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It is generally accepted that mutation rates of RNA viruses are inherently high due to the lack of proofreading mechanisms. However, direct estimates of mutation rate are surprisingly scarce, in particular for plant viruses. Here, based on the analysis of in vivo mutation frequencies in tobacco etch virus, we calculate an upper-bound mutation rate estimation of 3x10(-5) per site and per round of replication; a value which turns out to be undistinguishable from the methodological error. Nonetheless, the value is barely on the lower side of the range accepted for RNA viruses, although in good agreement with the only direct estimate obtained for other plant viruses. These observations suggest that, perhaps, differences in the selective pressures operating during plant virus evolution may have driven their mutation rates towards values lower than those characteristic of other RNA viruses infecting bacteria or animals.

Viruses adapt to their hosts by evading defense mechanisms and taking over cellular metabolism for their own benefit. Alterations in cell metabolism as well as side-effects of antiviral responses contribute to symptoms development and virulence. Sometimes, a virus may spill over from its usual host species into a novel one, where usually will fail to successfully infect and further transmit to new host. However, in some cases, the virus transmits and persists after fixing beneficial mutations that allow for a better exploitation of the new host. This situation would represent a case for a new emerging virus. Here we report results from an evolution experiment in which a plant virus was allowed to infect and evolve on a naïve host. After 17 serial passages, the viral genome has accumulated only five changes, three of which were non-synonymous. An amino acid substitution in the viral VPg protein was responsible for the appearance of symptoms, whereas one substitution in the viral P3 protein the epistatically contributed to exacerbate severity. DNA microarray analyses show that the evolved and ancestral viruses affect the global patterns of host gene expression in radically different ways. A major difference is that genes involved in stress and pathogen response are not activated upon infection with the evolved virus, suggesting that selection has favored viral strategies to escape from host defenses.

Undoubtedly, viruses represent a major threat faced by human and veterinary medicines and by agronomy. The rapid evolution of viruses enables them to escape from natural immunities and from state-of-the-art antiviral treatments, with new viruses periodically emerging with deadly consequences. Viruses have also become powerful and are increasingly used tools in the field of experimental evolution. A growing body of evidence points that the evolution of viruses is mainly determined by key features such as their compacted genomes, enormous population sizes, and short generation times. In addition, RNA viruses also present large selection coefficients, antagonistic epistasis, and high mutation rates. Most of this knowledge comes from studies that have used either bacteriophages or animal viruses in cell cultures as experimental systems. However, plant viruses provide almost identical advantages for evolutionary studies and, in addition, offer an invaluable tool for studying the interplay between viruses and pluricellular hosts. Without seeking to be exhaustive, here we summarize some peculiarities of plant viruses and review recent experiments that have explored important questions on evolution, such as the role of deleterious mutation and neutrality, the effect of different transmission modes in the evolution of virulence, and the heterogeneous selective constraints imposed by multiple hosts.

Abstract: Relative fitness determination has become a standard tool in experimental virus evolution studies. In this type of studies, the tested strain is mixed with a reference strain, which differs in an easy-to-score and genetically stable marker, and allowed to compete for a limited common pool of resources during a given number of generations. In this report, a TaqMan® real-time PCR methodology is proposed for quantifying the relative fitness of tobacco etch potyvirus strains (TEV) in in planta mixed infections with a reference TEV strain. Two different forward primers along with a common reverse one are used into separated reactions mixes from the same RNA preparation. The reference strain, named TEV-PC1, was genetically engineered to carry a neutral marker in a highly conserved region of the RNA polymerase NIb gene. This marker allows tracking the frequency of both competitors during competition experiments by real-time quantitative PCR using specific primers. Both the reproducibility and sensitivity of the method have been explored. Reproducibility was assessed by running multiple competition experiments for the same genotype. Sensitivity was assessed by comparing the results of competition experiments against TEV-PC1 of 24 single-nucleotide substitutions mutants. [Copyright &y& Elsevier]