Genetic Structure of the Population of Pepino mosaic virus Infecting Tomato Crops in Spain.

ABSTRACT The population structure of Pepino mosaic virus (PepMV), which has caused severe epidemics in tomato in Spain since 2000, was analyzed. Isolates were characterized by the nucleotide sequence of the triple gene block and coat protein gene and, for a subset of isolates, a part of the RNA-dependent RNA polymerase gene. The full-length sequence of the genomic RNA of a Solanum muricatum isolate from Peru also was determined. In spite of high symptom diversity, the Spanish population of PepMV mostly comprised highly similar isolates belonging to the strain reported in Europe (European tomato strain), which has been the most prevalent genotype in Spain. The Spanish PepMV population was not structured spatially or temporally. Also, isolates highly similar to those from nontomato hosts from Peru (Peruvian strain) or to isolate US2 from the United States (US2 strain) were detected at lower frequency relative to the European strain. These two strains were detected in peninsular Spain only in 2004, but the Peruvian strain has been detected in the Canary Islands since 2000. These results suggest that PepMV was introduced into Spain more than once. Isolates from the Peruvian and US2 strains always were found in mixed infections with the European tomato strain, and interstrain recombinants were detected. The presence of different strains of the virus, and of recombinant isolates, should be considered for the development of control strategies based on genetic resistance.

The rate and character of spontaneous mutation in an RNA virus.

Estimates of spontaneous mutation rates for RNA viruses are few and uncertain, most notably due to their dependence on tiny mutation reporter sequences that may not well represent the whole genome. We report here an estimate of the spontaneous mutation rate of tobacco mosaic virus using an 804-base cognate mutational target, the viral MP gene that encodes the movement protein (MP). Selection against newly arising mutants was countered by providing MP function from a transgene. The estimated genomic mutation rate was on the lower side of the range previously estimated for lytic animal riboviruses. We also present the first unbiased riboviral mutational spectrum. The proportion of base substitutions is the same as that in a retrovirus but is lower than that in most DNA-based organisms. Although the MP mutant frequency was 0.02-0.05, 35% of the sequenced mutants contained two or more mutations. Therefore, the mutation process in populations of TMV and perhaps of riboviruses generally differs profoundly from that in populations of DNA-based microbes and may be strongly influenced by a subpopulation of mutator polymerases.

An Analysis of Host Adaptation and Its Relationship with Virulence in Cucumber mosaic virus.

ABSTRACT The host range of a pathogen can have special consequences on its evolution and the evolution of its virulence. For generalists, adaptation to different hosts may be conditioned by different trade-offs in the pathogen's life history and be affected by evolutionary processes that shape pathogen populations. We have examined adaptation of Cucumber mosaic virus (CMV) to different hosts, and analyzed the relationship between host adaptation and virulence. For this, six CMV isolates from central Spain from three different hosts were compared for the ability to multiply and to affect host growth. These analyses were done before and after an experimental evolution process consisting of 10 serial passages in the original host of the isolate. The differential capacity to infect different hosts was compatible with host adaptation. However, the capacity to multiply in different hosts did not provide evidence of host adaptation and was not improved after 10 passages, suggesting that fitness of the natural population of CMV was at, or near to, its maximum. No relationship was found between capacity of multiplication and virulence in any of the three different hosts. These results suggest that the "trade-off" model for the evolution of virulence may not apply to CMV.

Association and host selectivity in multi-host pathogens.

The distribution of multi-host pathogens over their host range conditions their population dynamics and structure. Also, host co-infection by different pathogens may have important consequences for the evolution of hosts and pathogens, and host-pathogen co-evolution. Hence it is of interest to know if the distribution of pathogens over their host range is random, or if there are associations between hosts and pathogens, or between pathogens sharing a host. To analyse these issues we propose indices for the observed patterns of host infection by pathogens, and for the observed patterns of co-infection, and tests to analyse if these patterns conform to randomness or reflect associations. Applying these tests to the prevalence of five plant viruses on 21 wild plant species evidenced host-virus associations: most hosts and viruses were selective for viruses and hosts, respectively. Interestingly, the more host-selective viruses were the more prevalent ones, suggesting that host specialisation is a successful strategy for multi-host pathogens. Analyses also showed that viruses tended to associate positively in co-infected hosts. The developed indices and tests provide the tools to analyse how strong and common are these associations among different groups of pathogens, which will help to understand and model the population biology of multi-host pathogens.

Role of recombination in the evolution of natural populations of Cucumber mosaic virus, a tripartite RNA plant virus.

The role of recombination in the evolution of Cucumber mosaic virus (CMV) was analyzed in a collection of Spanish isolates from 1989 to 2002. Isolates were characterized by ribonuclease protection assay using six RNA probes, two for each of the three genomic RNAs, which allowed the identification of the analyzed regions as belonging to CMV isolates in subgroups IA, IB, and II. Most isolates belonged to subgroups IA (64%) and IB (12%), 5% were reassortants among subgroups IA, IB, or II, and 17% were recombinants between these groups. Recombinants at RNA3 were significantly more frequent than recombinants at RNAs 1 and 2. One IB-IA recombinant RNA3 was as frequent in central Spain as the IA RNA3. The genetic structure of the virus population suggested that reassortants and most recombinant genotypes were selected against and was consistent with a higher biological cost of reassortment than recombination. Data also suggest that recombinants that encode hybrid proteins are at a higher disadvantage than recombinants that exchange whole ORFs.

Variation and evolution of plant virus populations.

Over the last 15 years, interest in plant virus evolution has re-emerged, as shown by the increasing number of papers published on this subject. In recent times, research in plant virus evolution has been viewed from a molecular, rather than populational, standpoint, and there is a need for work aimed at understanding the processes involved in plant virus evolution. However, accumulated data from analyses of experimental and natural populations of plant viruses are beginning to delineate some trends that often run contrary to accepted opinion: (1) high mutation rates are not necessarily adaptive, as a large fraction of the mutations are deleterious or lethal; (2) in spite of high potential for genetic variation, populations of plant viruses are not highly variable, and genetic stability is the rule rather than the exception; (3) the degree of constriction of genetic variation in virus-encoded proteins is similar to that in their eukaryotic hosts and vectors; and (4) in spite of huge census sizes of plant virus populations, selection is not the sole factor that shapes their evolution, and genetic drift may be important. Here, we review recent advances in understanding plant virus evolution, and describe the experimental and analytical methods most suited to this purpose.

Estimation of population bottlenecks during systemic movement of tobacco mosaic virus in tobacco plants.

More often than not, analyses of virus evolution have considered that virus populations are so large that evolution can be explained by purely deterministic models. However, virus populations could have much smaller effective numbers than the huge reported census numbers, and random genetic drift could be important in virus evolution. A reason for this would be population bottlenecks during the virus life cycle. Here we report a quantitative estimate of population bottlenecks during the systemic colonization of tobacco leaves by Tobacco mosaic virus (TMV). Our analysis is based on the experimental estimation of the frequency of different genotypes of TMV in the inoculated leaf, and in systemically infected leaves, of tobacco plants coinoculated with two TMV genotypes. A simple model, based on the probability that a leaf in coinoculated plants is infected by just one genotype and on the frequency of each genotype in the source, was used to estimate the effective number of founders for the populations in each leaf. Results from the analysis of three leaves per plant in plants inoculated with different combinations of three TMV genotypes yielded highly consistent estimates. Founder numbers for each leaf were small, in the order of units. This would result in effective population numbers much smaller than the census numbers and indicates that random effects due to genetic drift should be considered for understanding virus evolution within an infected plant.