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

Brackney DE, Beane JE, Ebel GD - PLoS Pathog. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USA.

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.

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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).
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ppat-1000502-g005: 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).

Mentions: Next, we tested the association between viRNA coverage and mutation frequency directly. To accomplish this, we identified regions in NS5 and the 3′-UTR of the WNV genome that had varying degrees of viRNA coverage and sampled WNV genomes from the same RNA specimens used to generate sRNA libraries. viRNA coverage per nucleotide along the entire genome was used to determine the frequency distribution of number of viRNA hits per nucleotide position. Positions were then ranked into quartiles and the interquartile range combined. At 7 days there were three mutations identified in NS5 and no mutations in the 3′-UTR. These mutations were evenly distributed across the three frequency distribution classifications. In WNV genomes sampled at 14 days, mutations were detected in both NS5 and 3′-UTR with increasing viRNA coverage associated with increasing mutation frequency. (Chi-squared test for trend, p = 0.0393) (Figure 5).


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

Brackney DE, Beane JE, Ebel GD - PLoS Pathog. (2009)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2698148&req=5

ppat-1000502-g005: 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).
Mentions: Next, we tested the association between viRNA coverage and mutation frequency directly. To accomplish this, we identified regions in NS5 and the 3′-UTR of the WNV genome that had varying degrees of viRNA coverage and sampled WNV genomes from the same RNA specimens used to generate sRNA libraries. viRNA coverage per nucleotide along the entire genome was used to determine the frequency distribution of number of viRNA hits per nucleotide position. Positions were then ranked into quartiles and the interquartile range combined. At 7 days there were three mutations identified in NS5 and no mutations in the 3′-UTR. These mutations were evenly distributed across the three frequency distribution classifications. In WNV genomes sampled at 14 days, mutations were detected in both NS5 and 3′-UTR with increasing viRNA coverage associated with increasing mutation frequency. (Chi-squared test for trend, p = 0.0393) (Figure 5).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, University of New Mexico School of Medicine, Albuquerque, NM, USA.

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.

Show MeSH
Related in: MedlinePlus