Identification of two novel putative satellite RNAs with hammerhead structures in the virome of French and Spanish carrot samples.

Carrot virome analysis using high-throughput sequencing revealed the presence of two RNA molecules with properties of satellite RNAs that are homologous to the satellite RNA of cereal yellow dwarf virus-RPV (CYDV-RPV). Satellite 1 is 298 nt long, while satellite 2 is 368 nt long. Their positive and negative genome strands contain hammerhead ribozymes similar to those found in other self-cleaving satellite RNAs. While both satellites were detected in Spanish carrot populations, only satellite 2 was found in French carrot populations. The most likely helper virus for these two satellites is carrot red leaf virus (CtRLV), which, like CYDV-RPV, is a polerovirus.

Landscape heterogeneity shapes host-parasite interactions and results in apparent plant-virus codivergence.

Knowledge on how landscape heterogeneity shapes host-parasite interactions is central to understand the emergence, dynamics and evolution of infectious diseases. However, this is an underexplored subject, particularly for plant-virus systems. Here, we analyse how landscape heterogeneity influences the prevalence, spatial genetic structure, and temporal dynamics of Pepper golden mosaic and Pepper huasteco yellow vein begomoviruses infecting populations of the wild pepper Capsicum annuum glabriusculum (chiltepin) in Mexico. Environmental heterogeneity occurred at different nested spatial scales (host populations within biogeographical provinces), with levels of human management varying among host population within a province. Results indicate that landscape heterogeneity affects the epidemiology and genetic structure of chiltepin-infecting begomoviruses in a scale-specific manner, probably related to conditions favouring the viruses' whitefly vector and its dispersion. Increased levels of human management of the host populations were associated with higher virus prevalence and erased the spatial genetic structure of the virus populations. Also, environmental heterogeneity similarly shaped the spatial genetic structures of host and viruses. This resulted in the congruence between host and virus phylogenies, which does not seem to be due to host-virus co-evolution. Thus, results provide evidence of the key role of landscape heterogeneity in determining plant-virus interactions.

Time--the emerging dimension of plant virus studies.

Recent research has revealed that some plant viruses, like many animal viruses, have measurably evolving populations. Most of these viruses have single-stranded positive-sense RNA genomes, but a few have single-stranded DNA genomes. The studies show that extant populations of these viral species are only decades to centuries old. The genera in which they are placed have diverged since agriculture was invented and spread around the world during the Holocene period. We suggest that this is not mere coincidence but evidence that the conditions generated by agriculture during this era have favoured particular viruses. There is also evidence, albeit less certain, that some plant viruses, including a few shown to have measurably evolving populations, have much more ancient origins. We discuss the possible reasons for this clear discordance between short- and long-term evolutionary rate estimates and how it might result from a large timescale dependence of the evolutionary rates. We also discuss briefly why it is useful to know the rates of evolution of plant viruses.

Potential involvement of a cucumber homolog of phloem protein 1 in the long-distance movement of Cucumber mosaic virus particles.

The systemic movement of Cucumber mosaic virus (CMV) in cucumber plants was analyzed. The structure that is translocated and its putative interactions with phloem components were analyzed in phloem exudate (PE) samples, which reflect sieve tubes stream composition. Rate zonal centrifugation and electron-microscopy analyses of PE from CMV-infected plants showed that CMV moves through sieve tubes as virus particles. Gel overlay assays revealed that CMV particles interact with a PE protein, p48. The amino-acid sequence of several tryptic peptides of p48 was determined. Partial amino-acid sequence of p48 showed it was a cucumber homolog of phloem protein 1 (PP1) from pumpkin, with which p48 also shares several chemical properties. PP1 from pumpkin has plasmodesmata-gating ability and translocates in sieve tubes. Encapsidated CMV RNA in PE samples from infected plants was less accessible to digestion by RNase A than RNA in purified CMV particles, a property that was reconstituted by the in vitro interaction of purified CMV particles and protein p48. These results indicate that the interaction with p48 modifies CMV particle structure and suggest that CMV particles interact with the cucumber homolog of PP1 during translocation in the sieve tubes.

Variability and genetic structure of plant virus populations.

Populations of plant viruses, like all other living beings, are genetically heterogeneous, a property long recognized in plant virology. Only recently have the processes resulting in genetic variation and diversity in virus populations and genetic structure been analyzed quantitatively. The subject of this review is the analysis of genetic variation, its quantification in plant virus populations, and what factors and processes determine the genetic structure of these populations and its temporal change. The high potential for genetic variation in plant viruses, through either mutation or genetic exchange by recombination or reassortment of genomic segments, need not necessarily result in high diversity of virus populations. Selection by factors such as the interaction of the virus with host plants and vectors and random genetic drift may in fact reduce genetic diversity in populations. There is evidence that negative selection results in virus-encoded proteins being not more variable than those of their hosts and vectors. Evidence suggests that small population diversity, and genetic stability, is the rule. Populations of plant viruses often consist of a few genetic variants and many infrequent variants. Their distribution may provide evidence of a population that is undifferentiated, differentiated by factors such as location, host plant, or time, or that fluctuates randomly in composition, depending on the virus.

Secondary structure as a constraint on the evolution of a plant viral satellite RNA.

The genetic variability and evolution of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) was analyzed. Twenty-five CMV-satRNAs compared clustered into three main groups, and no correlation was found between genetic proximity and other characteristics (pathogenicity, geographical origin) of the satRNAs. Values for the number of nucleotide substitutions per site between any two satRNAs suggest that divergence is checked by functional constraints. The analysis of mutations relative to an ancestral sequence, and the number of substitutions per site at first, second and third positions of codons in putative open reading frames, show that the variation of CMV-satRNAs does not follow a pattern typical of coding sequences, and indicates that preservation of the sequence of encoded products is not a constraint to evolution. On the other hand, when the observed variation was analyzed relative to a secondary structure model proposed for CMV-satRNAs, several lines of evidence indicated that the maintenance of the secondary structure is a constraint to evolution: the number of substitutions per site, the number of point insertions and deletions and the number of base substitutions that would disrupt base-pairing were significantly higher for unpaired than for base-paired positions. Also, compensatory mutations at base-paired positions occurred more frequently than expected from random. The results suggest that CMV-satRNAs are non-coding, functional RNAs whose biology would be determined by their direct interaction with components of the host and/or the helper virus.

Virus particles of cucumber green mottle mosaic tobamovirus move systemically in the phloem of infected cucumber plants.

Systemic movement through the phloem of infected host plants is a key process in the life cycle of plant viruses, knowledge of which is scant. A main point to be elucidated is the structural form in which virus infection moves within the phloem. Indirect evidence suggests that virions might be the viral structure that moves in the phloem, but data from direct analysis in phloem sap have not been reported. We have done such analysis in the system cucumber (from which phloem exudate can be collected)/cucumber green mottle mosaic tobamovirus (CGMMV). CGMMV has structurally well-characterized particles. Both CGMMV coat protein and RNA were found in phloem exudate from infected cucumbers. Analysis of the accessibility of CGMMV RNA in phloem exudate to RNase A indicates that it is protected within a ribonucleoprotein structure. The accessibility to RNase A of the RNA in these structures was as in virus particles. Centrifugation analyses showed that the ribonucleoprotein structures in the phloem exudate have the same mass and isopycnic density as virions. Virus particles indistinguishable from purified virions were detected by electron microscopy in phloem exudate. No evidence of free RNA or other CGMMV-related structure was found in phloem exudate of infected plants. These results indicate that CGMMV movement in the phloem occurs mainly, if not exclusively, in the form of virus particles.

The expression level of the 3a movement protein determines differences in severity of symptoms between two strains of tomato aspermy cucumovirus.

Two strains of tomato aspermy cucumovirus, 1-TAV and V-TAV, differ in the severity of the symptoms induced in Nicotiana tabacum: 1-TAV induces a severe chlorotic mottle that appears 5 days post inoculation (d.p.i.) in the second systemic leaf, while V-TAV-infected plants show a mild chlorotic mottle, unevenly distributed in the leaf lamina, that appears 7 d.p.i. in the third or fourth systemic leaf. The manipulation of full-length cDNA clones giving infectious transcripts of V-TAV RNAs 1, 2, and 3 and 1-TAV RNA 3 revealed that the slow, mild phenotype of V-TAV maps to the movement protein (MP) gene. By site-directed mutagenesis it was further shown that this phenotype co-segregates with a single nucleotide substitution that introduces an in-frame UAA stop codon at the fourth position of the MP open reading frame of V-TAV. The presence of this stop codon results in a diminished expression of the MP in both tobacco protoplasts and leaves. Analyses of the progress of infection and of the time course of MP and coat protein accumulation show that the low level of MP in V-TAV-infected leaves limits the rate of cell-to-cell movement and leads to the mild phenotype. Data from the infectivity of RNA 3 transcripts with or without this stop codon, plus data from in vitro translation of virion or transcript RNA 3, suggest that the small amount of MP observed in V-TAV-infected leaves is expressed from a minor RNA 3 subpopulation lacking the stop codon.

Genetic variability of natural populations of cotton leaf curl geminivirus, a single-stranded DNA virus

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.

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.

Transmissibility of Cucumber mosaic virus by Aphis gossypii Correlates with Viral Accumulation and Is Affected by the Presence of Its Satellite RNA.

ABSTRACT Satellite RNAs (satRNAs) are associated with Cucumber mosaic virus (CMV) in tomato, most often causing severe epidemics of necrotic plants, and not associated with specific host symptoms. Laboratory studies on virus transmission by the aphid vector Aphis gossypii were performed to better understand the dynamics of field populations of CMV. The presence of satRNAs correlated with lower concentrations of virus in infected plants and with a decrease in the efficiency of transmission from satRNA-infected plants. Both the concentration of virus in CMV-infected tomato and the efficiency of transmission varied more extensively with nonnecrogenic satRNAs than with necrogenic satRNAs. A negative effect of satRNAs on virus accumulation can account, in part, for a decrease in the field transmission and recovery of CMV + satRNAs. Aphids behaved differently and probed less readily on plants infected with CMV + necrogenic satRNAs compared with plants containing non-necrogenic satRNAs. Aphid-mediated satRNA-free CMV infections were observed in test plants when aphids were fed on source plants containing CMV + nonnecrogenic satRNA; no comparable satRNA-free test plants occurred when aphids were fed on source plants containing necrogenic satRNAs. These results indicate that factors associated with transmission can be a determinant in the evolution of natural populations of CMV and its satRNA.

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.

Satellite RNA of cucumber mosaic cucumovirus spreads epidemically in natural populations of its helper virus.

ABSTRACT Three hundred thirty-eight isolates of cucumber mosaic cucumovirus (CMV), sampled from natural populations in six areas of Spain between 1989 and 1996, were screened for the presence of satellite RNA (satRNA). The frequency of CMV isolates with satRNA approached 1.00 in Valencia (east Spain) between 1990 and 1994 where a tomato necrosis epidemic induced by CMV+satRNA had started in 1986 and was smaller north and west of this area in 1992 and 1993. After 1994, satRNA almost disappeared from all CMV populations. Genetic typing of satRNA variantswas done by ribonuclease protection assay, and from these data, genetic distances were estimated for any pair of satRNA variants. CMV-satRNA populations were highly diverse, containing 0.07865 nucleotide substitutions per site on average. Data also showed that the whole compared set of 100 satRNA variants form a single population that is not structured according to place, year, host plant, or strain of helper virus (HV). This is in sharp contrast with the metapopulation structure of the Spanish CMV population. Thus, the genetic structure and dynamics of populations of CMV and its satRNA are not coupled. This shows that CMV-satRNA spreads epidemically, as a hyperparasite, in the population of its HV. This conclusion is relevant to the use of CMV-satRNA as a biocontrol agent of CMV.

[Influence of the permeability of the artery responsible for the infarction on the variability of heart rate and late potentials. Its importance in the risk stratification after myocardial infarction]. / Influencia de la permeabilidad de la arteria responsable del infarto sobre la variabilidad de la frecuencia cardíaca y potenciales tardíos. Su importancia en la estratificación del riesgo postinfarto de miocardio.

The use of the heart rate variability for the study of the Autonomic nervous system has been well established. We analyzed late potentials and heart rate variability in 29 control patients and in 102 consecutive patients with a first myocardial infarction. The data obtained were analyzed with both, the medical treatment (thrombolysis and beta-blockers) and the patency of the infarct related vessel. Patients with an infarct had diminished vagal tone as compared with the control group. Those patients with occluded related arteries showed higher incidence of late potentials; interestingly patients with late potentials also had diminished vagal tone. Without looking at the patency of the infarct related artery, thrombolitic and betablocker therapy did not have any effect on vagal tone. All the variables were correlated with the patency of the infarct related artery. Those patients with patent arteries had a preservation of the vagal tone; this was independent of the treatment received and the presence of late potentials. We concluded that the patency of the infarct related artery determines the absence of late potentials and preservation of the vagal tone. This might be one of the mechanisms of how thrombolitic therapy decreases the incidence of cardiac death.

Dynamics of nonpersistent aphid-borne viruses in lettuce crops covered with UV-absorbing nets.

Aphid-transmitted viruses frequently cause severe epidemics in lettuce grown under Mediterranean climates. Spatio-temporal dynamics of aphid-transmitted viruses and its vector were studied on lettuce (Lactuca sativa L.) grown under tunnels covered by two types of nets: a commercial UV-absorbing net (Bionet) and a Standard net. A group of plants infected by Cucumber mosaic virus (CMV, family Bromoviridae, genus Cucumovirus) and Lettuce mosaic virus (LMV, family Potyviridae, genus Potyvirus) was transplanted in each plot. The same virus-infected source plants were artificially infested by the aphid Macrosiphum euphorbiae (Thomas). Secondary spread of insects was weekly monitored and plants were sampled for the detection of viruses every two weeks. In 2008, the infection rate of both CMV and LMV were lower under the Bionet than under the Standard cover, probably due to the lower population density and lower dispersal rate achieved by M. euphorbiae. However, during spring of 2009, significant differences in the rate of infection between the two covers were only found for LMV six weeks after transplant. The spatial distribution of the viruses analysed by SADIE methodology was "at random", and it was not associated to the spatial pattern of the vector. The results obtained are discussed analyzing the wide range of interactions that occurred among UV-radiation, host plant, viruses, insect vector and environmental conditions. Our results show that UV-absorbing nets can be recommended as a component of an integrated disease management program to reduce secondary spread of lettuce viruses, although not as a control measure on its own.

Sequence and structure at the genome 3' end of the U2-strain of tobacco mosaic virus, a histidine-accepting tobamovirus.

The primary sequence of the 3' noncoding region of U2-TMV RNA was determined. A structural model was proposed based on chemical and enzymatic structure mapping as well as on analyses of nuclease protection by aminoacyl-tRNA-synthetase. The model agrees with those proposed for TMV "vulgare" RNA and confirms their general validity for the tobamoviruses. The RNA appears to have a tRNA-like, L-shaped structure at the 3' terminus, linked to a quasi-continuous double-helical stalk, with five pseudoknots involved in the formation of the whole structure. However, the structure of U2-TMV RNA is less stringently conserved than the 3' termini of "vulgare" and other histidine-accepting tobamoviruses. This difference is reflected in the kinetics of aminoacylation of the RNA.

Complex interactions among several nucleotide positions determine phenotypes defective for long-distance transport in the satellite RNA of cucumber mosaic virus.

Ix-satRNA is a CMV-satRNA necrogenic on tomato that is defective for long-distance systemic movement when coinoculated in tobacco with V-TAV. To analyze the determinants for this defective phenotype, full-length cDNA clones of Ix-satRNA and of the closely related, nondefective I17N-satRNA were obtained. Infectious transcripts from these clones (Ix5-satRNA and I17N1-satRNA) had the same phenotypes as the original satRNAs and differed only in four positions: positions 20 (Ix5, C; I17N1, U), 102 (Ix5, C; I17N1, U), 224 (Ix5, deleted; I17N1, A), 327 (Ix5, G; I17N1, A). By site-directed mutagenesis at positions 224 and 327 and by recombination using two common restriction sites, satRNAs in which the bases at these four positions were changed from the composition at Ix5-satRNA to the composition at I17N1-satRNA were obtained. A comparison of the phenotypes of the 13 single, double, and triple mutants (respective to Ix5-satRNA) showed that the defective phenotype of Ix5-satRNA is determined by the nature of the four positions analyzed; mutants at any of the positions 102, 224, and 327 accumulated as efficiently as I17N1-satRNA in systemically infected tobacco leaves when coinoculated with V-TAV. The change C20-->U also restored systemic movement, albeit imperfectly, giving rise to a phenotype that moved systemically less efficiently than I17N1-satRNA. This phenotype determined by U20 is expressed irrespective of the base at position 102, indicating an epistatic interaction between determinants 20 and 102; this interaction does not occur with position 224 or 327. That differences in the analyzed satRNAs are due to their being able, or not, to move systemically is shown by the fact that all of them (including Ix5-satRNA) accumulated to the same high level in directly inoculated leaves. The similarity in the sequences of the analyzed satRNAs and the complexity of the interactions among the effects of base changes at four positions scattered over the satRNA molecule suggest that the observed movement phenotypes may depend on conformational changes in the satRNAs.

Role of cucumovirus capsid protein in long-distance movement within the infected plant.

Direct evidence is presented for a host-specific role of the cucumovirus capsid protein in long-distance movement within infected plants. Cucumber (Cucumis sativus L.) is a systemic host for cucumber mosaic cucumovirus (CMV). Tomato aspermy cucumovirus, strain 1 (1-TAV), multiplied to the levels of CMV (i.e., replicated, moved from cell to cell, and formed infectious particles) in the inoculated leaves of cucumbers but was completely unable to spread systemically. The defective long-distance systemic movement of 1-TAV was complemented by CMV in mixed infections. Coinfection of cucumbers with 1-TAV RNA with various combinations of transcripts from full-length cDNA clones of CMV genomic RNA 1, RNA2, and RNA3 showed that CMV RNA3 alone complemented 1-TAV long-distance movement. We obtained mutants containing mutations in the two open reading frames in CMV RNA3 encoding the 3a protein and the capsid protein (CP), both of which are necessary for cell-to-cell movement of CMV. Complementation experiments with mutant CMV RNA3 showed that only 3a protein mutants, i.e., those with an intact CP, complemented the long-distance movement of 1-TAV in cucumbers. Since CMV and TAV have common systemic host plants, the results presented here are strong evidence for an active, host-specific function of the CPs of these two cucumoviruses for long-distance spread in the phloem. The results also suggest that the plasmodesmata in the vascular system and/or at the boundary between the mesophyll and the vascular system, involved in long-distance movement through the phloem, and those in the mesophyll, involved in cell-to-cell movement, differ functionally.

Analysis of the determinants of the satellite RNA of cucumber mosaic cucumovirus for high accumulation in squash.

Ix-satRNA is a 334-nt cucumber mosaic cucomovirus satellite RNA (CMV-satRNA) with the unusual property of accumulating to high levels in host plants of the family Cucurbitaceae. To identify the determinants for this unusual phenotype, recombinant satRNAs were obtained, by in vitro manipulation of full-length cDNA clones between clone-derived Ix5-satRNA and two other CMV-satRNAs that do not accumulate efficiently in squash: D4-satRNA, which differs in 7 positions from Ix5-satRNA, and B21-satRNA, which differs in 45 positions. The analysis of the accumulation in squash of these hybrid satRNAs showed that determinants for high accumulation in squash occur in three molecular domains: nucleotides 1-46 (a C at position 20), 47-185 (a C at position 102), and 185-334 (not identified). None of these determinants seemed to be necessary or sufficient to confer a phenotype of high accumulation in squash. They resulted, or did not result, in such a phenotype according to the sequences that occur in other parts of the satRNA molecule. Not all of the identified determinants were equally affected by sequence context. These results may suggest that the phenotype in squash is determined by the conformation of the satRNA.