Plant viruses, as any other living organisms, differ genetically from each other as a result of processes (such as mutation, recombination and other forms of genetic exchange) that generate genetic variation in each generation during their reproduction and processes (such as selection, migration and genetic drift) that modulate this variation, determine the distribution of the genetic variants within a population (i.e., the genetic structure of the population) and how it changes with time, in a dynamical phenomenon called evolution. For plant viruses, evolutionary forces that generate and modulate the genetic diversity of their populations are often associated to different phases in their biology and ecology, such as virus-host interactions and host to host transmission. Forces that shape the evolution of plant viruses are at the same time key factors affecting their pathogenic properties, including their ability to cause diseases (an aspect that is studied in the field of epidemiology). The present chapter aims to illustrate how measurement and analysis of genetic diversity and structure of plant virus populations are essential to the current knowledge on the evolutionary biology of plant viruses and how evolutionary factors have a relevant role in the dynamics of virus populations and therefore, in the epidemiology of plant virus diseases.
- genetic diversity
- genetic structure
- plant virus evolution
- plant virus epidemiology
- plant virus resistance
- plant virus emergence
Evolution is defined as the change with time of the frequency distribution of genetic variants in the population of an organism, what is called the genetic structure of the population. In this context, a population of plant viruses may be considered as the group of individuals of the same viral species living and reproducing in a particular and sufficiently restricted environment, so that it represents a single evolving unit (similarly as local interbreeding units of mating organisms, also called local populations or demes, are considered the fundamental evolving units in population genetics; see , pp. 45–46). Two different phases may be identified along the evolutionary process: in the first one, genetic variation is generated during reproduction and the second one consists of the modulation of that variability as the result of driving evolutionary forces acting along the organism's life cycle, which are associated to its biological and ecological interactions in a particular environment.
The present chapter does not seek to be a comprehensive review on the evolutionary mechanisms that shape the diversity and genetic structure of plant virus populations, nor on the broad knowledge derived from the significant number of scientific reports published on this subject during the last decades for different groups of plant viruses. There are a number of excellent reviews covering these topics. In the first part of the chapter, these reviews will be cited in relation to the most relevant concepts that are essential to understand evolution of virus populations. This first part is aimed to serve as a general guide for the readers, who if interested, will be easily addressed to the most relevant literature on the topic. The second part of the chapter will emphasize the important implications of these evolutionary mechanisms in the population dynamics of viruses, that is, the epidemiology of virus diseases, which finally determines their development and distribution in the field.
2. Generation and modulation of genetic diversity: driving forces in evolution of plant virus populations
2.1. Generation of genetic diversity in plant virus populations
Genetic diversity of a population can be defined as the probability that two genetic variants randomly chosen from the population are different . Two mechanisms are the main sources of genetic variation in viruses during their evolutionary process: mutation and genetic exchange. Both of them are presented in the following paragraphs.
Once some initial level of genetic variation is created by mutations, the opportunities of genetic exchange between different genetic variants may increase, contributing to the generation of new variability. Recombination and reassortment are frequent in populations of plant-infecting viruses with either RNA or DNA genomes [2, 5, 10]. Analysis of their sequences indicates that both mechanisms contribute significantly to the generation of variability in the evolution and diversification of certain taxonomic groups [11–16]. Recombination and reassortment events may involve members of the same plant virus species [17–19], members of different species [20–24] or even genus . Genetic exchange by recombination or reassortment may have important epidemiological implications of practical relevance, even more than mutation, as it has been associated to host jumps, host range expansion, changes in virulence, breaking of host resistance and finally, the emergence of new viral plant diseases. Outstanding examples of that are the contribution of recombination and reassortment in the development of a severe epidemic of Cassava mosaic disease in Uganda  and the appearance of several new recombinant species of begomoviruses in the Mediterranean Basin associated to the Tomato yellow leaf curl disease in tomato . However, in spite of the relevant epidemiological role of genetic exchange, till date, little information is available on the rates with which it occurs in plant viruses in the absence of selection. Recombination rates have been experimentally estimated in coinfections of different genotypes of
It has been exposed above that creation of variability is an initial and required step in the evolution of populations. On the other hand, variability may also contribute to effects of evolution that may be detrimental for populations. For instance, a population may become extinct because of an excessive accumulation of mutations, a phenomenon known as lethal mutagenesis , which also takes place in viruses and is an interesting mechanism for antiviral therapies . Also, high mutation rates combined to small sizes of asexual populations (as a result of genetic bottlenecks, see below) may led to the progressive accumulation of deleterious mutations and the loss of mutation-free individuals, with a consequent reduction in fitness in populations, which is called the "Muller's ratchet" [9, 38]. In addition to the adaptive relevance of genetic exchange to create beneficial genomic combinations, recombination and reassortment may represent a sexual mechanism contributing to compensate the accumulation of deleterious mutations and the effect of "Muller's ratchet" in populations, and it has been postulated as a theory of evolution of sex in RNA viruses [9, 39]. Alternatively, it could be that recombination, together with mutation, had evolved as consequences of the fast incorporation rate of RNA-dependent RNA polymerases in RNA viruses. It might be the case at least for several RNA viruses, including TEV, for which a highly significant correlation was found between their recombination and mutation rates . Possibilities for evolution of recombination in RNA viruses were reviewed in .
2.2. Evolutionary forces that determine the genetic diversity of populations of plant viruses
A key concept to understand evolution, that is, the change with time of the genetic structure of a population, is the fitness of an individual or genetic variant.
Fitness, the parameter that determines selection, is dependent on the environment. Therefore, changes in environmental conditions (for instance, a change of host) may be determinant for a variant generated by mutation to be eliminated from or fixed in the population, giving it a chance for adaptation. In a population, the proportion of mutations that are beneficial, neutral, deleterious or lethal is known as the
Comparative analysis of genetic diversity of populations in different phases of the infection cycle of plant viruses has allowed the identification of selective pressures associated to each of them, although selection is often difficult to distinguish from genetic drift, as both mechanisms result in the decrease in population diversity. Phases of viral cycle associated to selection were reviewed in  and . In summary, selective pressures are related to: i) the maintenance of functional structures, that is, certain amino acids involved in the stability of viral particles or in the secondary and tertiary molecular structures required for replication or other interactions; ii) interactions of viruses with their hosts, resulting in the genetic differentiation of natural populations according to the host, the overcoming of host resistance genes, changes in virulence and co-evolution of plants and viruses ; iii) interactions between viruses and their vectors for transmission and between hosts and vectors. The sequence analysis of genes related to some of the functions mentioned above for several plant DNA and RNA viruses indicated that selection on plant virus encoded proteins is mostly negative, as measured by the ratio between nucleotide diversities at non-synonymous and synonymous positions (
Observation of changes in the genetic structure of within-host populations that are associated to different degree of compatibility between the virus and the host provides insights into the host-adaptive process (reviewed in ). In compatible interactions of highly host-adapted viruses, negative selection tends to maintain virus population in equilibrium, resulting in a high stability of its genetic structure, as found in intra-host populations of
Another interesting topic on host-adaptive process is that concerning host-range evolution for those viruses that behave as multi-host parasites. Multi-host parasitism is common among plant viruses, leading to the consideration of generalist and specialist plant viruses [46, 47]. Different hosts represent, indeed, different environments for viruses and, accordingly, fitness differences should be expected for viruses across their host range. Genetic differentiation of virus populations according to the host may indicate host adaptation and detailed analysis show evidence of host adaptation in populations of a particular virus sampled from different hosts, or even from new hosts in which the virus has acquired the capacity to infect . More clear indication is obtained when a virus from an original host is serially transferred to other host and it is observed virus adaptation to the new host but associated to a fitness loss in the original host. This type of host selectivity has been shown even in cases of generalist viruses, supporting the theory of trade-offs across hosts, that is, the virus cannot simultaneously maximize its fitness in all of its alternative hosts . Antagonistic pleiotropy, that is, mutation with positive effects in a given host are deleterious in another one, seems to be the major cause of across-host fitness trade-offs, as reviewed in [48, 49].
In a population, the success in the adaptive process to a new host, that means that any beneficial mutation in the new host become fixed, depends on the distribution of mutational fitness effects (see above), which is highly affected by the environment (the new host species), so that there is a larger proportion of beneficial mutations as the taxonomic relatedness of the new host to the original one decreases. This process is in part explained by antagonistic pleiotropy and may be significantly sensitive to genetic drift . Adaptation is also dependent on
Selection pressures are also associated to the transmission process. Many plant viruses depend on vectors for transmission. Thus, virus-vector interaction is a probable source of selective pressures. Evidence of virus-vector selection comes from the loss of vector transmission capacity for viruses that have been mechanically transmitted in experiments of serial inoculations (reviewed in ). This phenomenon also suggests that might exist trade-offs for adaptation to transmission, as already commented for host adaptation. Virus-vector negative selection is supported by the lower
2.2.2. Genetic drift
Genetic drift refers to the random effects that result from reductions in the population size. In that context, one must consider what is called the effective population size, defined as the number of individuals that give rise the next generation. Plant viruses exhibit high replication rates; therefore, they may reach large population sizes in infected cells or plants. Though, the effective population size of their populations may be several orders of magnitude smaller, as estimated for TMV, TMGMV and WSMV [2, 44]. This may be considered a probable reason to explain the low genetic diversity commonly found in their natural populations, in spite of their high replication and mutation rates .
As shown for selection, genetic drift may be associated to almost every step of the virus life cycle and genetic bottlenecks, which are severe reductions in the effective population size, have been shown to operate during virus colonization of a host and transmission between hosts [58–65]. The multiplicity of infection (MOI), that is, the number of virus particles or genomes that infect a cell, has been estimated for some plant viruses either in local infections or along the systemic colonization of a host [66–68], giving roughly similar results, and showing that MOI may vary during systemic infection. Also, estimates of the size of genetic bottlenecks have been reported to be very low, associated to the systemic invasion of leaves , aphid transmission [58, 69] and contact transmission  between hosts. In addition, the size of bottlenecks is dependent on the viral load, at least during colonization of the host [70, 71].
Above estimates of MOI and of size of genetic bottlenecks are highly relevant to many important processes in virus evolution. For instance, MOI influence the opportunities for genetic exchange to take place, which at least require two different genotypes coinfecting a cell. Also, the efficiency of complementation of defective genomes may occur at high MOI levels, which may be particularly important when deleterious mutants have pleiotropic effects on other viral functions or on other environments (host jumps) . The direct consequence of a severe population bottleneck (an effective population size very low compared to the total census population, which may be very large ) is known as "founder effect", an extreme type of genetic drift implying that the new population (generation) is started (founded) by a few genetic variants randomly sampled from the original population. The overall evolutionary consequence of reductions in the effective population size is a decrease in the genetic diversity within each founded population and a strong spatial structure, derived from the increase in genetic diversity between daughter populations, as observed for TMV even at relatively high MOI , with a stochastic spatial distribution of genotypes [73, 74]. Finally, as already indicated in the end of Section 2.1, when bottlenecks lead the population to an effective size below the threshold needed for selection to eliminate deleterious mutations and ensure the transmission of the fittest variants, the fitness of the population may decrease by progressive accumulation of deleterious mutants, leading the population to extinction by mutational meltdown (the "Muller's ratchet" phenomenon) [9, 36, 75]. Interactions between viruses may promote extinction of one of the viruses by mutational meltdown: coinfection of TMV with TMGMV in
3. Dynamics of genetic diversity and structure of plant virus populations: implications of evolutionary factors in the epidemiology of diseases
The term evolutionary epidemiology has been coined to denote the link between evolutionary biology and epidemiology [44, 77], which basically describes the integration of ecological and evolutionary concepts, as those already reviewed in this chapter, to better understand specific epidemiological components of host-parasite interactions. Conversely, epidemiological dynamics is also an important factor influencing evolutionary dynamics. This framework is at the base of the most recent advances in the areas of evolutionary biology and epidemiology, some of which are commented below, pointing to the most relevant reviews covering those subjects.
Two of the most important properties of pathogens, with evident implications in epidemiology of diseases, are pathogenicity and virulence. Pathogenicity is defined as the qualitative capacity of a pathogen to infect and cause disease on a host. Virulence is the degree of damage that the pathogen infection causes to the host, but in the context of evolution, it refers to the decrease in fitness of the host caused by the pathogen. Hosts may exert selective pressures on both virulence and pathogenicity of the pathogen, as it happens in agricultural systems in which humans manipulate the host genetic structure by the deployment of host genetic resistance in the field, with the consequent risk of appearance of resistance-breaking variants. On the other hand, the genetic structure of host populations may change in response to the pathogen selective pressures, but mostly in natural ecosystems. The reciprocal evolutionary interaction between hosts and pathogens brings the concept of host-pathogen coevolution. In spite of a broad collection of theoretical models regarding host-parasite coevolution, experimental evidences are scant and some advances have been made in this field to test theoretical hypotheses, which have been recently reviewed [45, 78].
Other epidemiological implications of the evolutionary dynamics of plant viruses deal with the improvement of disease management strategies. Acosta-Leal  evaluated possible opportunities for virus-disease control, as resistance genes, natural plant resistance mechanisms, control of coinfection dynamics, modeling virus robustness, etc. and discussed about research advances and needs in relation to a simple theoretical model, which states that an assembly of management measures should be addressed to altogether reduce the effective population size of virus populations, increase their genetic diversity and maximize bottleneck effects, so that a virus population could be gradually excluded from its hosts species.
Finally, a highly relevant epidemiological consequence of plant virus evolutionary dynamics is the risk of emergence of virus diseases, which seriously compromise agriculture production worldwide. Emergence of new diseases may occur either by appearance of new virus species that spill over from wild plant reservoirs or of well known viruses that suddenly show new pathogenic and epidemiological properties (host jumps, resistance-breaking variants). The risk of resistance-breaking was evaluated for a set of representative plant viruses and pathosystems in relation to an index of evolutionary potential, based on the effective population size, the degree of genetic exchange and the amount of gene flow, which was proposed as an important determinant of the durability of resistance against plant viruses . Ecological and epidemiological factors of plant virus emergence often have their origin at the interface between managed and natural ecosystems and are mostly related to a rapid expansion of human activity, including the worldwide distribution of crop species far from their geographic origins, the intensiveness of agricultural practices and the international trade facilitating the spread of damaging viral species, all of them under the effect of global climate change . Factors favoring emergence derive from complex interactions among host plants, viruses and their vectors (for vector-borne viruses) and have been analyzed in the context of evolutionary ecology, genetics and epidemiology . In summary, they result in changes in the ecology and genetic composition of host plant, virus and vector populations during three different temporal phases that describe the process of emergence. In a first phase, viruses spill over from host reservoirs in which they are well-adapted (often reservoirs of wild plants) and jump to the same host species in a new ecological environment or to a new host species. In this phase, ecological conditions for plant hosts, viruses (and vectors) must favor the contact between the original and the new host populations for emergence to occur. This includes the introduction of hosts, viruses (and vectors), often by human activity, in areas where they were not present before. Other factor facilitating new contacts is ecosystem simplification , characterized by reduced species diversity in agricultural compared to natural ecosystems, a concomitant reduction in the genetic diversity of crops compared to wild populations and a higher host density. A second phase consists on the evolutionary process of virus adaptation to the new host or environment to the point that new infections and transmission in the new host is ensured, making between-host transmission independent from the original reservoir. As indicated in Section 2.2.1, adaptation to a new host is a process governed by an assembly of evolutionary factors, such the generation of beneficial mutations (and genetic exchange in cases of cellular coinfection), the interaction between beneficial mutations (epistasis) in a favorable environment (a particular new host) that may results in trade-offs across hosts and obviously, stochastic effects. Here, it is important to stress that the symplast, where plant viruses must replicate and evolve, is a high structured environment where virus populations adopt a metapopulation structure, a set of subpopulations, each one occupying different tissues and organs. This metapopulation structure is probably generated by the effect of genetic bottlenecks and might affect the efficiency of natural selection . In the third phase, an efficient epidemiology should optimize between-host transmission in the new host and environment, which implies new adaptation to vectors in the case of vectored viruses. As predicted by theoretical models, the epidemiological potential of a pathogen depends on its basic reproductive ratio (
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