Incomplete bunyavirus particles can cooperatively support virus infection and spread.

Bunyaviruses lack a specific mechanism to ensure the incorporation of a complete set of genome segments into each virion, explaining the generation of incomplete virus particles lacking one or more genome segments. Such incomplete virus particles, which may represent the majority of particles produced, are generally considered to interfere with virus infection and spread. Using the three-segmented arthropod-borne Rift Valley fever virus as a model bunyavirus, we here show that two distinct incomplete virus particle populations unable to spread autonomously are able to efficiently complement each other in both mammalian and insect cells following co-infection. We further show that complementing incomplete virus particles can co-infect mosquitoes, resulting in the reconstitution of infectious virus that is able to disseminate to the mosquito salivary glands. Computational models of infection dynamics predict that incomplete virus particles can positively impact virus spread over a wide range of conditions, with the strongest effect at intermediate multiplicities of infection. Our findings suggest that incomplete particles may play a significant role in within-host spread and between-host transmission, reminiscent of the infection cycle of multipartite viruses.

Empirical estimates of the mutation rate for an alphabaculovirus.

Mutation rates are of key importance for understanding evolutionary processes and predicting their outcomes. Empirical mutation rate estimates are available for a number of RNA viruses, but few are available for DNA viruses, which tend to have larger genomes. Whilst some viruses have very high mutation rates, lower mutation rates are expected for viruses with large genomes to ensure genome integrity. Alphabaculoviruses are insect viruses with large genomes and often have high levels of polymorphism, suggesting high mutation rates despite evidence of proofreading activity by the replication machinery. Here, we report an empirical estimate of the mutation rate per base per strand copying (s/n/r) of Autographa californica multiple nucleopolyhedrovirus (AcMNPV). To avoid biases due to selection, we analyzed mutations that occurred in a stable, non-functional genomic insert after five serial passages in Spodoptera exigua larvae. Our results highlight that viral demography and the stringency of mutation calling affect mutation rate estimates, and that using a population genetic simulation model to make inferences can mitigate the impact of these processes on estimates of mutation rate. We estimated a mutation rate of µ = 1×10-7 s/n/r when applying the most stringent criteria for mutation calling, and estimates of up to µ = 5×10-7 s/n/r when relaxing these criteria. The rates at which different classes of mutations accumulate provide good evidence for neutrality of mutations occurring within the inserted region. We therefore present a robust approach for mutation rate estimation for viruses with stable genomes, and strong evidence of a much lower alphabaculovirus mutation rate than supposed based on the high levels of polymorphism observed.

Biological Characterization of Physostegia Chlorotic Mottle Virus, an Emergent Virus Infecting Vegetables in Diversified Production Systems.

In 2014, Physostegia chlorotic mottle virus (PhCMoV) was discovered in Austria in Physostegia virginiana. Subsequent collaborative efforts established a link between the virus and severe fruit symptoms on important crops such as tomato, eggplant, and cucumber across nine European countries. Thereafter, specific knowledge gaps, which are crucial to assess the risks PhCMoV can pose for production and how to manage it, needed to be addressed. In this study, the transmission, prevalence, and disease severity of PhCMoV were examined. This investigation led to the identification of PhCMoV presence in a new country, Switzerland. Furthermore, our research indicates that the virus was already present in Europe 30 years ago. Bioassays demonstrated PhCMoV can result in up to 100% tomato yield losses depending on the phenological stage of the plant at the time of infection. PhCMoV was found to naturally infect 12 new host plant species across eight families, extending its host range to 21 plant species across 15 plant families. The study also identified a polyphagous leafhopper (genus Anaceratagallia) as a natural vector of PhCMoV. Overall, PhCMoV was widespread in small-scale diversified vegetable farms in Belgium where tomato is grown in soil under tunnels, occurring in approximately one-third of such farms. However, outbreaks were sporadic and were associated at least once with the cultivation in tomato tunnels of perennial plants that can serve as a reservoir host for the virus and its vector. To further explore this phenomenon and manage the virus, studying the ecology of the vector would be beneficial.

Plant neighbours can make or break the disease transmission chain of a fungal root pathogen.

Biodiversity can reduce or increase disease transmission. These divergent effects suggest that community composition rather than diversity per se determines disease transmission. In natural plant communities, little is known about the functional roles of neighbouring plant species in belowground disease transmission. Here, we experimentally investigated disease transmission of a fungal root pathogen (Rhizoctonia solani) in two focal plant species in combinations with four neighbour species of two ages. We developed stochastic models to test the relative importance of two transmission-modifying mechanisms: (1) infected hosts serve as nutrient supply to increase hyphal growth, so that successful disease transmission is self-reinforcing; and (2) plant resistance increases during plant development. Neighbouring plants either reduced or increased disease transmission in the focal plants. These effects depended on neighbour age, but could not be explained by a simple dichotomy between hosts and nonhost neighbours. Model selection revealed that both transmission-modifying mechanisms are relevant and that focal host-neighbour interactions changed which mechanisms steered disease transmission rate. Our work shows that neighbour-induced shifts in the importance of these mechanisms across root networks either make or break disease transmission chains. Understanding how diversity affects disease transmission thus requires integrating interactions between focal and neighbour species and their pathogens.

Second compartment widens plasmid invasion conditions: Two-compartment pair-formation model of conjugation in the gut.

Understanding under which conditions conjugative plasmids encoding antibiotic resistance can invade bacterial communities in the gut is of particular interest to combat the spread of antibiotic resistance within and between animals and humans. We extended a one-compartment model of conjugation to a two-compartment model, to analyse how differences in plasmid dynamics in the gut lumen and at the gut wall affect the invasion of plasmids. We compared scenarios with one and two compartments, different migration rates between the lumen and wall compartments, and different population dynamics. We focused on the effect of attachment and detachment rates on plasmid dynamics, explicitly describing pair formation followed by plasmid transfer in the pairs. The parameter space allowing plasmid invasion in the one-compartment model is affected by plasmid costs and intrinsic conjugation rates of the transconjugant, but not by these characteristics of the donor. The parameter space allowing plasmid invasion in the two-compartment model is affected by attachment and detachment rates in the lumen and wall compartment, and by the bacterial density at the wall. The one- and two-compartment models predict the same parameter space for plasmid invasion if the conditions in both compartments are equal to the conditions in the one-compartment model. In contrast, the addition of the wall compartment widens the parameter space allowing invasion compared with the one-compartment model, if the density at the wall is higher than in the lumen, or if the attachment rate at the wall is high and the detachment rate at the wall is low. We also compared the pair-formation models with bulk-conjugation models that describe conjugation by instantaneous transfer of the plasmid at contact between cells, without explicitly describing pair formation. Our results show that pair-formation and bulk-conjugation models predict the same parameter space for plasmid invasion. From our simulations, we conclude that conditions at the gut wall should be taken into account to describe plasmid dynamics in the gut and that transconjugant characteristics rather than donor characteristics should be used to parameterize the models.

Chicken gut microbiome members limit the spread of an antimicrobial resistance plasmid in Escherichia coli.

Plasmid-mediated antimicrobial resistance is a major contributor to the spread of resistance genes within bacterial communities. Successful plasmid spread depends upon a balance between plasmid fitness effects on the host and rates of horizontal transmission. While these key parameters are readily quantified in vitro, the influence of interactions with other microbiome members is largely unknown. Here, we investigated the influence of three genera of lactic acid bacteria (LAB) derived from the chicken gastrointestinal microbiome on the spread of an epidemic narrow-range ESBL resistance plasmid, IncI1 carrying blaCTX-M-1, in mixed cultures of isogenic Escherichia coli strains. Secreted products of LAB decreased E. coli growth rates in a genus-specific manner but did not affect plasmid transfer rates. Importantly, we quantified plasmid transfer rates by controlling for density-dependent mating opportunities. Parametrization of a mathematical model with our in vitro estimates illustrated that small fitness costs of plasmid carriage may tip the balance towards plasmid loss under growth conditions in the gastrointestinal tract. This work shows that microbial interactions can influence plasmid success and provides an experimental-theoretical framework for further study of plasmid transfer in a microbiome context.

Effect of donor-recipient relatedness on the plasmid conjugation frequency: a meta-analysis.

BACKGROUND: Conjugation plays a major role in the transmission of plasmids encoding antibiotic resistance genes in both clinical and general settings. The conjugation efficiency is influenced by many biotic and abiotic factors, one of which is the taxonomic relatedness between donor and recipient bacteria. A comprehensive overview of the influence of donor-recipient relatedness on conjugation is still lacking, but such an overview is important to quantitatively assess the risk of plasmid transfer and the effect of interventions which limit the spread of antibiotic resistance, and to obtain parameter values for conjugation in mathematical models. Therefore, we performed a meta-analysis on reported conjugation frequencies from Escherichia coli donors to various recipient species. RESULTS: Thirty-two studies reporting 313 conjugation frequencies for liquid broth matings and 270 conjugation frequencies for filter matings were included in our meta-analysis. The reported conjugation frequencies varied over 11 orders of magnitude. Decreasing taxonomic relatedness between donor and recipient bacteria, when adjusted for confounding factors, was associated with a lower conjugation frequency in liquid matings. The mean conjugation frequency for bacteria of the same order, the same class, and other classes was 10, 20, and 789 times lower than the mean conjugation frequency within the same species, respectively. This association between relatedness and conjugation frequency was not found for filter matings. The conjugation frequency was furthermore found to be influenced by temperature in both types of mating experiments, and in addition by plasmid incompatibility group in liquid matings, and by recipient origin and mating time in filter matings. CONCLUSIONS: In our meta-analysis, taxonomic relatedness is limiting conjugation in liquid matings, but not in filter matings, suggesting that taxonomic relatedness is not a limiting factor for conjugation in environments where bacteria are fixed in space.

Adaptive benefits from small mutation supplies in an antibiotic resistance enzyme.

Populations with large mutation supplies adapt via the "greedy" substitution of the fittest genotype available, leading to fast and repeatable short-term responses. At longer time scales, smaller mutation supplies may in theory lead to larger improvements when distant high-fitness genotypes more readily evolve from lower-fitness intermediates. Here we test for long-term adaptive benefits from small mutation supplies using in vitro evolution of an antibiotic-degrading enzyme in the presence of a novel antibiotic. Consistent with predictions, large mutant libraries cause rapid initial adaptation via the substitution of cohorts of mutations, but show later deceleration and convergence. Smaller libraries show on average smaller initial, but also more variable, improvements, with two lines yielding alleles with exceptionally high resistance levels. These two alleles share three mutations with the large-library alleles, which are known from previous work, but also have unique mutations. Replay evolution experiments and analyses of the adaptive landscape of the enzyme suggest that the benefit resulted from a combination of avoiding mutational cohorts leading to local peaks and chance. Our results demonstrate adaptive benefits from limited mutation supplies on a rugged fitness landscape, which has implications for artificial selection protocols in biotechnology and argues for a better understanding of mutation supplies in clinical settings.

Going, going, gone: predicting the fate of genomic insertions in plant RNA viruses.

Horizontal gene transfer is common among viruses, while they also have highly compact genomes and tend to lose artificial genomic insertions rapidly. Understanding the stability of genomic insertions in viral genomes is therefore relevant for explaining and predicting their evolutionary patterns. Here, we revisit a large body of experimental research on a plant RNA virus, tobacco etch potyvirus (TEV), to identify the patterns underlying the stability of a range of homologous and heterologous insertions in the viral genome. We obtained a wide range of estimates for the recombination rate-the rate at which deletions removing the insertion occur-and these appeared to be independent of the type of insertion and its location. Of the factors we considered, recombination rate was the best predictor of insertion stability, although we could not identify the specific sequence characteristics that would help predict insertion instability. We also considered experimentally the possibility that functional insertions lead to higher mutational robustness through increased redundancy. However, our observations suggest that both functional and non-functional increases in genome size decreased the mutational robustness. Our results therefore demonstrate the importance of recombination rates for predicting the long-term stability and evolution of viral RNA genomes and suggest that there are unexpected drawbacks to increases in genome size for mutational robustness.

Unraveling the causes of adaptive benefits of synonymous mutations in TEM-1 ß-lactamase.

While synonymous mutations were long thought to be without phenotypic consequences, there is growing evidence they can affect gene expression, protein folding, and ultimately the fitness of an organism. In only a few cases have the mechanisms by which synonymous mutations affect the phenotype been elucidated. We previously identified 48 mutations in TEM-1 ß-lactamase that increased resistance of Escherichia coli to cefotaxime, 10 of which were synonymous. To better understand the molecular mechanisms underlying the beneficial effect of these synonymous mutations, we made a series of measurements for a panel containing the 10 synonymous together with 10 non-synonymous mutations as a reference. Whereas messenger levels were unaffected, we found that total and functional TEM protein levels were higher for 5 out of 10 synonymous mutations. These observations suggest that some of these mutations act on translation or a downstream process. Similar effects were observed for some small-benefit non-synonymous mutations, suggesting a similar causal mechanism. For the synonymous mutations, we found that the cost of resistance scales with TEM protein levels. A resistance landscape for four synonymous mutations revealed strong epistasis: none of the combinations of mutations exceeded the resistance of the largest-effect mutation and there were synthetically neutral combinations. By considering combined effects of these mutations, we could infer that functional TEM protein level is a multi-dimensional phenotype. These results suggest that synonymous mutations may have beneficial effects by increasing the expression of an enzyme with low substrate activity, which may be realized via multiple, yet unknown, post-transcriptional mechanisms.

Quantitative analysis of the dose-response of white spot syndrome virus in shrimp.

White spot syndrome virus (WSSV) is an important cause of mortality and economic losses in shrimp farming. Although WSSV-induced mortality is virus dose dependent and WSSV infection does not necessarily lead to mortality, the relationships between virus-particle dose, infection and mortality have not been analysed quantitatively. Here, we explored WSSV dose-response by a combination of experiments, modelling and meta-analysis. We performed dose-response experiments in Penaeus vannamei postlarvae, recorded host mortality and detected WSSV infection. When we fitted infection models to these data, two models-differing in whether they incorporated heterogeneous host susceptibility to the virus or not-were supported for two independent experiments. To determine the generality of these results, we reanalysed published data sets and then performed a meta-analysis. We found that WSSV dose-response kinetics is indeed variable over experiments. We could not clearly identify which specific infection model has the most support by meta-analysis, but we argue that these results also are most concordant with a model incorporating varying levels of heterogeneous host susceptibility to WSSV. We have identified suitable models for analysing WSSV dose-response, which can elucidate the most basic virus-host interactions and help to avoid underestimating WSSV infection at low virus doses.

High virulence does not necessarily impede viral adaptation to a new host: a case study using a plant RNA virus.

BACKGROUND: Theory suggests that high virulence could hinder between-host transmission of microparasites, and that virulence therefore will evolve to lower levels. Alternatively, highly virulent microparasites could also curtail host development, thereby limiting both the host resources available to them and their own within-host effective population size. In this case, high virulence might restrain the mutation supply rate and increase the strength with which genetic drift acts on microparasite populations. Thereby, this alternative explanation limits the microparasites' potential to adapt to the host and ultimately the ability to evolve lower virulence. As a first exploration of this hypothesis, we evolved Tobacco etch virus carrying an eGFP fluorescent marker in two semi-permissive host species, Nicotiana benthamiana and Datura stramonium, for which it has a large difference in virulence. We compared the results to those previously obtained in the natural host, Nicotiana tabacum, where we have shown that carriage of eGFP has a high fitness cost and its loss serves as a real-time indicator of adaptation. RESULTS: After over half a year of evolution, we sequenced the genomes of the evolved lineages and measured their fitness. During the evolution experiment, marker loss leading to viable virus variants was only observed in one lineage of the host for which the virus has low virulence, D. stramonium. This result was consistent with the observation that there was a fitness cost of eGFP in this host, while surprisingly no fitness cost was observed in the host for which the virus has high virulence, N. benthamiana. Furthermore, in both hosts we observed increases in viral fitness in few lineages, and host-specific convergent evolution at the genomic level was only found in N. benthamiana. CONCLUSIONS: The results of this study do not lend support to the hypothesis that high virulence impedes microparasites' evolution. Rather, they exemplify that jumps between host species can be game changers for evolutionary dynamics. When considering the evolution of genome architecture, host species jumps might play a very important role, by allowing evolutionary intermediates to be competitive.

Mutation supply and the repeatability of selection for antibiotic resistance.

Whether evolution can be predicted is a key question in evolutionary biology. Here we set out to better understand the repeatability of evolution, which is a necessary condition for predictability. We explored experimentally the effect of mutation supply and the strength of selective pressure on the repeatability of selection from standing genetic variation. Different sizes of mutant libraries of antibiotic resistance gene TEM-1 ß-lactamase in Escherichia coli, generated by error-prone PCR, were subjected to different antibiotic concentrations. We determined whether populations went extinct or survived, and sequenced the TEM gene of the surviving populations. The distribution of mutations per allele in our mutant libraries followed a Poisson distribution. Extinction patterns could be explained by a simple stochastic model that assumed the sampling of beneficial mutations was key for survival. In most surviving populations, alleles containing at least one known large-effect beneficial mutation were present. These genotype data also support a model which only invokes sampling effects to describe the occurrence of alleles containing large-effect driver mutations. Hence, evolution is largely predictable given cursory knowledge of mutational fitness effects, the mutation rate and population size. There were no clear trends in the repeatability of selected mutants when we considered all mutations present. However, when only known large-effect mutations were considered, the outcome of selection is less repeatable for large libraries, in contrast to expectations. We show experimentally that alleles carrying multiple mutations selected from large libraries confer higher resistance levels relative to alleles with only a known large-effect mutation, suggesting that the scarcity of high-resistance alleles carrying multiple mutations may contribute to the decrease in repeatability at large library sizes.

Within-host spatiotemporal dynamics of plant virus infection at the cellular level.

A multicellular organism is not a monolayer of cells in a flask; it is a complex, spatially structured environment, offering both challenges and opportunities for viruses to thrive. Whereas virus infection dynamics at the host and within-cell levels have been documented, the intermediate between-cell level remains poorly understood. Here, we used flow cytometry to measure the infection status of thousands of individual cells in virus-infected plants. This approach allowed us to determine accurately the number of cells infected by two virus variants in the same host, over space and time as the virus colonizes the host. We found a low overall frequency of cellular infection (<0.3), and few cells were coinfected by both virus variants (<0.1). We then estimated the cellular contagion rate (R), the number of secondary infections per infected cell per day. R ranged from 2.43 to values not significantly different from zero, and generally decreased over time. Estimates of the cellular multiplicity of infection (MOI), the number of virions infecting a cell, were low (<1.5). Variance of virus-genotype frequencies increased strongly from leaf to cell levels, in agreement with a low MOI. Finally, there were leaf-dependent differences in the ease with which a leaf could be colonized, and the number of virions effectively colonizing a leaf. The modeling of infection patterns suggests that the aggregation of virus-infected cells plays a key role in limiting spread; matching the observation that cell-to-cell movement of plant viruses can result in patches of infection. Our results show that virus expansion at the between-cell level is restricted, probably due to the host environment and virus infection itself.

Temporal dynamics of intrahost molecular evolution for a plant RNA virus.

Populations of plant RNA viruses are highly polymorphic in infected plants, which may allow rapid within-host evolution. To understand tobacco etch potyvirus (TEV) evolution, longitudinal samples from experimentally evolved populations in the natural host tobacco and from the alternative host pepper were phenotypically characterized and genetically analyzed. Temporal and compartmental variabilities of TEV populations were quantified using high throughput Illumina sequencing and population genetic approaches. Of the two viral phenotypic traits measured, virulence increased in the novel host but decreased in the original one, and viral load decreased in both hosts, though to a lesser extent in the novel one. Dynamics of population genetic diversity were also markedly different among hosts. Population heterozygosity increased in the ancestral host, with a dominance of synonymous mutations fixed, whereas it did not change or even decreased in the new host, with an excess of nonsynonymous mutations. All together, these observations suggest that directional selection is the dominant evolutionary force in TEV populations evolving in a novel host whereas either diversifying selection or random genetic drift may play a fundamental role in the natural host. To better understand these evolutionary dynamics, we developed a computer simulation model that incorporates the effects of mutation, selection, and drift. Upon parameterization with empirical data from previous studies, model predictions matched the observed patterns, thus reinforcing our idea that the empirical patterns of mutation accumulation represent adaptive evolution.

Experimental evolution of pseudogenization and gene loss in a plant RNA virus.

Viruses have evolved highly streamlined genomes and a variety of mechanisms to compress them, suggesting that genome size is under strong selection. Horizontal gene transfer has, on the other hand, played an important role in virus evolution. However, evolution cannot integrate initially nonfunctional sequences into the viral genome if they are rapidly purged by selection. Here we report on the experimental evolution of pseudogenization in virus genomes using a plant RNA virus expressing a heterologous gene. When long 9-week passages were performed, the added gene was lost in all lineages, whereas viruses with large genomic deletions were fixed in only two out of ten 3-week lineages and none in 1-week lineages. Illumina next-generation sequencing revealed considerable convergent evolution in the 9- and 3-week lineages with genomic deletions. Genome size was correlated to within-host competitive fitness, although there was no correlation with virus accumulation or virulence. Within-host competitive fitness of the 3-week virus lineages without genomic deletions was higher than for the 1-week lineages. Our results show that the strength of selection for a reduced genome size and the rate of pseudogenization depend on demographic conditions. Moreover, for the 3-week passage condition, we observed increases in within-host fitness, whereas selection was not strong enough to quickly remove the nonfunctional heterologous gene. These results suggest a demographically determined "sweet spot" might exist, where heterologous insertions are not immediately lost while evolution can act to integrate them into the viral genome.

Relocation of the NIb gene in the tobacco etch potyvirus genome.

Potyviruses express most of their proteins from a long open reading frame that is translated into a large polyprotein processed by three viral proteases. To understand the constraints on potyvirus genome organization, we relocated the viral RNA-dependent RNA polymerase (NIb) cistron to all possible intercistronic positions of the Tobacco etch virus (TEV) polyprotein. Only viruses with NIb at the amino terminus of the polyprotein or in between P1 and HC-Pro were viable in tobacco plants.

A viral protein mediates superinfection exclusion at the whole-organism level but is not required for exclusion at the cellular level.

UNLABELLED: Superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by the same or a closely related virus, has been described for different viruses, including important pathogens of humans, animals, and plants. Citrus tristeza virus (CTV), a positive-sense RNA virus, represents a valuable model system for studying SIE due to the existence of several phylogenetically distinct strains. Furthermore, CTV allows SIE to be examined at the whole-organism level. Previously, we demonstrated that SIE by CTV is a virus-controlled function that requires the viral protein p33. In this study, we show that p33 mediates SIE at the whole-organism level, while it is not required for exclusion at the cellular level. Primary infection of a host with a fluorescent protein-tagged CTV variant lacking p33 did not interfere with the establishment of a secondary infection by the same virus labeled with a different fluorescent protein. However, cellular coinfection by both viruses was rare. The obtained observations, along with estimates of the cellular multiplicity of infection (MOI) and MOI model selection, suggested that low levels of cellular coinfection appear to be best explained by exclusion at the cellular level. Based on these results, we propose that SIE by CTV is operated at two levels--the cellular and the whole-organism levels--by two distinct mechanisms that could function independently. This novel aspect of viral SIE highlights the intriguing complexity of this phenomenon, further understanding of which may open up new avenues to manage virus diseases. IMPORTANCE: Many viruses exhibit superinfection exclusion (SIE), the ability of an established virus infection to interfere with a secondary infection by related viruses. SIE plays an important role in the pathogenesis and evolution of virus populations. The observations described here suggest that SIE could be controlled independently at different levels of the host: the whole-organism level or the level of individual cells. The p33 protein of citrus tristeza virus (CTV), an RNA virus, was shown to mediate SIE at the whole-organism level, while it appeared not to be required for exclusion at the cellular level. SIE by CTV is, therefore, highly complex and appears to use mechanisms different from those proposed for other viruses. A better understanding of this phenomenon may lead to the development of new strategies for controlling viral diseases in human populations and agroecosystems.

Testing the independent action hypothesis of plant pathogen mode of action: a simple and powerful new approach.

The independent action hypothesis is a simple model of pathogen infection that can make many useful predictions on infection kinetics and, therefore, a number of different tests of independent action have been developed. However, some of these analyses are rather sophisticated, limiting their appeal to experimentalists, and it is also unclear how well the different tests perform. Here, we developed and evaluated a simple and robust new test of independent action. Our new test is based on using a constant inoculum dose of one pathogen variant, varying the dose of a second variant, and then quantifying the infection response for the first variant. We simulated infection data in which we introduced deviations from independent action, experimental variation, or both. Simulations showed that our new procedure has many advantages over the existing tests of independent action, especially if only systemic-infection data are available. We also performed experimental tests of our new procedure using two marked Tobacco etch virus (TEV) variants. We found minor deviations from the independent action model, which were not detected by previous tests using existing methods, exemplifying the utility of this approach. We discuss the implications for TEV infection kinetics and consider how to reconcile different dose-dependent effects.

Estimation of the in vivo recombination rate for a plant RNA virus.

Phylogenomic evidence suggested that recombination is an important evolutionary force for potyviruses, one of the larger families of plant RNA viruses. However, mixed-genotype potyvirus infections are marked by low levels of cellular coinfection, precluding template switching and recombination events between virus genotypes during genomic RNA replication. To reconcile these conflicting observations, we evaluated the in vivo recombination rate (rg) of Tobacco etch virus (TEV; genus Potyvirus, family Potyviridae) by coinfecting plants with pairs of genotypes marked with engineered restriction sites as neutral markers. The recombination rate was then estimated using two different approaches: (i) a classical approach that assumed recombination between marked genotypes can occur in the whole virus population, rendering an estimate of rg = 7.762 × 10(-8) recombination events per nucleotide site per generation, and (ii) an alternative method that assumed recombination between marked genotypes can occur only in coinfected cells, rendering a much higher estimate of rg = 3.427 × 10(-5) recombination events per nucleotide site per generation. This last estimate is similar to the TEV mutation rate, suggesting that recombination should be at least as important as point mutation in creating variability. Finally, we compared our mutation and recombination rate estimates to those reported for animal RNA viruses. Our analysis suggested that high recombination rates may be an unavoidable consequence of selection for fast replication at the cost of low fidelity.