Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti

Genotyping of a microsatellite locus to differentiate clinical Ostreid herpesvirus 1 specimens

International audienceOstreid herpesvirus 1 (OsHV-1) is a DNA virus belonging to the Malacoherpesviridae family from the Herpesvirales order. OsHV-1 has been associated with mortality outbreaks in different bivalve species including the Pacific cupped oyster, Crassostrea gigas. Since 2008, massive mortality events have been reported among C. gigas in Europe in relation to the detection of a variant of OsHV-1, called μVar. Since 2009, this variant has been mainly detected in France. These results raise questions about the emergence and the virulence of this variant. The search for association between specific virus genetic markers and clinical symptoms is of great interest and the characterization of the genetic variability of OsHV-1 specimens is an area of growing interest. Determination of nucleotide sequences of PCR-amplified virus DNA fragments has already been used to characterize OsHV-1 specimens and virus variants have thus been described. However, the virus DNA sequencing approach is time-consuming in the high-scale format. Identification and genotyping of highly polymorphic microsatellite loci appear as a suitable approach. The main objective of the present study was the development of a genotyping method in order to characterise clinical OsHV-1 specimens by targeting a particular microsatellite locus located in the ORF4 area. Genotyping results were compared to sequences already available. An excellent correlation was found between the detected genotypes and the corresponding sequences showing that the genotyping approach allowed an accuraté discrimination between virus specimens

Detection of dsRNA and DENV2-derived small RNAs in infected HWE mosquitoes.

<p>(A) Detection of dsRNA in midguts of mock infected (a) and DENV2 infected (b) <i>Ae. aegypti</i> using the dsRNA-specific J2 antibody in indirect IFA. (B) Detection of small RNAs in total RNA from DENV2 infected mosquito midguts at various time-points after infection by hybridization with a ³²P-labeled 22 nt RNA probe complementary (antisense) to the DENV2 prM gene in an RNase protection assay <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000299#ppat.1000299-Franz1" target="_blank">[23]</a>. (C) Northern blot analysis of small RNAs in whole mosquitoes 14 days post infection with DENV2 using a ³²P-labeled antisense RNA probe from a 298 nt region of the DENV2 prM gene.</p

Primers used for the generation of dsRNAs targeting RNAi pathway genes.

<p>T7 promoter (not shown) was added to the 5′ end of each primer for in vitro transcription of dsRNA.</p

DENV2 infection of Aag2 cells.

<p>(A) Growth curve of DENV2 in Aag2 cells. Virus titers in cell culture medium were determined by plaque assay. (B) Detection of DENV2 genomic RNA in Aag2 cells by northern blot analysis (5 µg total RNA/lane). Stained ribosomal RNA is shown as loading control. (C) Detection of dsRNA in mock infected (a) and DENV2 infected (b) Aag2 cells using the dsRNA-specific J2 antibody in an indirect immunofluorescence assay (IFA). (D) Detection of DENV2 genome-derived small RNAs consistent in size with siRNAs on successive days after infection. Small RNAs were detected using sense (a) and antisense (b) riboprobes from the prM gene of DENV2.</p

Transmission of DENV2 by <i>Ae. aegypti</i> at 7, 10, and 12 days post infection.

<p>Groups of 200 mosquitoes were non-injected or injected with PBS or 500 ng dsRNA.βGAL, dsRNA.ago2, dsRNA.r2d2, or dsRNA.dcr2. Two days later all mosquito groups were orally infected with DENV2. At 7 (A), 10 (B) and 12 (C) dpi multiple batches of 10 mosquitoes were allowed to probe artificial feeding solutions. The feeding solutions were assayed for virus titer (* indicate P<0.05).</p

DENV2 infection of <i>Aedes aegypti</i> HWE mosquitoes.

<p>(A) DENV2 titers in midguts (red symbols/line) and remaining tissues (carcass; blue symbols/line) of individual mosquitoes at pre-determined days post-infection (dpi). (B) Detection of DENV2 E antigen by indirect IFA in midgut (a and b) and salivary gland (c and d) tissue at 7 (a and c) and 14 (b and d) dpi. Insets in panels a and c show IFAs of midguts and salivary glands from non-infected mosquitoes. (C) Northern blot hybridization to determine accumulation of DENV2 genomic RNA in midguts and carcasses from 5 to 14 days post-infection (5 µg total RNA/lane). Ethidium bromide-stained rRNA shown as loading controls. NBF = non-bloodfed (non-infected).</p

Effects of silencing expression of key RNAi genes prior to DENV2 infection.

<p>(A) Northern blot analysis showing RNA silencing of RNAi component genes in individual mosquitoes (3 µg total RNA/lane). Blots were hybridized with antisense ³²P-dCTP-labeled DNA probes amplified from <i>Aa-ago2</i> (a), <i>Aa-r2d2</i> (b) and <i>Aa-dcr2</i> (c) genes. Ribosomal RNAs are shown as loading controls. (B) Mean DENV2 titers 7 dpi in individual <i>Ae. aegypti</i> with an impaired RNAi pathway. Mosquitoes were either non-injected or injected with 500 ng of dsRNA.βGAL, dsRNA.ago2, dsRNA.r2d2, or dsRNA.dcr2 and allowed to recover for 2 days, then given a blood meal containing ∼10⁷ pfu/ml of DENV2. (Bars indicate mean values of titers ±SEM, * indicate P<0.05). (C) Detection of DENV2 genomic RNA by northern blot analysis in whole <i>Ae. aegypti</i> at 14 days after virus challenge. Approximately 5 µg of the total RNA extracted from 60 mosquitoes were loaded in each lane. Blots were hybridized with a ³²P-labeled cDNA probe derived from the prM sequence of DENV2 RNA. Ribosomal RNAs are shown as loading controls. Quantitation of RNA signal was performed using a phosphorimager.</p