Hongye Li and Marilyn J. Roossinck report on an experiment to show how genetic bottlenecks work in a virus population.

Academic / Technical Report
Hongye Li

Hongye Li and Marilyn J. Roossinck, "Genetic Bottlenecks Reduce Population Variation in an Experimental RNA Virus Population," Journal of Virology 78, no. 19 (October 2004): 10582–10587

Journal of Virology
Hongye Li, Marilyn J. Roossinck
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Genetic bottlenecks are stochastic events that limit genetic variation in a population and result in founding populations that can lead to genetic drift. Evidence of past genetic bottlenecks in numerous biological systems, from mammals to viruses, has been described. In this study, we used an artificial population of Cucumber mosaic virus consisting of 12 restriction enzyme marker-bearing mutants. This population was inoculated onto young leaves of tobacco plants and monitored throughout the course of systemic infection. We show here that the genetic variation in a defined population of an RNA virus is significantly, stochastically, and reproducibly reduced during the systemic infection process, providing clear evidence of a genetic bottleneck.

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In this study, we compared the number of genetically marked mutants of CMV present in the inoculated tobacco leaves with those in the primary and secondary systemically infected leaves. Our results showed that the population diversity decreased significantly when the population moved from the inoculated leaves to primary systemic leaves and decreased further as the systemic infection progressed. During this process, the elimination of a majority of the mutants was stochastic. Hence, while the population is affected by selection, there is a significant stochastic bottleneck present during virus movement from the inoculated leaves to uninoculated young leaves by which the CMV population variation is reduced. This finding indicates that systemic movement plays an important role in the genetic structure of RNA virus population in infected plants. This finding also provides a plausible explanation for the observed difference in mutation frequency of CMV in infected tobacco plants versus in protoplasts and the level of genetic variation that was lower than expected (43).

Plant cells are connected by numerous plasmodesmata (PD) through which the virions or nucleic acid of plant viruses can pass (26). Plant viruses encode movement proteins that have the capacity to increase the plasmodesmatal molecular size exclusion limit and facilitate virus movement between cells (26, 28). For a successful systemic infection of a plant virus, the virus must establish an infection in epidermal cells, move through several mesophyll cells followed by vascular bundle sheath (BS) and vascular parenchyma (VP) cells, and then enter into the companion cell-sieve element (CC-SE) complex within the inoculated leaves (Fig. ​(Fig.1B).1B). Once inside the CC-SE complex, the viruses are transported along with the photoassimilates towards sink tissues (8, 21, 28). After viruses reach a systemic leaf, they exit from the phloem cells of major veins, followed by an invasion of BS cells and then mesophyll cells (5, 8, 44).

It is generally accepted that there are restrictions at the interface between the VP and CC-SE complex and that these restrictions may affect systemic movement of the virus in the plant (8, 10, 21, 28, 47). The restriction at the interface of the VP and the CC/SE complex may be associated with the frequency of PD, which are abundant in cell walls between mesophyll cells but are limited at the interface between the VP and the CC/SE complex for plants of apoplastic phloem loaders, including the Solanaceae (46). The restriction of virus entry into the phloem may also be associated with different mechanisms that viruses use to move into the vascular tissues (8, 21).

Details of the dynamics of virus loading into and unloading from the phloem are unknown. Inoculation of a small area of the fifth leaf followed by excision at 2 dpi and inoculation of the whole leaf followed by excision at 6 dpi resulted in similar populations in the systemic tissue (Table ​(Table2),2), implying that virus loading into the phloem of the initially infected leaf may occur over a short period of time rather than as a continuous process. It is also possible that infected mesophyll cells exclude superinfection, and this could result in subpopulations in different areas of systemically infected leaves. However, this type of distribution is not seen in inoculated leaves, and given the abundance of PD between mesophyll cells, it seems likely that numerous virions enter a cell simultaneously. Hall et al. (22) found that the coinfection of two related Wheat streak mosaic virus strains resulted in spatial subdivisions of virus strains within individual systemic leaves or tillers of plants, with some leaves containing either strain alone and some containing both. An analogous phenomenon was shown recently by fluorescent labeling of potyviruses in both inoculated leaves and systemic leaves of Nicotiana benthamiana plants by confocal laser scanning microscopy (9). However, the spatial separation of populations did not occur when two unrelated viruses coinfected the same hosts (9, 22). These results were interpreted as being the result of virus-induced gene silencing at a cellular level. Our findings showed that the compositions of populations in different areas of the same systemic leaf varied from each other (Table ​(Table2),2), indicating that the distribution of the mutants in the systemic leaf occurs in patches. Retaining the inoculated leaf on the plant did not increase the number of mutants recovered from the systemic leaf (Table ​(Table2).2). Hence, the most plausible explanation of the bottlenecks found in this study is that only a subset of genotypes in a virus quasispecies are randomly loaded into vascular tissue in a loading event due to the physical and/or functional restriction of PD at the interface of the VP and the CC-SE complex. Once this population of genotypes reaches the systemic leaf, they exit and initiate infection. Since different areas of the systemically infected leaves contain different populations, it is plausible that the exit from the phloem is also restricted. Consequently, the populations in the inoculated leaves are segregated and subpopulations are formed in the systemic leaf after the systemic movement.

The presence of bottlenecks associated with human immunodeficiency virus has been deduced by the significant reduction of genetic diversity (19, 24, 29). The most important implication of genetic bottlenecks is genetic drift, which can drive changes in an RNA virus population and the emergence of new virus strains (3, 16, 48). In addition, repeated bottlenecks can result in a loss of fitness, as demonstrated by several experimental systems (4, 12, 31, 32, 49). The influence of bottlenecks on the fitness of the RNA virus population is associated with the bottleneck size. Novella et al. (30) showed that the population will gain fitness in large population transmissions due to competition and optimization of the quasispecies. CMV may be considered an extremely fit virus since it has the largest host range of any known virus, infecting over 1,200 species of plants (13). Clearly, the bottlenecks described in this study are not restrictive enough to cause an overall loss in fitness. They may, however, be sufficient to limit the mutation frequency in systemically infected plants.

Fny CMV did not exhibit any changes in the consensus sequence over multiple passages in several different host species (43). Although this lack of genetic drift under the influence of significant bottlenecks suggests that the majority of individuals do not contain mutations, in some cases (e.g., pepper), every individual virus examined had at least one mutation (43). Hence, negative selection is also an important factor that contributes to the remarkable stability of the CMV genomic sequence.

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