordinarily successful in protecting against many diseases. The main drawbacks in their development and use have been reliance on an unpredictable method of attenuation and the potential for evolutionary reversion to high virulence. Methods of genetic engineering now provide many safer alternatives to live vaccines, so if live vaccines are to compete with these alternatives in the future, they must either have superior immunogenicity or they must be able to overcome these former disadvantages. Several live vaccine designs that were historically inaccessible are now feasible because of advances in genome synthesis. Some of those methods are addressed here, with an emphasis on whether they enable predictable levels of attenuation and whether they are stable against evolutionary reversion. These new designs overcome many of the former drawbacks and position live vaccines to be competitive with alternatives. Not only do new methods appear to retard evolutionary reversion enough to prevent vaccine-derived epidemics, but it may even be possible to permanently attenuate live vaccines that are transmissible but cannot evolve to higher virulence under prolonged adaptation.

Tobamoviruses have probably co-diverged with their eudicotyledonous hosts for at least 110 million years.

Abstract: A phylogeny has been calculated by maximum likelihood comparisons of the concatenated consensus protein sequences of 29 tobamoviruses shown to be non-recombinant. This phylogeny has statistically significant support throughout, including its basal branches. The viruses form eight lineages that are congruent with the taxonomy of the hosts from which each was first isolated and, with the exception of three of the twenty-nine species, all fall into three clusters that have either asterid or rosid or caryophyllid hosts (i.e. the major subdivisions of eudicotyledonous plants). A modified Mantel permutation test showed that the patristic distances of virus and host phylogenies are significantly correlated, especially when the three anomalously placed viruses are removed. When the internal branches of the virus phylogeny were collapsed the congruence decreased. The simplest explanation of this congruence of the virus and host phylogenies is that most tobamovirus lineages have co-diverged with their primary plant hosts for more than 110 million years, and only the brassica-infecting lineage originated from a major host switch from asterids to rosids. Their co-divergence seems to have been 'fuzzy' rather than 'strict', permitting viruses to switch hosts within major host clades. Our conclusions support those of a coalesence analysis of tobamovirus sequences, that used proxy node dating, but not a similar analysis of nucleotide sequences from dated samples, which concluded that the tobamoviruses originated only 100 thousand years ago.

Time dependence of evolutionary metrics during the 2009 pandemic influenza virus outbreak

Abstract: With the expansion of DNA sequencing technology, quantifying evolution during emerging viral outbreaks has become an important tool for scientists and public health officials. Although it is known that the degree of sequence divergence significantly affects the calculation of evolutionary metrics in viral outbreaks, the extent and duration of this effect during an actual outbreak remains unclear. We have analyzed how limited divergence time during an early viral outbreak affects the accuracy of molecular evolutionary metrics. Using sequence data from the first 25 months of the 2009 pandemic H1N1 (pH1N1) outbreak, we calculated each of three different standard evolutionary metrics—molecular clock rate (i.e., evolutionary rate), whole-gene dN/dS, and site-wise dN/dS—for hemagglutinin and neuraminidase, using increasingly longer time windows, from 1 month to 25 months. For the molecular clock rate, we found that at least 3–4 months of temporal divergence from the start of sampling was required to make precise estimates that also agreed with long-term values. For whole-gene dN/dS, we found that at least 2 months of data were required to generate precise estimates, but 6–9 months were required for estimates to approach their long term values. For site-wise dN/dS estimates, we found that at least 6 months of sampling divergence was required before the majority of sites had at least one mutation and were thus evolutionarily informative. Furthermore, 8 months of sampling divergence was required before the site-wise estimates appropriately reflected the distribution of values expected from known protein-structure-based evolutionary pressure in influenza. In summary, we found that evolutionary metrics calculated from gene sequence data in early outbreaks should be expected to deviate from their long-term estimates for at least several months after the initial emergence and sequencing of the virus.

The effect of ecosystem biodiversity on virus genetic diversity depends on virus species: A study of chiltepin-infecting begomoviruses in Mexico.

Abstract: Current declines in biodiversity put at risk ecosystem services that are fundamental for human welfare. Increasing evidence indicates that one such service is the ability to reduce virus emergence. It has been proposed that the reduction of virus emergence occurs at two levels: through a reduction of virus prevalence/transmission and, as a result of these epidemiological changes, through a limitation of virus genetic diversity. Although the former mechanism has been studied in a few host-virus interactions, very little is known about the association between ecosystem biodiversity and virus genetic diversity. To address this subject, we estimated genetic diversity, synonymous and non-synonymous nucleotide substitution rates, selection pressures, and frequency of recombinants and re-assortants in populations of Pepper golden mosaic virus (PepGMV) and Pepper huasteco yellow vein virus (PHYVV) that infect chiltepin plants in Mexico. We then analyzed how these parameters varied according to the level of habitat anthropization, which is the major cause of biodiversity loss. Our results indicated that genetic diversity of PepGMV (but not of PHYVV) populations increased with the loss of biodiversity at higher levels of habitat anthropization. This was mostly the consequence of higher rates of synonymous nucleotide substitutions, rather than of adaptive selection. The frequency of recombinants and re-assortants was higher in PepGMV populations infecting wild chiltepin than in those infecting cultivated ones, suggesting that genetic exchange is not the main mechanism for generating genetic diversity in PepGMV populations. These findings provide evidence that biodiversity may modulate the genetic diversity of plant viruses, but it may differentially affect even two closely related viruses. Our analyses may contribute to understanding the factors involved in virus emergence.

The genomes of many yam species contain transcriptionally active endogenous geminiviral sequences that may be functionally expressed.

Abstract: Endogenous viral sequences are essentially 'fossil records' that can sometimes reveal the genomic features of long extinct virus species. Although numerous known instances exist of single-stranded DNA (ssDNA) genomes becoming stably integrated within the genomes of bacteria and animals, there remain very few examples of such integration events in plants. The best studied of these events are those which yielded the geminivirus-related DNA elements found within the nuclear genomes of various Nicotiana species. Although other ssDNA virus-like sequences are included within the draft genomes of various plant species, it is not entirely certain that these are not contaminants. The Nicotiana geminivirus-related DNA elements therefore remain the only definitively proven instances of endogenous plant ssDNA virus sequences. Here, we characterize two new classes of endogenous plant virus sequence that are also apparently derived from ancient geminiviruses in the genus Begomovirus. These two endogenous geminivirus-like elements (EGV1 and EGV2) are present in the Dioscorea spp. of the Enantiophyllum clade. We used fluorescence in situ hybridization to confirm that the EGV1 sequences are integrated in the D. alata genome and showed that one or two ancestral EGV sequences likely became integrated more than 1.4 million years ago during or before the diversification of the Asian and African Enantiophyllum Dioscorea spp. Unexpectedly, we found evidence of natural selection actively favouring the maintenance of EGV-expressed replication-associated protein (Rep) amino acid sequences, which clearly indicates that functional EGV Rep proteins were probably expressed for prolonged periods following endogenization. Further, the detection in D. alata of EGV gene transcripts, small 21-24 nt RNAs that are apparently derived from these transcripts, and expressed Rep proteins, provides evidence that some EGV genes are possibly still functionally expressed in at least some of the Enantiophyllum clade species.

CodonShuffle: a tool for generating and analyzing synonymously mutated sequences.

Abstract: Because synonymous mutations do not change the amino acid sequence of a protein, they are generally considered to be selectively neutral. Empiric data suggest, however, that a significant fraction of viral mutational fitness effects may be attributable to synonymous mutation. Bias in synonymous codon usage in viruses may result from selection for translational efficiency, mutational bias, base pairing requirements in RNA structures, or even selection against specific dinucleotides by innate immune effectors. Experimental analyses of codon usage and genome evolution have been facilitated by advances in synthetic biology, which now make it feasible to generate viral genomes that contain large numbers of synonymous mutations. The generally pleiotropic effects of synonymous mutation on viral fitness have, at times, made it difficult to define the mechanistic basis for the observed attenuation of these heavily mutated viruses. We have addressed this problem by developing a bioinformatic tool for the generation and analysis of viral sequences with large-scale synonymous mutation. A variety of permutation strategies are applied to shuffle codons within an open reading frame. After measuring the dinucleotide frequency, codon usage, codon pair bias, and free energy of RNA folding for each permuted genome, we used z -score normalization and a least squares regression model to quantify their overall distance from the starting sequence. Using this approach, the user can easily identify a large number of synonymously mutated sequences with varying similarity to a wild-type genome across a range of nucleic-acid-based determinants of viral fitness. We believe that this tool will be useful in designing genomes for subsequent experimental studies of the fitness impacts of synonymous mutation.

Connecting within-host dynamics to the rate of viral molecular evolution

Abstract: Viruses evolve rapidly, providing a unique system for understanding the processes that influence rates of molecular evolution. Neutral theory posits that the evolutionary rate increases linearly with the mutation rate. The occurrence of deleterious mutations causes this relationship to break down at high mutation rates. Previous studies have identified this as an important phenomenon, particularly for RNA viruses which can mutate at rates near the extinction threshold. We propose that in addition to mutation dynamics, viral within-host dynamics can also affect the between-host evolutionary rate. We present an analytical model that predicts the neutral evolution rate for viruses as a function of both within-host parameters and deleterious mutations. To examine the effect of more detailed aspects of the virus life cycle, we also present a computational model that simulates acute virus evolution using target cell-limited dynamics. Using influenza A virus as a case study, we find that our simulation model can predict empirical rates of evolution better than a model lacking within-host details. The analytical model does not perform as well as the simulation model but shows how the within-host basic reproductive number influences evolutionary rates. These findings lend support to the idea that the mutation rate alone is not sufficient to predict the evolutionary rate in viruses, instead calling for improved models that account for viral within-host dynamics.

Determinants of taxonomic composition of plant viruses at the Nature Conservancy's Tallgrass Prairie Preserve, Oklahoma.

Abstract: The role of biotic and abiotic factors in shaping the diversity and composition of communities of plant viruses remain understudied, particularly in natural settings. In this study, we test the effects of host identity, location, and sampling year on the taxonomic composition of plant viruses in six native plant species [Ambrosia psilostachya (Asteraceae), Vernonia baldwinii (Asteraceae), Asclepias viridis (Asclepiadaceae), Ruellia humilis (Acanthaceae), Panicum virgatum (Poaceae) and Sorghastrum nutans (Poaceae)] from the Nature Conservancy's Tallgrass Prairie Preserve in northeastern Oklahoma. We sampled over 400 specimens of the target host plants from twenty sites (plots) in the Tallgrass Prairie Preserve over 4 years and tested them for the presence of plant viruses applying virus-like particle and double-stranded RNA enrichment methods. Many of the viral sequences identified could not be readily assigned to species, either due to their novelty or the shortness of the sequence. We thus grouped our putative viruses into operational viral taxonomic units for further analysis. Partial canonical correspondence analysis revealed that the taxonomic composition of plant viruses in the target species had a significant relationship with host species (P value: 0.001) but no clear relation with sampling site or year. Variation partitioning further showed that host identity explained about 2-5 per cent of the variation in plant virus composition. We could not interpret the significant relationship between virus composition and host plants with respect to host taxonomy or ecology. Only six operational viral taxonomic units had over 5 per cent incidence over a 4-year period, while the remainder exhibited sporadic infection of the target hosts. This study is the first of its kind to document the dynamics of the entire range of viruses in multiple plant species in a natural setting.

Experimental evolution of an RNA virus in cells with innate immunity defects

Abstract: Experimental evolution studies have shown that RNA viruses respond rapidly to directional selection and thus can adapt efficiently to changes in host cell tropism, antiviral drugs, or other imposed selective pressures. However, the evolution of RNA viruses under relaxed selection has been less extensively explored. Here, we evolved vesicular stomatitis virus in mouse embryonic fibroblasts knocked-out for PKR, a protein with a central role in antiviral innate immunity. Vesicular stomatitis virus adapted to PKR-negative mouse embryonic fibroblasts in a gene-specific manner, since the evolved viruses exhibited little or no fitness improvement in PKR-positive cells. Full-length sequencing revealed the presence of multiple parallel nucleotide substitutions arising in independent evolution lines. However, site-directed mutagenesis showed that the effects of these substitutions were not PKR dependent. In contrast, we found evidence for sign epistasis, such that a given substitution which was positively selected was strongly deleterious when tested as a single mutation. Our results suggest that virus evolution in cells with specific innate immunity defects may drive viral specialization. However, this process is not deterministic at the molecular level, probably because the fixation of mutations which are tolerated under a relaxed selection regime is governed mainly by random genetic drift.

Effect of mismatch repair on the mutation rate of bacteriophage ϕX174.

Abstract: Viral mutation rates vary widely in nature, yet the mechanistic and evolutionary determinants of this variability remain unclear. Small DNA viruses mutate orders of magnitude faster than their hosts despite using host-encoded polymerases for replication, which suggests these viruses may avoid post-replicative repair. Supporting this, the genome of bacteriophage ϕX174 is completely devoid of GATC sequence motifs, which are required for methyl-directed mismatch repair in Escherichia coli . Here, we show that restoration of the randomly expected number of GATC sites leads to an eightfold reduction in the rate of spontaneous mutation of the phage, without severely impairing its replicative capacity over the short term. However, the efficacy of mismatch repair in the presence of GATC sites is limited by inefficient methylation of the viral DNA. Therefore, both GATC avoidance and DNA under-methylation elevate the mutation rate of the phage relative to that of the host. We also found that the effects of GATC sites on the phage mutation rate vary extensively depending on their specific location within the phage genome. Finally, the mutation rate reduction afforded by GATC sites is fully reverted under stress conditions, which up-regulate repair pathways and expression of error-prone host polymerases such as heat and treatment with the base analog 5-fluorouracil, suggesting that access to repair renders the phage sensitive to stress-induced mutagenesis.

Co-infection with two strains of Brome mosaic bromovirus reveals common RNA recombination sites in different hosts.

Abstract: We have previously reported intra-segmental crossovers in Brome mosaic virus (BMV) RNAs. In this work, we studied the homologous recombination of BMV RNA in three different hosts: barley (Hordeum vulgare), Chenopodium quinoa, and Nicotiana benthamiana that were co-infected with two strains of BMV: Russian (R) and Fescue (F). Our work aimed at (1) establishing the frequency of recombination, (2) mapping the recombination hot spots, and (3) addressing host effects. The F and R nucleotide sequences differ from each other at many translationally silent nucleotide substitutions. We exploited this natural variability to track the crossover sites. Sequencing of a large number of cDNA clones revealed multiple homologous crossovers in each BMV RNA segment, in both the whole plants and protoplasts. Some recombination hot spots mapped at similar locations in different hosts, suggesting a role for viral factors, but other sites depended on the host. Our results demonstrate the chimeric ('mosaic') nature of the BMV RNA genome.