Aurora Fraile

Estimation of Population Bottlenecks during Systemic Movement of Tobacco Mosaic Virus in Tobacco Plants

ABSTRACT 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.

Multiple infection, recombination and genome relationships among begomovirus isolates found in cotton and other plants in Pakistan

Begomoviruses occur in many plant species in Pakistan and are associated with an epidemic of cotton leaf curl disease that has developed since 1985. PCR analysis with primer pairs specific for each of four already sequenced types of DNA-A of cotton leaf curl virus (CLCuV-PK types a, 26, 72b and 804a), or for okra yellow vein mosaic virus (OYVMV), indicated that many individual naturally infected plants of cotton and other malvaceous species contained two or three begomovirus sequences. Similarly, sequence differences among overlapping fragments of begomovirus DNA-A, amplified from individual naturally infected plants, indicated much multiple infection in malvaceous and non-malvaceous species. Some cotton plants contained DNA-A sequences typical of begomoviruses from non-malvaceous species, and some non-malvaceous plants contained sequences typical of CLCuV-PK. Some DNA-A sequences were chimaeric; they each included elements typical of different types of CLCuV-PK, or of different malvaceous and/or non-malvaceous begomoviruses. Often an apparent recombination site occurred at the origin of replication. No complete CLCuV-PK DNA-A sequence was found in malvaceous or non-malvaceous species collected in Pakistan outside the area of the cotton leaf curl epidemic but chimaeric sequences, including a part that was typical of CLCuV-PK DNA-A, did occur there. We suggest that recombination among such pre-existing sequences was crucial for the emergence of CLCuV-PK. Recombination, following multiple infection, could also explain the network of relationships among many of the begomoviruses found in the Indian subcontinent, and their evolutionary divergence, as a group, from begomoviruses causing similar diseases in other geographical regions.

Genetic Variability of Natural Populations of Cotton Leaf Curl Geminivirus, a Single-Stranded DNA Virus

Abstract. Reports on the genetic variability and evolution of natural populations of DNA viruses are scarce in comparison with the abundant information on the variability of RNA viruses. Geminiviruses are plant viruses with circular ssDNA genomes that are replicated by the host plant DNA polymerases. Whitefly-transmitted geminiviruses (WTG) are the agents of important diseases of crop plants and best exemplify emerging plant viruses. In this report we have analyzed the genetic diversity of cotton leaf curl geminivirus (CLCuV), a typical emerging WTG. No genetic differentiation was observed between isolates from different host plant species or geographic regions. Thus, the analyzed isolates represented a unique, undifferentiated population. Genetic variability, estimated as nucleotide diversities at synonymous positions in open reading frames (ORFs) for the AC1 (=replication) protein and coat protein (CP = AV1), was very high, exceeding the values reported for different genes in several plant and animal RNA viruses. This was unexpected in a virus that uses the DNA replication machinery of its eukaryotic host. Diversities at nonsynonymous positions, on the other hand, indicated that variability may be constrained in the genome of CLCuV. The ratio of nonsynonymous-to-synonymous substitutions varied for the different ORFs: they were higher for CP than for AC1 and lower still for the AC4 and AV2 ORFs, which overlap AC1 and CP ORFs, respectively. Analysis of nucleotide diversities at synonymous and nonsynonymous positions of the AC4 and AV2 ORFs suggest that their evolution is constrained by AC1 and CP, respectively. Data suggest that AC4 and AV2 are new genes that may have originated by overprinting on the preexistent AC1 and CP genes. Evidence for recombination was found for the AC1 and CP ORFs and for the noncoding intergenic region (IR). Data indicate that the origin of replication is a major recombination point in the IR, but not the only one. Analyses of the IR also suggest that recombinants may be frequent in the population and that recombination may have an important role in the generation of CLCuV variability.

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.

Estimation of the Effective Number of Founders That Initiate an Infection after Aphid Transmission of a Multipartite Plant Virus

ABSTRACT The fecundity of RNA viruses can be very high. Thus, it is often assumed that viruses have large populations, and RNA virus evolution has been mostly explained using purely deterministic models. However, population bottlenecks during the virus life cycle could result in effective population numbers being much smaller than reported censuses, and random genetic drift could be important in virus evolution. A step at which population bottlenecks may be severe is host-to-host transmission. We report here an estimate of the size of the population that starts a new infection when Cucumber mosaic virus (CMV) is transmitted by the aphid Aphis gossypii, based on the segregation of two CMV genotypes in plants infected by aphids that acquired the virus from plants infected by both genotypes. Results show very small effective numbers of founders, between one and two, both in experiments in which the three-partite genome of CMV was aphid transmitted and in experiments in which a fourth RNA, CMV satellite RNA, was also transmitted. These numbers are very similar to those published for Potato virus Y, which has a monopartite genome and is transmitted by aphids according to a different mechanism than CMV. Thus, the number of genomic segments seems not to be a major determinant of the effective number of founders. Also, our results suggest that the occurrence of severe bottlenecks during horizontal transmission is general for viruses nonpersistently transmitted by aphids, indicating that random genetic drift should be considered when modeling virus evolution.

The coevolution of plants and viruses: resistance and pathogenicity.

Virus infection may damage the plant, and plant defenses are effective against viruses; thus, it is currently assumed that plants and viruses coevolve. However, and despite huge advances in understanding the mechanisms of pathogenicity and virulence in viruses and the mechanisms of virus resistance in plants, evidence in support of this hypothesis is surprisingly scant, and refers almost only to the virus partner. Most evidence for coevolution derives from the study of highly virulent viruses in agricultural systems, in which humans manipulate host genetic structure, what determines genetic changes in the virus population. Studies have focused on virus responses to qualitative resistance, either dominant or recessive but, even within this restricted scenario, population genetic analyses of pathogenicity and resistance factors are still scarce. Analyses of quantitative resistance or tolerance, which could be relevant for plant-virus coevolution, lag far behind. A major limitation is the lack of information on systems in which the host might evolve in response to virus infection, that is, wild hosts in natural ecosystems. It is presently unknown if, or under which circumstances, viruses do exert a selection pressure on wild plants, if qualitative resistance is a major defense strategy to viruses in nature, or even if characterized genes determining qualitative resistance to viruses did indeed evolve in response to virus infection. Here, we review evidence supporting plant-virus coevolution and point to areas in need of attention to understand the role of viruses in plant ecosystem dynamics, and the factors that determine virus emergence in crops.

Constraints to Genetic Exchange Support Gene Coadaptation in a Tripartite RNA Virus

Genetic exchange by recombination, or reassortment of genomic segments, has been shown to be an important process in RNA virus evolution, resulting often in important phenotypic changes affecting host range and virulence. However, data from numerous systems indicate that reassortant or recombinant genotypes could be selected against in virus populations and suggest that there is coadaptation among viral genes. Little is known about the factors affecting the frequency of reassortants and recombinants along the virus life cycle. We have explored this issue by estimating the frequency of reassortant and recombinant genotypes in experimental populations of Cucumber mosaic virus derived from mixed infections with four different pairs of isolates that differed in about 12% of their nucleotide sequence. Genetic composition of progeny populations were analyzed at various steps of the virus life cycle during host colonization: infection of leaf cells, cell-to-cell movement within the inoculated leaf, encapsidation of progeny genomes, and systemic movement to upper noninoculated leaves. Results indicated that reassortant frequencies do not correspond to random expectations and that selection operates against reassortant genotypes. The intensity of selection, estimated through the use of log-linear models, increased as host colonization progressed. No recombinant was detected in any progeny. Hence, results showed the existence of constraints to genetic exchange linked to various steps of the virus life cycle, so that genotypes with heterologous gene combinations were less fit and disappeared from the population. These results contribute to explain the low frequency of recombinants and reassortants in natural populations of many viruses, in spite of high rates of genetic exchange. More generally, the present work supports the hypothesis of coadaptation of gene complexes within the viral genomes.

Molecular epidemiology of Cucumber mosaic virus and its satellite RNA.

Molecular analysis of viral isolates can yield information that facilitates an understanding of virus epidemiology and has been termed molecular epidemiology. This approach has only recently been applied to plant viruses. Results on the molecular epidemiology of Cucumber mosaic virus (CMV) and its satellite RNA (satRNA) in Spain, where CMV is endemic in vegetable crops are presented here. To characterise the genetic structure of CMV populations, c. 300 isolates, representing 17 outbreaks (i.e. sub-populations) in different crops, regions and years, were compared. Genetic analyses of CMV isolates were done by ribonuclease protection assay of cRNA probes representing RNA1, RNA2 and the two open reading frames in RNA3. All isolates belonged to one of three genetic types: Sub-group II and two types of Sub-group I. The genetic structure of the 17 sub-populations varied randomly, without correlation with location, year, or host plant species. Thus, CMV in Spain shows a metapopulation structure with local extinction and random recolonisation from local or distant virus reservoirs. The frequency of mixed infections and of new genetic types generated by reassortment of genomic segments or by recombination was also estimated. Results indicate that heterologous genetic combinations are not favoured. About 30% of CMV isolates were supporting a satRNA. The frequency of CMV isolates with a satRNA differed for each sub-population, being c. 1 in eastern Spain in 1990 and decreasing to c. 0 in distant regions and in subsequent years. Molecular analyses of CMV-satRNA isolates show high genetic diversity, due both to the accumulation of point mutations and to recombination. The CMV-satRNA population is a single, unstructured one. Thus, the CMV-satRNA population has a genetic structure and dynamics different from those of its helper virus. This indicates that CMV-satRNA has spread epidemically on the extant virus population from an original reservoir in eastern Spain. The relevance of these results for the control of CMV infections is discussed.

THE EVOLUTION OF VIRULENCE IN A PLANT VIRUS

Abstract The evolution of virulence is a rapidly growing field of research, but few reports deal with the evolution of virulence in natural populations of parasites. We present here an observational and experimental analysis of the evolution of virulence of the plant virus Cucumber mosaic virus (CMV) during an epidemic on tomato in eastern Spain. Three types of CMV isolates were found that caused in tomato plants either a systemic necrosis (N isolates), stunting and a severe reduction of leaf lamina (Y isolates), or stunting and leaf curl (A isolates). These phenotypes were due to the presence of satellite RNAs (satRNAs) necrogenic (in N isolates) or attenuative (in A isolates) of the symptoms caused by CMV without satRNA (Y isolates). For these three types of isolates, parameters of virulence and transmission were estimated experimentally. For virulence the ranking of isolates was N < Y < A, for trans‐missibility, Y < A < N. The predictions of theoretical models for the evolution of virulence were analyzed with these parameters and compared with observations from the field. A single‐infection model predicted adequately the observed long‐term evolution of the CMV population to intermediate levels of virulence. A coinfection model that considered competition between isolates with an effect on transmission explained the invasion of the CMV population by N isolates at the beginning of the epidemic, and its predictions also agreed with field data on the long‐term evolution of the CMV population. An important conclusion from both models was that the density of the aphid vectors population is a major factor in the evolution of CMV virulence. This may be relevant for the design of control strategies for CMV‐induced diseases.

Evolution of Virulence in Natural Populations of the Satellite RNA of Cucumber mosaic virus

From 1986 to 1992, an epidemic of tomato necrosis caused by Cucumber mosaic virus (CMV) plus CMV satellite RNAs (satRNAs) occurred in eastern Spain. From 1989 onward, the frequency of tomato necrosis di-minshed, and it almost completely disappeared after 1991. Analyses of plants infected with CMV and with CMV satRNA and of the phenotype (necrogenic or nonnecrogenic for tomato) induced by some CMV satRNA variants, showed that the disappearance of tomato necrosis was due to changes in the genetic composition of the satRNA population (i.e., to its evolution toward decreased virulence). Analysis of components of the fitness of satRNA variants, necrogenic or nonnecrogenic for tomato, showed that necrogenic and nonnecrogenic variants did not differ in infectivity or in their accumulation level in tomato and that they represented the same fraction of encapsidated RNA. Other fitness components were positively correlated with the greater virulence of necrogenic variants, in that they were favored in mixed infections with nonnecrogenic variants and were more effectively passed into CMV progeny than were nonnecrogenic variants. On the other hand, necrogenic CMV satRNA variants caused a more pronounced depression in the accumulation of CMV than did nonnecro-genic variants, which could affect the efficiency of aphid transmission. Thus, the evolution of virulence in the CMV satRNA population can be explained by trade-offs between factors that determine virulence and factors that affect transmission, as predicted by theoretical models on the evolution of virulence in parasites.