Fig 1.
Detection of sfRNA in mosquito saliva.
(A) sfRNA species detected by Northern blotting in infected mosquitoes. (B) sfRNA1 detected by northern blotting in infected pooled mosquito saliva. (C) gRNA; (D) sfRNA; and (E) sfRNA:gRNA ratio in salivary glands and saliva determined by qRT-PCR. Points represent a pair of salivary glands, or one saliva collected from one mosquito. (C-D) Bars represent geometric means ± 95% C.I. and dashed lines indicate limit of detections which were 133 copies in gRNA (C) and 31,216 copies in 3’ UTR/ sfRNA1 (D). (E) Bars represent arithmetic means ± s.e.m. N salivary glands, 43; N saliva, 71; from two mosquito batches. (F) Sequences of sfRNA species found in saliva.
Fig 2.
Salivary sfRNA is sensitive to nuclease degradation only after detergent treatment.
(A-C) Impact of Triton X-100 treatment on nuclease resistance for in vitro transcribed sfRNA1 in uninfected saliva (A) and for sfRNA1 (B) and gRNA (C) from infected saliva. (D-F) Impact of DMSO and Proteinase K treatment on nuclease resistance for in vitro transcribed sfRNA1 in uninfected saliva (D) and for sfRNA1 (E) and gRNA (F) from infected saliva. Compare the third and fourth group in E to note that unless inhibited by PMSF PK decreased RNase activity. Symbol colors indicate biological repeats. Saliva samples were collected from three mosquito batches. Dashed lines indicate limits of detection which were 133 copies in gRNA and 31,216 copies in 3’ UTR/ sfRNA1. u, uninfected. i, infected. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001, ****, p-value <0.0001 as determined by Fisher’s LSD test.
Fig 3.
Mosquito salivary EVs are enriched in DENV2 3’UTR RNA, relative to infected cells.
(A) Representative images of nuclei (blue), DENV2 gRNA (green), and DENV2 3’UTR (magenta) from C6/36 cells 24 h.p.i. with DENV2 NGC at an MOI of 0.3. Scale bar, 50 μm. (B) Representative images of EVs imaged in brightfield (grey), DENV2 gRNA (green), and DENV2 3’UTR (magenta) from Ae. aegypti saliva collected 10 days post inoculation with DENV2 NGC. Scale bar, 2 μm. (A & B) RNA-FISH of DENV2 gRNA and 3’UTR labelled with Oligopaints probes. (C) Ratios of DENV2 3’UTR: gRNA fluorescence signals as detected via RNA-FISH in DENV2 NGC infected C6/36 cells and EVs from Ae. aegypti saliva (left). Ratios of DENV2 3’UTR: gRNA fluorescence signals as detected via RNA-FISH on EVs from Ae. aegypti saliva from the three independent labelling experiments (right). ****, p ≤ 10−4 as determined by a two-sided Mann-Whitney U test. Exact p-value = 1.79 x 10−12. NCells = 102; NEVs = 35.
Table 1.
Higher sfRNA:gRNA ratio in mosquito saliva increases DENV2 infection of human cells.
The experiment number, identity of the virus strain, concentration of gRNA and sfRNA, and sfRNA:gRNA ratio for pools of salivas collected from DENV2 infected mosquitoes are shown in the first five columns. The total volume of salivary inoculum (volume of infected saliva (inf.) + volume of uninfected saliva (uninf.)) used to infect Huh-7 cells and the gRNA copies added per well of Huh-7 cells are indicated in columns six and seven. The geometric mean, lower and upper 95% CI of dsRNA foci per cell at 2 days post infection are in column eight.
Fig 4.
Higher sfRNA:gRNA ratio in mosquito saliva increases DENV2 infection.
(A) Representative pictures of nucleus (DAPI, blue) and dsRNA (green) from Huh-7 cells supplemented with uninfected saliva. (B-E) Representative pictures of nucleus and dsRNA from (B, D) and number of dsRNA foci per (C, E) Huh-7 cell supplemented with saliva infected with DENV2 PR6452 or PR1940 containing either high or low sfRNA:gRNA ratio. (B, D) Scale bar, 50 μm. (C, E) Each point indicates dsRNA foci in one cell. Bars show geometric means ± 95% C.I. *, p-value < 0.05; **, p-value < 0.01 as determined using a Mann-Whitney U test.
Fig 5.
DENV2 3’UTR enhances DENV2 gRNA viral levels while inhibiting innate immunity.
Huh-7 cells were either regularly infected (non-treated), or 2 hours prior to infection transfected with sense DENV2 3’UTR, antisense DENV2 3’UTR or lipofectamine alone. 17 hours post infection total cellular RNA was extracted and (A) Absolute copy number of intracellular DENV2 gRNA was measured by qRT-PCR. **, p-value ≤ 0.005, ****, p-value ≤ 0.0001 as determined by a Fisher’s LSD test. (B) Relative mRNA transcript levels of IFN-β and IFN-λ1 measured at 17 hours post infection, normalized first to GAPDH mRNA levels, and then to regular infection (“none” treatment post infection). *, p-value ≤ 0.05, as determined by unpaired t-test with Welch’s correction. (C) Relative mRNA transcript levels of interferon stimulated genes, ISG15 and MX-1 measured at 17 hours post-infection, normalized first to GAPDH mRNA levels, and then to regular infection (“none” treatment post infection). *, p-value≤0.05, **, p-value ≤ 0.005. For ISG15, p-value was determined by a Dunn’s test. For MX-1 mRNA levels, p-value was determined by an unpaired t-test with Welch’s correction. Points demonstrate replicate values from one independent experiment. Bars demonstrate arithmetic means ± s.e.m.
Fig 6.
Model: sfRNA in salivary vesicles is transported into cells to enhance infection at the biting site.
sfRNA packaged inside extracellular vesicles in infectious mosquito saliva is transferred to human skin cells during biting. sfRNA inhibits the antiviral innate immune response (interferon (IFN) system) to enhance skin cell virus infection. Created with BioRender.com.