Endogenous non-retroviral elements in genomes of Aedes mosquitoes and vector competence

International audienceRecent extensive (re)emergences of arthropod-borne viruses (arboviruses) such as chikungunya (CHIKV), zika (ZIKV) and dengue (DENV) viruses highlight the role of the epidemic vectors, Aedes aegypti and Aedes albopictus, in their spreading. Differences of vector competence to arboviruses highlight different virus/vector interactions. While both are highly competent to transmit CHIKV (Alphavirus,Togaviridae), only Ae. albopictus is considered as a secondary vector for DENV (Flavivirus, Flaviviridae). Among other factors such as environmental temperature, mosquito antiviral immunity and microbiota, the presence of non-retroviral integrated RNA virus sequences (NIRVS) in both mosquito genomes may modulate the vector competence. Here we review the current knowledge on these elements, highlighting the mechanisms by which they are produced and endogenized into Aedes genomes. Additionally, we describe their involvement in antiviral immunity as a stimulator of the RNA interference pathways and in some rare cases, as producer of viral-interfering proteins. Finally, we mention NIRVS as a tool for understanding virus/vector co-evolution. The recent discovery of endogenized elements shows that virus/vector interactions are more dynamic than previously thought, and genetic markers such as NIRVS could be one of the potential targets to reduce arbovirus transmission

Contrasting silencing mechanisms of the same target mRNA by two regulatory RNAs in Escherichia coli

International audienceSmall RNAs are key components of complex regulatory networks. These molecules can integrate multiple cellular signals to control specific target mR-NAs. The recent development of high-throughput methods tremendously helped to characterize the full targetome of sRNAs. Using MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we reveal the targetomes of two sRNAs, CyaR and RprA. Interestingly, both CyaR and RprA interact with the 5-UTR of hdeD mRNA, which encodes an acid-resistance membrane protein. We demonstrate that CyaR classically binds to the RBS of hdeD, interfering with translational initiation. We identified an A/U-rich motif on hdeD, which is bound by the RNA chaperone Hfq. Our results indicate that binding of this motif by Hfq is required for CyaR-induced degradation of hdeD mRNA. Additional data suggest that two molecules of RprA must bind the 5-UTR of hdeD to block translation initiation. Surprisingly, while both CyaR and RprA sRNAs bind to the same motif on hdeD mRNA, RprA solely acts at the translational level, leaving the target RNA intact. By interchanging the seed region of CyaR and RprA sRNAs, we also swap their regulatory behavior. These results suggest that slight changes in the seed region could modulate the regulation of target mRNAs

Combining Wolbachia-induced sterility and virus protection to fight Aedes albopictus-borne viruses

International audienceAmong the strategies targeting vector control, the exploitation of the endosymbiont Wolba-chia to produce sterile males and/or invasive females with reduced vector competence seems to be promising. A new Aedes albopictus transinfection (ARwP-M) was generated by introducing wMel Wolbachia in the ARwP line which had been established previously by replacing wAlbA and wAlbB Wolbachia with the wPip strain. Various infection and fitness parameters were studied by comparing ARwP-M, ARwP and wild-type (S ANG population) Ae. albopictus sharing the same genetic background. Moreover, the vector competence of ARwP-M related to chikungunya, dengue and zika viruses was evaluated in comparison with ARwP. ARwP-M showed a 100% rate of maternal inheritance of wMel and wPip Wolba-chia. Survival, female fecundity and egg fertility did not show to differ between the three Ae. albopictus lines. Crosses between ARwP-M males and S ANG females were fully unfertile regardless of male age while egg hatch in reverse crosses increased from 0 to about 17% with S ANG males aging from 3 to 17 days. When competing with S ANG males for S ANG females, ARwP-M males induced a level of sterility significantly higher than that expected for an equal mating competitiveness (mean Fried index of 1.71 instead of 1). The overall Wolbachia density in ARwP-M females was about 15 fold higher than in ARwP, mostly due to the wMel infection. This feature corresponded to a strongly reduced vector competence for chikungunya and dengue viruses (in both cases, 5 and 0% rates of transmission at 14 and 21 days post infection) with respect to ARwP females. Results regarding Zika virus did not highlight significant differences between ARwP-M and ARwP. However, none of the tested ARwP-M females was capable at transmitting ZIKV. These findings are expected to promote the exploitation of Wolbachia to suppress the wild-type Ae. albopictus populations. PLOS Neglected Tropical Diseases | https://doi

Profile of Small RNAs, vDNA Forms and Viral Integrations in Late Chikungunya Virus Infection of Aedes albopictus Mosquitoes

International audienceThe Asian tiger mosquito Aedes albopictus is contributing to the (re)-emergence of Chikungunya virus (CHIKV). To gain insights into the molecular underpinning of viral persistence, which renders a mosquito a life-long vector, we coupled small RNA and whole genome sequencing approaches on carcasses and ovaries of mosquitoes sampled 14 days post CHIKV infection and investigated the profile of small RNAs and the presence of vDNA fragments. Since Aedes genomes harbor nonretroviral Endogenous Viral Elements (nrEVEs) which confers tolerance to cognate viral infections in ovaries, we also tested whether nrEVEs are formed after CHIKV infection. We show that while small interfering (si)RNAs are evenly distributed along the full viral genome, PIWI-interacting (pi)RNAs mostly arise from a ~1000 bp window, from which a unique vDNA fragment is identified. CHIKV infection does not result in the formation of new nrEVEs, but piRNAs derived from existing nrEVEs correlate with differential expression of an endogenous transcript. These results demonstrate that all three RNAi pathways contribute to the homeostasis during the late stage of CHIKV infection, but in different ways, ranging from directly targeting the viral sequence to regulating the expression of mosquito transcripts and expand the role of nrEVEs beyond immunity against cognate viruses

Maternal inheritance efficiency of the <i>w</i>Mel infection in the AR<i>w</i>P-M <i>Ae</i>. <i>albopictus</i> line.

<p>The data sheet shows the number (N) of analyzed and the percentage of infected male and female individuals at each generation following the <i>w</i>Mel transinfection.</p

Crosses between AR<i>w</i>P-M and wild-type <i>Ae</i>. <i>albopictus</i> (S_ANG) to measure the level of induced cytoplasmic incompatibility and compare the male mating competitiveness.

<p>In all of the crosses, females were 2±1 days old. The CI_corr level in the CI crosses was measured at three different male ages. Competition crosses consisted of young (3 ±1 days old) AR<i>w</i>P-M and S_ANG males at 1:1 ratio.</p

Combining <i>Wolbachia</i>-induced sterility and virus protection to fight <i>Aedes albopictus</i>-borne viruses - Fig 2

<p><b>Female fecundity (left) and hatch rate (right) in AR<i>w</i>P-M <i>Ae</i>. <i>albopictus</i> in comparison with recipient AR<i>w</i>P and wild-type <i>Ae</i>. <i>albopictus</i>.</b> S = S_ANG wild-type <i>Ae</i>. <i>albopictus</i>; AR<i>w</i>P = <i>w</i>Pip infected <i>Ae</i>. <i>albopictus</i>; AR<i>w</i>P-M <i>w</i>Pip + <i>w</i>Mel infected <i>Ae</i>. <i>albopictus</i>. Error bars show the SEM of three biological replicates, each containing 17–20 fed females. In both cases, values are not significantly different by ANOVA-Bonferroni (P > 0.05).</p

<i>w</i>Mel and <i>w</i>Pip <i>Wolbachia</i> density in AR<i>w</i>P and AR<i>w</i>P-M females and males measured by using <i>Ae</i>. <i>albopictus</i> actin gene as reference.

<p><i>w</i>Mel and <i>w</i>Pip <i>Wolbachia</i> density in AR<i>w</i>P and AR<i>w</i>P-M females and males measured by using <i>Ae</i>. <i>albopictus</i> actin gene as reference.</p

Titration of the viral particles of CHIKV, DENV and ZIKV in body and saliva of AR<i>w</i>P and AR<i>w</i>P-M <i>Ae</i>. <i>Albopictus</i>.

<p>The number of viral particles in the body and saliva of both mosquito lines were titrated for evaluating the viral load in each mosquito line. A: the number of CHIKV viral particles in the body and saliva of AR<i>w</i>P and AR<i>w</i>P-M at 7 and 14 dpi; B and C: the number of DENV-1 (B) and ZIKV (C) viral particles in the body and saliva of AR<i>w</i>P and AR<i>w</i>P-M at 14 and 21 dpi. Differences between <i>Ae</i>. <i>albopictus</i> lines were not statistically significant (Kruskal–Wallis test: P > 0.05).</p

Rates of infection, dissemination efficiency and transmission efficiency for CHIKV, DENV and ZIKV in AR<i>w</i>P and AR<i>w</i>P-M <i>Ae</i>. <i>Albopictus</i>.

<p>IR = Infection rate; DE = Dissemination rate; TE = transmission rate; A: the differences between <i>Ae</i>. <i>albopictus</i> lines are significant with respect to all of the three parameters and at both time intervals (7, 14 dpi) post the infection (Fisher exact test, P < 0.05); B: AR<i>w</i>P and AR<i>w</i>P-M significantly differed with regard to IR and DE at 14 dpi (Fisher exact test, P < 0.05); C: AR<i>w</i>P and AR<i>w</i>P-M did not significantly differ with regard to any of the evaluated parameters.</p