RNAi targeting of West Nile virus in mosquito midguts promotes virus diversification

Abstract

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification.

Figure 1. Size and Abundance of Small RNA Reads Mapping to the WNV Genome.

The total abundance of sRNAs mapping to the WNV genome based on sRNA length. Black and grey bars correspond to 7 and 14 days post infection, respectively.

Figure 2. viRNA Coverage of WNV Genome at 7 and 14 Days Post Infection.

Complete genome of WNV showing the intensity of viRNA coverage at each nucleotide of the genome at 7 (A) and 14 (B) days postfeeding. Reads originating from the positive strand are shown in blue, above the axis, and those originating from the negative strand are shown in red, below the axis. Particular regions appear to be preferentially targeted by the RNAi response, including the 5′ aspect of the C coding region and the 3′ non-coding region. Peaks are generally higher at 14 than 7 days post infection (dpi), with notable exceptions in NS2a, at the NS4b/NS5 junction and the 3′-UTR. Both positive and negative polarity RNA is targeted by viRNAs generated in mosquito midgut cells. At 14 dpi, although true for both 7 and 14 dpi, the focus of viRNA targeting in the C coding sequence (C) is characterized by a relatively intense targeting of the negative strand compared to the 3′-UTR (D). Notably, well documented imperfect stem-loops and other critical functional RNA structures within the 3′-UTR do not appear to be intensely targeted by the RNAi response.

Figure 3. A Small Subset of viRNA Reads Is Highly Abundant.

viRNA abundance observed at 7 (A) and 14 (B) days post infection (black bars) compared to the null expectation of equal abundance of all reads (grey bars). Expected values were obtained through permutation analysis with replacement sampling of n = 2544 (day 7) or n = 4419 (day 14) random genome positions. 100,000 permutations were conducted for each dataset. Expected and observed distributions of read abundances were statistically compared by the Kolmogorov-Smirnov test (P<0.001 at both timepoints).

Figure 4. Targeting of the WNV Genome by RNAi at 7 and 14 Days Post Infection Is Correlated.

(A) Count of reads aligning to each genome position at 7 and 14 days, n = 11,029, Spearman r = 0.7610, p<0.0001. (B) Count of abundant individual sequence reads in libraries obtained at 7 and 14 days, n = 433, Pearson R-squared = 0.03674, p<0.0001.

Figure 5. Intense Targeting of WNV Genome Is Associated with High Mutational Diversity.

viRNA coverage intensity was classified according to quartiles. Interquartile ranges (min, max) at 7 and 14 days were 1–6 (0, 64) and 3–11 (0, 120), respectively. The total number of nucleotides sequenced in each coverage class was 10,225, 5,062 and 7,475 at 7 dpi and 5,990, 7,442 and 7,438 at 14 dpi. Statistical significance was determined by chi squared test for trend. A significant association between viRNA coverage was detected at 14 dpi (p = 0.0393) but not at 7 dpi (p = 0.8107).