Deep sequencing reveals persistence of intra- and inter-host genetic diversity in natural and greenhouse populations of zucchini yellow mosaic virus

Abstract

The genetic diversity present in populations of RNA viruses is likely to be strongly modulated by aspects of their life history, including mode of transmission. However, how transmission mode shapes patterns of intra- and inter-host genetic diversity, particularly when acting in combination with de novo mutation, population bottlenecks and the selection of advantageous mutations, is poorly understood. To address these issues, this study performed ultradeep sequencing of zucchini yellow mosaic virus in a wild gourd, Cucurbita pepo ssp. texana, under two infection conditions: aphid vectored and mechanically inoculated, achieving a mean coverage of approximately 10 ,000×. It was shown that mutations persisted during inter-host transmission events in both the aphid vectored and mechanically inoculated populations, suggesting that the vector-imposed transmission bottleneck is not as extreme as previously supposed. Similarly, mutations were found to persist within individual hosts, arguing against strong systemic bottlenecks. Strikingly, mutations were seen to go to fixation in the aphid-vectored plants, suggestive of a major fitness advantage, but remained at low frequency in the mechanically inoculated plants. Overall, this study highlights the utility of ultradeep sequencing in providing high-resolution data capable of revealing the nature of virus evolution, particularly as the full spectrum of genetic diversity within a population may not be uncovered without sequence coverage of at least 2500-fold.

Fig. 1.

Effect of coverage in the probability (estimated assuming a binomial distribution) of detecting the ZYMV CP variants uncovered in this study. Each colour represents a different mutation, labelled with their nucleotide position in the genome and variant frequency in parentheses. Six CP mutations were uncovered in the current study. Four of these mutations were sampled only once and ranged from 3 to 4.8 % in variant frequency: nt 8547 (3 %), 8631 (4.8 %), 8971 and 9009 (3.7 %). The other two were found in more than one sample with variant frequencies averaging 11.7 % (nt 8715) and 3.7 % (nt 8971).

Fig. 2.

Variation in variant frequency over time and space of ZYMV variants in the field experiment. The three-dimensional graphics show changes in variant frequency, with each colour representing a different plant. The y-axis shows how the variant frequency increases over the course of the infection. The x-axis shows variation over time within each plant or intra-host variation (moving from left to right). The z-axis shows variation over space, or between each plant (moving from the front to the back of the graph). These four graphs depict fluctuations in variant frequencies at nt 2205 (a), 7317 (b), 4626 (c) and 1254 (d).

Fig. 3.

Fluctuations in variant frequency (nt 7317) in aphid-transmitted versus mechanically transmitted plants. The variant frequency in the first infected field plant (▪; F8) increased from >1 % to 98.7 % over the course of infection. In contrast, in the greenhouse plants (⧫), the frequency in the first plant was 10 % and dropped to 1.7 % by the last host.

Fig. 4.

Distribution of mutations across the ZYMV genome under field (below) and greenhouse (above) conditions. The length of the lines indicates the relative number of samples with that particular mutation, and the schematic of the viral genome indicates in which gene each mutation occurred.