Selective Factors Associated with the Evolution of Codon Usage in Natural Populations of Arboviruses.

Arboviruses (arthropod borne viruses) have life cycles that include both vertebrate and invertebrate hosts with substantial differences in vector and host specificity between different viruses. Most arboviruses utilize RNA for their genetic material and are completely dependent on host tRNAs for their translation, suggesting that virus codon usage could be a target for selection. In the current study we analyzed the relative synonymous codon usage (RSCU) patterns of 26 arboviruses together with 25 vectors and hosts, including 8 vertebrates and 17 invertebrates. We used hierarchical cluster analysis (HCA) and principal component analysis (PCA) to identify trends in codon usage. HCA demonstrated that the RSCU of arboviruses reflects that of their natural hosts, but not that of dead-end hosts. Of the two major components identified by PCA, the first accounted for 62.1% of the total variance, and among the 59 codons analyzed in this study, the leucine codon CTG had the highest correlation with the first principal component, however isoleucine had the highest correlation during amino acid analysis. Nucleotide and dinucleotide composition were the variables that explained most of the total codon usage variance. The results suggest that the main factors driving the evolution of codon usage in arboviruses is based on the nucleotide and dinucleotide composition present in the host. Comparing codon usage of arboviruses and potential vector hosts can help identifying potential vectors for emerging arboviruses.

Antigenic diversification is correlated with increased thermostability in a mammalian virus.

The theory of plastogenetic congruence posits that ultimately, the pressure to maintain function in the face of biomolecular destabilization produces robustness. As temperature goes up so does destabilization. Thus, genetic robustness, defined as phenotypic constancy despite mutation, should correlate with survival during thermal challenge. We tested this hypothesis using vesicular stomatitis virus (VSV). We produced two sets of evolved strains after selection for higher thermostability by either preincubation at 37°C or by incubation at 40°C during infection. These VSV populations became more thermostable and also more fit in the absence of thermal selection, demonstrating an absence of tradeoffs. Eleven out of 12 evolved populations had a fixed, nonsynonymous substitution in the nucleocapsid (N) open reading frame. There was a partial correlation between thermostability and mutational robustness that was observed when the former was measured at 42°C, but not at 37°C. These results are consistent with our earlier work and suggest that the relationship between robustness and thermostability is complex. Surprisingly, many of the thermostable strains also showed increased resistance to monoclonal antibody and polyclonal sera, including sera from natural hosts. These data suggest that evolved thermostability may lead to antigenic diversification and an increased ability to escape immune surveillance in febrile hosts, and potentially to an improved robustness. These relationships have important implications not only in terms of viral pathogenesis, but also for the development of vaccine vectors and oncolytic agents.

RNA Viruses and RNAi: Quasispecies Implications for Viral Escape.

Due to high mutation rates, populations of RNA viruses exist as a collection of closely related mutants known as a quasispecies. A consequence of error-prone replication is the potential for rapid adaptation of RNA viruses when a selective pressure is applied, including host immune systems and antiviral drugs. RNA interference (RNAi) acts to inhibit protein synthesis by targeting specific mRNAs for degradation and this process has been developed to target RNA viruses, exhibiting their potential as a therapeutic against infections. However, viruses containing mutations conferring resistance to RNAi were isolated in nearly all cases, underlining the problems of rapid viral evolution. Thus, while promising, the use of RNAi in treating or preventing viral diseases remains fraught with the typical complications that result from high specificity of the target, as seen in other antiviral regimens.

RNA replication errors and the evolution of virus pathogenicity and virulence.

RNA viruses of plants and animals have polymerases that are error-prone and produce complex populations of related, but non-identical, genomes called quasispecies. While there are vast variations in mutation rates among these viruses, selection has optimized the exact error rate of each species to provide maximum speed of replication and amount of variation without losing the ability to replicate because of excessive mutation. High mutation rates result in the selection of populations increasingly robust, which means they are increasingly resistant to show phenotypic changes after mutation. It is possible to manipulate the mutation rate, either by the use of mutagens or by selection (or genetic manipulation) of fidelity mutants. These polymerases usually, but not always, perform as well as wild type (wt) during cell infection, but show major phenotypic changes during in vivo infection. Both high and low fidelity variants are attenuated when the wt virus is virulent in the host. Alternatively when wt infection is non-apparent, the variants show major restrictions to spread in the infected host. Manipulation of mutation rates may become a new strategy to develop attenuated vaccines for humans and animals.

Congruent evolution of fitness and genetic robustness in vesicular stomatitis virus.

Quasispecies theory is a case of mutation-selection balance for evolution at high mutation rates, such as those observed in RNA viruses. One of the main predictions of this model is the selection for robustness, defined as the ability of an organism to remain phenotypically unchanged in the face of mutation. We have used a collection of vesicular stomatitis virus strains that had been evolving either under positive selection or under random drift. We had previously shown that the former increase in fitness while the latter have overall fitness decreases (I. S. Novella, J. B. Presloid, T. Zhou, S. D. Smith-Tsurkan, B. E. Ebendick-Corpus, R. N. Dutta, K. L. Lust, and C. O. Wilke, J. Virol. 84:4960-4968, 2010). Here, we determined the robustness of these strains and demonstrated that strains under positive selection not only increase in fitness but also increase in robustness. In contrast, strains under drift not only decreased in fitness but also decreased in robustness. There was a good overall correlation between fitness and robustness. We also tested whether there was a correlation between fitness and thermostability, and we observed that the correlation was imperfect, indicating that the fitness effects of mutations are exerted in part at a level other than changing the resistance of the protein to temperature.

The role of environmental factors on the evolution of phenotypic diversity in vesicular stomatitis virus populations.

Virus adaptation to an ever-changing environment requires the availability of variants with phenotypes that can fulfil new requirements for replication. High mutation rates result in the generation of these variants. The factors that contribute to the maintenance or elimination of this diversity, however, are not fully understood. This study used a collection of vesicular stomatitis virus strains generated under different conditions to measure the extent of variation within each population, and tested the effects of several environmental factors on diversity. It was found that the host-cell type used for selection sometimes had an effect on the extent of variation and that there may be different levels of variation over time. Persistent infections promoted higher levels of diversity than acute infections, presumably due to complementation. In contrast, environmental heterogeneity, host breadth and the cell type used for testing (as opposed to the cell type used for selection) did not seem to have an effect on the amount of phenotypic diversity observed.

Specific and nonspecific host adaptation during arboviral experimental evolution.

During the past decade or so, there has been a substantial body of work to dissect arboviral evolution and to develop models of adaptation during host switching. Regardless of what species serve as host or vectors, and of the geographic distribution and the mechanisms of replication, arboviruses tend to have slow evolutionary rates in nature. The hypothesis that this is the result of replication in the disparate environments provided by host and vector did not receive solid experimental support in any of the many viral species tested. Instead, it seems that from the virus's point of view, either the two environments are sufficiently similar or one of the environments so dominates viral evolution that there is tolerance for suboptimal adaptation to the other environment. Replication in alternating environments has an unexpected cost in that there is decreased genetic variance that translates into a compromised adaptability for bypassed environments. Arboviruses under strong and continuous positive selection may have unusual patterns of genomic changes, with few or no mutations accumulated in the consensus sequence or with dN/dS values typically consistent with random drift in DNA-based organisms.

Genomic evolution of vesicular stomatitis virus strains with differences in adaptability.

Virus strains with a history of repeated genetic bottlenecks frequently show a diminished ability to adapt compared to strains that do not have such a history. These differences in adaptability suggest differences in either the rate at which beneficial mutations are produced, the effects of beneficial mutations, or both. We tested these possibilities by subjecting four populations (two controls and two mutants with lower adaptabilities) to multiple replicas of a regimen of positive selection and then determining the fitnesses of the progeny through time and the changes in the consensus, full-length sequences of 56 genomes. We observed that at a given number of passages, the overall fitness gains observed for control populations were larger than fitness gains in mutant populations. However, these changes did not correlate with differences in the numbers of mutations accumulated in the two types of genomes. This result is consistent with beneficial mutations having a lower beneficial effect on mutant strains. Despite the overall fitness differences, some replicas of one mutant strain at passage 50 showed fitness increases similar to those observed for the wild type. We hypothesized that these evolved, high-fitness mutants may have a lower robustness than evolved, high-fitness controls. Robustness is the ability of a virus to avoid phenotypic changes in the face of mutation. We confirmed our hypothesis in mutation-accumulation experiments that showed a normalized fitness loss that was significantly larger in mutant bottlenecked populations than in control populations.

Incongruent fitness landscapes, not tradeoffs, dominate the adaptation of vesicular stomatitis virus to novel host types.

Host radiation refers to the ability of parasites to adapt to new environments and expand or change their niches. Adaptation to one specific environment may involve a loss in adaptation to a second environment. Thus, fitness costs may impose limits to niche expansion and constitute the cost of specialization. Several reports have addressed the cost of host radiation in vesicular stomatitis virus (VSV), but in some cases the experimental setup may have resulted in the overestimation of fitness costs. To clarify this issue, experiments were carried out in which a reference strain of VSV was allowed to adapt to HeLa, MDCK and BHK-21 cells, and to a regime of alternation between HeLa and Madin-Darby canine kidney (MDCK) cells. Measurement of viral fitness on each cell type showed that most virus populations behaved as generalists, and increased in fitness in all environments. Tradeoffs, where a fitness increase in one environment led to a fitness decrease in another environment, were rare. These results highlight the importance of using appropriate methods to measure fitness in evolved virus populations, and provide further support to a model of evolutionary dynamics in which costs due to incongruent landscapes provided by different environments are more common than tradeoffs.

A linear relationship between fitness and the logarithm of the critical bottleneck size in vesicular stomatitis virus populations.

We explored the relationship between fitness change and population size during transmission in vesicular stomatitis populations of very high fitness. The results show a linear correlation between the logarithm of the critical bottleneck size (population size at which there are no significant fitness changes after 20 passages) and the initial fitness of the population. In addition, limits to fitness increases during large-population passages of very-high-fitness strains were abolished by increasing the population size during transmission, indicating that beneficial variation is still available in these populations.

Antagonistic pleiotropy involving promoter sequences in a virus.

Selection of specialist genotypes, that is, populations with limited niche width, promotes the maintenance of diversity. Specialization to a particular environment may have a cost in other environments, including fitness tradeoffs. When the tradeoffs are the result of mutations that have a beneficial effect in the selective environment but a deleterious effect in other environments, we have antagonistic pleiotropy. Alternatively, tradeoffs can result from the fixation of mutations that are neutral in the selective environment but have a negative effect in other environments, and thus the tradeoff is due to mutation accumulation. We tested the mechanisms underlying the fitness tradeoffs observed during adaptation to persistent infection of vesicular stomatitis virus in insect cells by sequencing the full-length genomes of 12 strains with a history of replication in a single niche (acute mammalian infection or persistent insect infection) or in temporally heterogeneous niches and correlated genetic and fitness changes. Ecological theory predicts a correlation between the selective environment and the niche width of the evolved populations, such that adaptation to single niches should lead to the selection of specialists and niche cycling should result in the selection of generalists. Contrary to this expectation, adaptation to one of the single niches resulted in a generalist and adaptation to a heterogeneous environment led to the selection of a specialist. Only one-third of the mutations that accumulated during persistent infection had a fitness cost that could be explained in all cases by antagonistic pleiotropy. Mutations involved in fitness tradeoffs included changes in regulatory sequences, particularly at the 3' termini of the genomes, which contain the single promoter that controls viral transcription and replication.

Rapid adaptive amplification of preexisting variation in an RNA virus.

The amount and nature of preexisting variation in a population of RNA viruses is an important determinant of the virus's ability to adapt rapidly to a changed environment. However, direct quantification of this preexisting variation may be cumbersome, because potentially beneficial alleles are typically rare, and isolation of a large number of subclones is required. Here, we propose a simpler method. We infer the initial population structure of vesicular stomatitis virus (VSV) by fitting a mathematical model of asexual evolution to an extensive set of measurements of VSV fitness dynamics under various conditions, including new and previously published data. The inferred variation of fitness in the initial population agrees very well with the results of direct experiments with subclone fitness quantification. From the same procedure, we also estimate the mean fitness effect of beneficial mutations (selection coefficient s), the percentage of sites in the genome that are under moderate positive or negative selection, and the percentage of sites where beneficial mutations may potentially occur. For VSV strain MARM U evolving in BHK-21 cells, the three parameters have values of 0.39, 9%, and 0.06%, respectively. The method can be generalized and applied easily to other rapidly evolving microbes, including both asexual microorganisms and those with recombination.

Emergence of mammalian cell-adapted vesicular stomatitis virus from persistent infections of insect vector cells.

Arboviruses (arthropod-borne viruses) represent quintessential generalists, with the ability to infect and perform well in multiple hosts. However, antagonistic pleiotropy imposed a cost during the adaptation to persistent replication of vesicular stomatitis virus in sand fly cells and resulted in strains that initially replicated poorly in hamster cells, even when the virus was allowed to replicate periodically in the latter. Once a debilitated strain started replicating continuously in mammalian cells, fitness increased significantly. Fitness recovery did not entail back mutations or compensatory mutations, but instead, we observed the replacement of persistence-adapted genomes by mammalian cell-adapted strains with a full set of new, unrelated sequence changes. These mammalian cell-adapted genomes were present at low frequencies in the populations with a history of persistence for up to a year and quickly became dominant during mammalian infection, but coexistence was not stable in the long term. Periodic acute replication in mammalian cells likely contributed to extending the survival of minority genomes, but these genomes were also found in strictly persistent populations.

Adaptability costs in immune escape variants of vesicular stomatitis virus.

We have used vesicular stomatitis virus (VSV) to determine the cost of antiserum resistance during escape from a polyclonal immune response. Replication of VSV in the presence of polyclonal antiserum resulted in the selection of antibody-escape mutants, as shown by increased fitness in the presence of antiserum and by increased resistance to neutralization. However, resistance came at a cost of overall fitness loss in the BHK-21 host cells. Sequencing of the surface G glycoprotein showed that two to four mutations were fixed in each population, most of which mapped in the A1 and A2 antigenic sites. Selected resistant populations were passaged as large populations in BHK-21 cells under constant conditions, which would normally lead to fitness increases. Nevertheless, many of the populations showed little or no sign of recovery, although the resistant phenotype was maintained. These results suggest that while antiserum resistance can develop, it may come at a cost in fitness and further limitations in the adaptability of the populations.

Vesicular stomatitis virus evolution during alternation between persistent infection in insect cells and acute infection in mammalian cells is dominated by the persistence phase.

Vesicular stomatitis virus has the potential for very rapid evolution in the laboratory, but like many other arboviruses, it evolves at a relatively slow rate in the natural environment. Previous work showed that alternating replication in different cell types does not promote stasis. In order to determine whether other factors promote stasis, we compared the fitness trajectories of populations evolving during acute infections in mammalian cells, populations evolving during persistent infections in insect cells, and populations evolving during alternating acute and persistent infection cycles. Populations evolving under constant conditions increased in fitness in the environment in which they replicated. An asymmetric trade-off was observed such that acute infection had no cost for persistence but persistent replication had a dramatic cost for acute infection in mammalian cells. After an initial period of increase, fitness remained approximately constant in all the populations that included persistent replication, but fitness continuously increased in populations evolving during acute infections. Determination of the consensus sequence of the genes encoding the N, P, M, and G proteins showed that the pattern of mutation accumulation was coherent with fitness changes during persistence so that once fitness reached a maximum, the rate of mutation accumulation dropped. Persistent replication dominated both the genetic and the phenotypic evolution of the populations that alternated between acute infection of mammalian cells and persistence in insect cells, and fitness loss was observed in the mammalian environment despite periodic replication in mammalian cells. These results show that stasis can be achieved without good levels of adaptation to both the mammalian and the insect environments.

Fitness analyses of vesicular stomatitis strains with rearranged genomes reveal replicative disadvantages.

Gene expression of the nonsegmented negative-strand RNA viruses is determined by the position of each gene relative to that the single 3' promoter. The general order of genes among all of the viruses of the order Mononegavirales is highly conserved. In previous work we generated recombinant viruses in which the order of the three central genes of the prototypical rhabdovirus, vesicular stomatitis virus, was rearranged to all six possible permutations. While some of these viruses replicated less well than the wild type when assayed by single-step growth analyses in BSC-1 cells, others replicated as well or slightly better. In the work reported here, we used competition assays to compare the fitness of the viruses with alternative gene orders to that of the wild-type (wt) virus. We found that the relative fitness of these recombinant viruses depended on the multiplicity of infection (MOI) but not on the population size. However, during competitions at low MOI, when complementation cannot compensate for the defects of the populations with rearranged genomes, the virus with the wt gene order was always the most fit.

Density-dependent selection in vesicular stomatitis virus.

We used vesicular stomatitis virus to test the effect of complementation on the relative fitness of a deleterious mutant, monoclonal antibody-resistant mutant (MARM) N, in competition with its wild-type ancestor. We carried out competitions of MARM N and wild-type populations at different multiplicities of infection (MOIs) and initial ratios of the wild type to the mutant and found that the fitness of MARM N relative to that of the wild type is very sensitive to changes in the MOI (i.e., the degree of complementation) but depends little, if at all, on the initial frequencies of MARM N and the wild type. Further, we developed a mathematical model under the assumption that during coinfection both viruses contribute to a common pool of protein products in the infected cell and that they both exploit this common pool equally. Under such conditions, the fitness of all virions that coinfect a cell is the average fitness in the absence of coinfection of that group of virions. In the absence of coinfection, complementation cannot take place and the relative fitness of each competitor is only determined by the selective value of its own products. We found good agreement between our experimental results and the model predictions, which suggests that the wild type and MARM N freely share all of their gene products under coinfection.

Replication at periodically changing multiplicity of infection promotes stable coexistence of competing viral populations.

RNA viruses are widely used to study evolution experimentally. Many standard protocols of virus propagation and competition are done at nominally low multiplicity of infection (m.o.i.), but lead during one passage to two or more rounds of infection, of which the later ones are at high m.o.i. Here, we develop a model of the competition between wild type (wt) and a mutant under a regime of alternating m.o.i. We assume that the mutant is deleterious when it infects cells on its own, but derives a selective advantage when rare and coinfecting with wt, because it can profit from superior protein products created by the wt. We find that, under these assumptions, replication at alternating low and high m.o.i. may lead to the stable coexistence of wt and mutant for a wide range of parameter settings. The predictions of our model are consistent with earlier observations of frequency-dependent selection in vesicular stomatitis virus and human immunodeficiency virus type 1. Our results suggest that frequency-dependent selection may be common in typical evolution experiments with viruses.

Contributions of vesicular stomatitis virus to the understanding of RNA virus evolution.

Vesicular stomatitis virus has been a preferred system to study evolution for several decades. New approaches to antiviral treatment, such as lethal mutagenesis, stem from investigations done with VSV. Recent work has shed new light in the way we view neutrality, a fundamental concept to understand the evolutionary history of RNA viruses.

Phenotypic mixing and hiding may contribute to memory in viral quasispecies.

BACKGROUND: In a number of recent experiments with food-and-mouth disease virus, a deleterious mutant, RED, was found to avoid extinction and remain in the population for long periods of time. Since RED characterizes the past evolutionary history of the population, this observation was called quasispecies memory. While the quasispecies theory predicts the existence of these memory genomes, there is a disagreement between the expected and observed mutant frequency values. Therefore, the origin of quasispecies memory is not fully understood. RESULTS: We propose and analyze a simple model of complementation between the wild type virus and a mutant that has an impaired ability of cell entry, the likely cause of fitness differences between wild type and RED mutants. The mutant will go extinct unless it is recreated from the wild type through mutations. However, under phenotypic mixing-and-hiding as a mechanism of complementation, the time to extinction in the absence of mutations increases with increasing multiplicity of infection (m.o.i.). If the RED mutant is constantly recreated by mutations, then its frequency at equilibrium under selection-mutation balance also increases with increasing m.o.i. At high m.o.i., a large fraction of mutant genomes are encapsidated with wild-type protein, which enables them to infect cells as efficiently as the wild type virions, and thus increases their fitness to the wild-type level. Moreover, even at low m.o.i. the equilibrium frequency of the mutant is higher than predicted by the standard quasispecies model, because a fraction of mutant virions generated from wild-type parents will also be encapsidated by wild-type protein. CONCLUSIONS: Our model predicts that phenotypic hiding will strongly influence the population dynamics of viruses, particularly at high m.o.i., and will also have important effects on the mutation-selection balance at low m.o.i. The delay in mutant extinction and increase in mutant frequencies at equilibrium may, at least in part, explain memory in quasispecies populations.