The error rate of RNA-dependent RNA polymerases (RdRp) affects the mutation frequency in a population of viral RNAs. Using chikungunya virus (CHIKV), we describe a unique arbovirus fidelity variant with a single C483Y amino acid change in the nsP4 RdRp that increases replication fidelity and generates populations with reduced genetic diversity. In mosquitoes, high fidelity CHIKV presents lower infection and dissemination titers than wild type. In newborn mice, high fidelity CHIKV produces truncated viremias and lower organ titers. These results indicate that increased replication fidelity and reduced genetic diversity negatively impact arbovirus fitness in invertebrate and vertebrate hosts. Studies with the first RNA-dependent RNA polymerase (RdRp) fidelity variant demonstrated that poliovirus RdRp error rate is a major contributor to population mutation frequency (1) and that reduced genetic diversity negatively impacts dissemination and pathogenesis in vivo (refs. 2-4; although the implications of these findings were limited by a relatively artificial model). The explanation for this reduced fitness is based on the idea that a diverse RNA virus population contains, by chance, more variants with potentially advantageous adaptive mutations, whereas a less diverse population is not as likely to possess such variants. Based on these observations, we hypothesized that RdRp fidelity of other RNA viruses can be modulated with similar fitness costs.Nucleoside analogs including ribavirin and 5-fluorouracil (5-FU) are mutagenic compounds that are misincorporated into viral genomes during RNA synthesis, resulting in deleterious mutations by mispairing in the following replication cycle (5-7). The consequent increase in the number of mutations places more genomic RNAs beyond a hypothetical error threshold: a mutation frequency at which the majority of the population harbors low fitness or lethal mutations (5,8). In this theoretical context, RNA virus populations lie close to this threshold-even moderate increases in mutation frequency could severely diminish infectivity. Increasing polymerase fidelity would potentially create a population farther from this error threshold that could better tolerate mutational input. Solid evidence supports this logic: DNA polymerase (9, 10) and reverse transcriptase (11-13) virus variants with antimutator phenotypes are linked to mutations that increase fidelity and reduce base analog incorporation. These observations present an interesting paradox: If point mutations in RNA viruses can increase RdRp fidelity, thereby decreasing replication mistakes, why is higher fidelity in RNA viruses not selected in nature? Is there a tradeoff between being error-prone but adaptable or being less adaptable but possessing higher fidelity? Does higher fidelity necessarily incur a phenotypic disadvantage in hosts, where a reduced ability to produce adaptive mutations impairs fitness, as was suggested by poliovirus studies?Arthropod borne (arbo-) viruses are unique among RNA viruses in that they obligatel...

Based on structural data of the RNA-dependent RNA polymerase, rational targeting of key residues, and screens for Coxsackievirus B3 fidelity variants, we isolated nine polymerase variants with mutator phenotypes, which allowed us to probe the effects of lowering fidelity on virus replication, mutability, and in vivo fitness. These mutator strains generate higher mutation frequencies than WT virus and are more sensitive to mutagenic treatments, and their purified polymerases present lower-fidelity profiles in an in vitro incorporation assay. Whereas these strains replicate with WT-like kinetics in tissue culture, in vivo infections reveal a strong correlation between mutation frequency and fitness. Variants with the highest mutation frequencies are less fit in vivo and fail to productively infect important target organs, such as the heart or pancreas. Furthermore, whereas WT virus is readily detectable in target organs 30 d after infection, some variants fail to successfully establish persistent infections. Our results show that, although mutator strains are sufficiently fit when grown in large population size, their fitness is greatly impacted when subjected to severe bottlenecking, which would occur during in vivo infection. The data indicate that, although RNA viruses have extreme mutation frequencies to maximize adaptability, nature has fine-tuned replication fidelity. Our work forges ground in showing that the mutability of RNA viruses does have an upper limit, where larger than natural genetic diversity is deleterious to virus survival.T hirty years ago, our regard of RNA viruses as simple, uniform organisms changed with the demonstration of the genetically heterogeneous composition of Qβ-phage populations (1), a concept that has been extended to all RNA viruses. Indeed, as early as 1965, the error-prone nature of RNA virus replication had been observed (2). Since that time, significant progress has been made in describing the highly polymorphic mutational distributions within RNA virus populations, the mechanisms responsible for their generation, and their implication in virus adaptability, evolution, and fitness (3). Important progress was made in recent studies using antimutator strains of RNA viruses presenting higher-fidelity polymerases that generate less diverse populations (4-6). These studies revealed that, although mutation rates can be reduced for these viruses, nature has seemingly selected for error-prone replication to maximize adaptability. This results in virus populations with extreme mutation frequencies approaching a maximum beyond which the likelihood of lethal mutations greatly diminishes virus viability (7). In recent years, the study of lethal mutagenesis as an antiviral approach, based on the accumulation of lethal mutations through treatment with mutagenic compounds, was pivotal in showing that RNA viruses are particularly sensitive to even moderate increases in their already elevated mutation frequencies (8-13). The strong correlation between decreased mutation frequencies and comp...

The high replication and mutation rates of RNA viruses can result in the emergence of new epidemic variants. Thus, the ability to follow host-specific evolutionary trajectories of viruses is essential to predict and prevent epidemics. By studying the spatial and temporal evolution of chikungunya virus during natural transmission between mosquitoes and mammals, we have identified viral evolutionary intermediates prior to emergence. Analysis of virus populations at anatomical barriers revealed that the mosquito midgut and salivary gland pose population bottlenecks. By focusing on virus subpopulations in the saliva of multiple mosquito strains, we recapitulated the emergence of a recent epidemic strain of chikungunya and identified E1 glycoprotein mutations with potential to emerge in the future. These mutations confer fitness advantages in mosquito and mammalian hosts by altering virion stability and fusogenic activity. Thus, virus evolutionary trajectories can be predicted and studied in the short term before new variants displace currently circulating strains.

Understanding how a pathogen colonizes and adapts to a new host environment is a primary aim in studying emerging infectious diseases. Adaptive mutations arise among the thousands of variants generated during RNA virus infection, and identifying these variants will shed light onto how changes in tropism and species jumps can occur. Here, we adapted Coxsackie virus B3 to a highly permissive and less permissive environment. Using deep sequencing and bioinformatics, we identified a multi-step adaptive process to adaptation involving residues in the receptor footprints that correlated with receptor availability and with increase in virus fitness in an environment-specific manner. We show that adaptation occurs by selection of a dominant mutation followed by group selection of minority variants that together, confer the fitness increase observed in the population, rather than selection of a single dominant genotype.