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. 2009 Mar 5:9:49.
doi: 10.1186/1471-2180-9-49.

Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

Chris M Cirimotich  1 , Jaclyn C Scott, Aaron T Phillips, Brian J Geiss, Ken E Olson
Affiliations

Suppression of RNA interference increases alphavirus replication and virus-associated mortality in Aedes aegypti mosquitoes

Chris M Cirimotich et al. BMC Microbiol. .

Abstract

Background: Arthropod-borne viruses (arboviruses) can persistently infect and cause limited damage to mosquito vectors. RNA interference (RNAi) is a mosquito antiviral response important in restricting RNA virus replication and has been shown to be active against some arboviruses. The goal of this study was to use a recombinant Sindbis virus (SINV; family Togaviridae; genus Alphavirus) that expresses B2 protein of Flock House virus (FHV; family Nodaviridae; genus Alphanodavirus), a protein that inhibits RNAi, to determine the effects of linking arbovirus infection with RNAi inhibition.

Results: B2 protein expression from SINV (TE/3'2J) inhibited the accumulation of non-specific small RNAs in Aedes aegypti mosquito cell culture and virus-specific small RNAs both in infected cell culture and Ae. aegypti mosquitoes. More viral genomic and subgenomic RNA accumulated in cells and mosquitoes infected with TE/3'2J virus expressing B2 (TE/3'2J/B2) compared to TE/3'2J and TE/3'2J virus expressing GFP. TE/3'2J/B2 exhibited increased infection rates, dissemination rates, and infectious virus titers in mosquitoes following oral bloodmeal. Following infectious oral bloodmeal, significantly more mosquitoes died when TE/3'2J/B2 was ingested. The virus was 100% lethal following intrathoracic inoculation of multiple mosquito species and lethality was dose-dependent in Ae. aegypti.

Conclusion: We show that RNAi is active in Ae. aegypti cell culture and that B2 protein inhibits RNAi in mosquito cells when expressed by a recombinant SINV. Also, SINV more efficiently replicates in mosquito cells when RNAi is inhibited. Finally, TE/3'2J/B2 kills mosquitoes in a dose-dependent manner independent of infection route and mosquito species.

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Figures

Figure 1
Figure 1
Detection of V5-B2 protein in mosquito cell culture. V5-B2 protein was detected by immunoblot using anti-V5 antibody in total protein from Aag2 cell culture. Molecular weights are indicated on the left side of each panel. Lane 1, protein molecular weight marker. Lane 2, TE/3'2J/B2-infected cells. Lane 3, TE/3'2J/GFP-infected cells. Lane 4, TE/3'2J-infected cells. Lane 5, Mock-infected cells.
Figure 2
Figure 2
Accumulation of Dicer cleavage products in cells infected with TE/3'2J/GFP or TE/3'2J/B2 virus. Cell lysates were generated from Aag2 cells 36 hours post mock-, TE/3'2J/GFP, or TE/3'2J/B2 virus-infection (MOI = 0.01) (indicated to left of each panel). A synthetic 500 bp biotinylated dsRNA product was introduced into the lysates and, at indicated time points, samples were taken and the presence of small RNAs was determined by Northern blot analysis. Ethidium bromide-stained ribosomal RNAs located below each blot serve as loading controls. Arrows indicate position of 25 and 19 nucleotide markers.
Figure 3
Figure 3
Detection of virus-specific siRNAs in Aag2 cells (A) and Ae. aegypti (Higgs White Eyes) mosquitoes (B). Monolayers of Aag2 cells were mock infected or infected with TE/3'2J, TE/3'2J/GFP, or TE/3'2J/B2 virus at MOI = 0.01. Mosquitoes were intrathoracically inoculated with cell culture medium from TE/3'2J, TE/3'2J/GFP, or TE/3'2J/B2 virus. At indicated times post infection, total RNA was isolated and probed using an E1-specific riboprobe for virus-derived siRNA. Ethidium bromide-stained ribosomal RNA below each blot serves as a loading control. Time in hours post infection is noted below ribosomal RNA controls. Arrows indicate position of 25 and 19 nucleotide markers.
Figure 4
Figure 4
Detection of viral RNAs in Aag2 cells (A) and Ae. aegypti mosquitoes (B). Monolayers of Aag2 cells were mock-infected or infected with TE/3'2J, TE/3'2J/GFP, or TE/3'2J/B2 virus at MOI = 0.01. Mosquitoes were intrathoracically-inoculated with cell culture medium, TE/3'2J, TE/3'2J/GFP, or TE/3'2J/B2 virus. At indicated times post infection, total RNA was isolated and an E1-specific riboprobe was used to detect virus genomic and subgenomic RNA. Ethidium bromide-stained ribosomal RNA below each blot serves as a loading control. Time post infection for each virus in (A) is 0, 24, 48, and 72 hrs, and in (B) 0, 48, and 96 hrs. G = genomic; S1 = first subgenomic; S2 = second subgenomic.
Figure 5
Figure 5
Growth of TE/3'2J, TE/3'2J/GFP, and TE/3'2J/B2 viruses in invertebrate and vertebrate cells. A) Triplicate flasks containing cell monolayers of Aag2 cells (A, top panel) and Vero cells (A, bottom panel) were infected at MOI = 0.01. Titers were determined by plaque formation on Vero cells. Black circles = TE/3'2J, Black squares = TE/3'2J/GFP, Black triangles = TE/3'2J/B2. B) Cytopathic effect of TE/3'2J, TE/3'2J/GFP, and TE/3'2J/B2 on Aag2 cells at 72 hrs post infection (MOI = 0.01).
Figure 6
Figure 6
Infection and dissemination of TE/3'2J, TE/3'2J/GFP, and TE/3'2J/B2 viruses in Ae. aegypti mosquitoes following oral bloodmeal. At the indicated day post-bloodmeal, viral titers were determined for A) midguts and remaining B) mosquito carcass. n = 48 per group. "TE/3"' = TE/3'2J, "GFP" = TE/3'2J-GFP, "B2" = TE/3'2J/B2. Horizontal line represents the mean for each data set. (*) above data set indicates that the mean TE/3'2J/B2 titer is significantly higher than TE/3'2J and TE/3'2J/GFP infections. (**) below the infection and dissemination rates indicates significantly higher infection and dissemination rates as compared to TE/3'2J virus infection. Due to the lack of dissemination positive mosquitoes in the Day 4 TE/3 and GFP samples (Figure B), statistical significance of the Day 4 B2 group as compared to the TE/3 and GFP groups could not be determined.
Figure 7
Figure 7
Virus associated mortality of Ae. aegypti HWE mosquitoes following infection by TE/3'J/B2 virus. A) Oral bloodmeal infection Mosquitoes were given an infectious oral bloodmeal containing 1 × 107 PFU of virus and kept at optimal rearing conditions. Mortality was monitored daily for a total of 21 days. n = 200 mosquitoes per group. B) Infection via intrathoracic injection Mosquitoes were injected with virus stock diluted to 1 × 107 PFU/ml and mortality was monitored daily. Day one mortality was not included. Black diamonds = Mock; Black circles = TE/3'2J; Black squares = TE/3'2J/GFP; Black triangles = TE/3'2J/B2. C) Determination of a mosquito 50 percent lethal dose for TE/3'2J-B2 infection. Groups of mosquitoes were intrathoracically injected with TE/3'2J/B2 virus diluted ten-fold and mortality was monitored daily. n = 50 mosquitoes/group. White bar indicates 50% mortality.
Figure 8
Figure 8
Virus-associated mortality in different mosquito species. Female Ae. albopictus (A) or Cx. tritaeniorhynchus (B) mosquitoes were injected with virus stock diluted to 1 × 107 PFU/ml and mortality was monitored daily. Day one mortality was not included. Black diamonds = Mock; Black circles = TE/3'2J; Black squares = TE/3'2J/GFP; Black triangles = TE/3'2J/B2.
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