Analysis of viral (zucchini yellow mosaic virus) genetic diversity during systemic movement through a Cucurbita pepo vine

Abstract

Determining the extent and structure of intra-host genetic diversity and the magnitude and impact of population bottlenecks is central to understanding the mechanisms of viral evolution. To determine the nature of viral evolution following systemic movement through a plant, we performed deep sequencing of 23 leaves that grew sequentially along a single Cucurbita pepo vine that was infected with zucchini yellow mosaic virus (ZYMV), and on a leaf that grew in on a side branch. Strikingly, of 112 genetic (i.e. sub-consensus) variants observed in the data set as a whole, only 22 were found in multiple leaves. Similarly, only three of the 13 variants present in the inoculating population were found in the subsequent leaves on the vine. Hence, it appears that systemic movement is characterized by sequential population bottlenecks, although not sufficient to reduce the population to a single virion as multiple variants were consistently transmitted between leaves. In addition, the number of variants within a leaf increases as a function of distance from the inoculated (source) leaf, suggesting that the circulating sap may serve as a continual source of virus. Notably, multiple mutational variants were observed in the cylindrical Inclusion (CI) protein (known to be involved in both cell-to-cell and systemic movement of the virus) that were present in multiple (19/24) leaf samples. These mutations resulted in a conformational change, suggesting that they might confer a selective advantage in systemic movement within the vine. Overall, these data reveal that bottlenecks occur during systemic movement, that variants circulate in the phloem sap throughout the infection process, and that important conformational changes in CI protein may arise during individual infections.

RNA Polymerase Slippage as a Mechanism for the Production of Frameshift Gene Products in Plant Viruses of the Potyviridae Family

“…Interestingly, the presence of an additional A residue in this motif was also detected in libraries of PPV-derived small RNAs (not shown). Besides, published data on another potyvirus, Zucchini yellow mosaic virus, showed a minor variant with an extra A in all samples of a Cucurbita pepo vine studied by deep sequencing of long RNAs (13), and our analysis located this modification in the PIPO GA 6 motif as well.…”

Transcriptional slippage in the positive‐sense RNA virus family Potyviridae

“…The mean frequency of an “A” insertion at the GA 6 site was 0.8%, with frequencies for individual samples ranging from 0.3 to 1.0% ( Appendix Fig S3 ). Similarly, a low level (1–2%) of frameshift mutations at position 2,891 (which corresponds to the GA 6 site) has been reported for Zucchini yellow mosaic virus 20 .…”

The Potyviruses: An Evolutionary Synthesis Is Emerging

“…These describe features of gene populations, using mathematical models, and compare observed features with those likely to result from sequential random changes. Popgen analyses have been used to study potyvirus populations within plant populations (e.g., Achon [64]; Li et al [65]; Wang et al [66]; Hajizadeh et al [67]) and, increasingly, virus populations within individual plants (e.g., Cuevas et al [68]; Domingo and Perales [69]; Dunham et al [70]; Kutnjak et al [71]; Rousseau et al [72]; Seo et al [73]). Such studies have shown that the effective populations of potyviruses are all around 10,000 [74], and the "The high potential for genetic variation in plant viruses need not necessarily result in high diversity of virus populations.…”