Characterization of <i>Aedes aegypti</i> innate-immune pathways that limit Chikungunya virus replication

Replication of arboviruses in their arthropod vectors is controlled by innate immune responses. The RNA sequence-specific break down mechanism, RNA interference (RNAi), has been shown to be an important innate antiviral response in mosquitoes. In addition, immune signaling pathways have been reported to mediate arbovirus infections in mosquitoes; namely the JAK/STAT, immune deficiency (IMD) and Toll pathways. Very little is known about these pathways in response to chikungunya virus (CHIKV) infection, a mosquito-borne alphavirus (Togaviridae) transmitted by aedine species to humans resulting in a febrile and arthralgic disease. In this study, the contribution of several innate immune responses to control CHIKV replication was investigated. In vitro experiments identified the RNAi pathway as a key antiviral pathway. CHIKV was shown to repress the activity of the Toll signaling pathway in vitro but neither JAK/STAT, IMD nor Toll pathways were found to mediate antiviral activities. In vivo data further confirmed our in vitro identification of the vital role of RNAi in antiviral defence. Taken together these results indicate a complex interaction between CHIKV replication and mosquito innate immune responses and demonstrate similarities as well as differences in the control of alphaviruses and other arboviruses by mosquito immune pathways

Evolution et adaptation du virus chikungunya vis-à-vis des ses hôtes vecteurs

Les arbovirus se caractérisent par un taux de mutation élevé mais leur cycle de transmission nécessitant à la fois une réplication chez un vertébré et un invertébré tend à limiter leur évolution. Un changement d hôtes peut favoriser l émergence de nouveaux variants. Ainsi, lors de l émergence du virus chikungunya (CHIKV) dans l Océan Indien en 2004, un nouveau variant épidémique portant un changement d acide aminé dans la glycoprotéine E1, a été associé à une meilleure transmission par le vecteur secondaire, Aedes albopictus. Nous avons montré que lorsque le variant d origine et le variant épidémique sont proposés à proportions égales dans un même repas sanguin, le variant épidémique était préférentiellement transmis chez Ae. albopictus. En revanche, lorsque les deux variants ont été inoculés par voie intrathoracique, permettant de court-circuiter la barrière intestinale, ce variant n était plus sélectionné chez Ae. albopictus. Ceci met en évidence le rôle clé de cette barrière dans la sélection du variant épidémique. D autres mutations pourraient être favorisées par le changement d hôtes. Nous avons donc évalué si l alternance d hôtes pouvait limiter l évolution du CHIKV compromettant sa fitness. Le variant épidémique du CHIKV a été passé en série ou en alternance sur des cellules de mammifère et de moustique. Après 30 passages, les souches virales ont été caractérisées génétiquement et phénotypiquement. Dans ces conditions expérimentales l alternance d hôtes ne limite pas l évolution du CHIKV. Par contre, nous avons identifié de nouvelles substitutions d acides aminés dans la glycoprotéine E2 qui pourraient moduler la compétence vectorielle du moustiqueArboviruses are characterized by high rates of mutation. However, it has been assumed that their evolution is constrained by requirement for alternate replication in vertebrate and invertebrate hosts. Host change would favor the emergence of new viral variants pre-existing in the viral population. Indeed, during the 2004 outbreak of chikungunya virus (CHIKV) in the Indian Ocean, a newly emerged epidemic variant harboring a single amino-acid substitution in the E1 glycoprotein was highly transmitted by an unusual mosquito vector, Aedes albopictus. We showed that when the original and the newly emerged epidemic variants were provided at equal titers in blood-meals, the epidemic variant was preferentially transmitted by Ae. albopictus. Interestingly, when inoculating both variants into mosquitoes bypassing the midgut barrier, the epidemic variant was no longer selected in Ae. albopictus. Our findings suggest that the midgut barrier plays a key role in the selection of the epidemic variant. Subsequent adaptive mutations in the CHIKV genome are likely to emerge questioning on the evolution of CHIKV. We evaluated if host alternation can limit CHIKV evolution and results in fitness trade-offs. To test this hypothesis, the newly emerging variant of CHIKV was serially or alternately passaged in mammalian or mosquito cells. After 30 passages, obtained CHIKV strains were genetically and phenotypically characterized. Our results were not in line with the general assumption stating that host alternation constrains CHIKV evolution. However, our experimental approach suggested that new amino-acid substitutions in the E2 glycoprotein could modulate the vector competence in mosquitoesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Dissemination and transmission of the E1-226V variant of chikungunya virus in Aedes albopictus are controlled at the midgut barrier level.

Emergence of arboviruses could result from their ability to exploit new environments, for example a new host. This ability is facilitated by the high mutation rate occurring during viral genome replication. The last emergence of chikungunya in the Indian Ocean region corroborates this statement since a single viral mutation at the position 226 on the E1 glycoprotein (E1-A226V) was associated with enhanced transmission by the mosquito Aedes albopictus in regions where the major mosquito vector, Aedes aegypti, is absent.We used direct competition assays in vivo to dissect out the mechanisms underlying the selection of E1-226V by Ae. albopictus. When the original variant E1-226A and the newly emerged E1-226V were provided in the same blood-meal at equal titers to both species of mosquitoes, we found that the proportion of both variants was drastically different in the two mosquito species. Following ingestion of the infectious blood-meal, the E1-226V variant was preferentially selected in Ae. albopictus, whereas the E1-226A variant was sometimes favored in Ae. aegypti. Interestingly, when the two variants were introduced into the mosquitoes by intrathoracic inoculations, E1-226V was no longer favored for dissemination and transmission in Ae. albopictus, showing that the midgut barrier plays a key role in E1-226V selection.This study sheds light on the role of the midgut barrier in the selection of novel arbovirus emerging variants. We also bring new insight into how the pre-existing variant E1-226V was selected among other viral variants including E1-226A. Indeed the E1-226V variant present at low levels in natural viral populations could rapidly emerge after being selected in Ae. albopictus at the midgut barrier level

A Wolbachia wMel transinfection in Aedes albopictus is not detrimental to host fitness and inhibits Chikungunya virus

BACKGROUND: Wolbachia inherited intracellular bacteria can manipulate the reproduction of their insect hosts through cytoplasmic incompatibility (CI), and certain strains have also been shown to inhibit the replication or dissemination of viruses. Wolbachia strains also vary in their relative fitness effects on their hosts and this is a particularly important consideration with respect to the potential of newly created transinfections for use in disease control. METHODOLOGY/PRINCIPAL FINDINGS: In Aedes albopictus mosquitoes transinfected with the wMel strain from Drosophila melanogaster, which we previously reported to be unable to transmit dengue in lab challenges, no significant detrimental effects were observed on egg hatch rate, fecundity, adult longevity or male mating competitiveness. All these parameters influence the population dynamics of Wolbachia, and the data presented are favourable with respect to the aim of taking wMel to high population frequency. Challenge with the chikungunya (CHIKV) virus, for which Ae. albopictus is an important vector, was conducted and the presence of wMel abolished CHIKV dissemination to the saliva. CONCLUSIONS/SIGNIFICANCE: Taken together, these data suggest that introducing wMel into natural Ae. albopictus populations using bidirectional CI could be an efficient strategy for preventing or reducing the transmission of arboviruses by this species

Effect of E1-A226V substitution on CHIKV dissemination to salivary glands of <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV). <i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with a blood-meal containing both E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL.

<p>Every day, 5 mosquitoes were sacrificed to isolate salivary glands. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

Effect of the E1-A226V substitution on CHIKV transmission in <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. At days 3, 5 and 7 post-infection, mosquitoes were prepared for salivation. Collected saliva was used to inoculate monolayers of Vero cells. After 3 days at 37°C, clones were identified by sequencing. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

Effect of E1-A226V substitution on CHIKV dissemination to wings of <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with a blood-meal containing both E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. Every day, 5 mosquitoes were sacrificed to isolate the wings. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p

Unbalanced proportions of E1-226V and E1-226A provided in blood-meals to <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV). <i>Ae. aegypti</i> and <i>Ae. albopictus</i> were given an infectious blood-meal containing unbalanced proportions of two clones E1-A and E1-V isolated from the strains E1-226A and E1-226V, respectively.

<p>Two mixes were tested: one containing 1∶9 (E1-A:E1-V) and one containing a mix of 9∶1 (E1-A:E1-V). At day 7 post-infection, dissemination (A) and transmission (B) were examined by estimating the proportion of E1-V in wings and saliva, respectively, of five individual females. Error bars show the confidence intervals (95%).</p

Effects of intrathoracic injection compared to blood-meal ingestion on CHIKV dissemination and transmission in <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> and <i>Ae. albopictus</i> were orally infected or inoculated with E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. At day 7 post-infection, dissemination (A) and transmission (B) were examined by estimating the proportion of E1-226V in wings and saliva. Error bars show the confidence intervals (95%).</p

Effect of E1-A226V substitution on CHIKV infection in <i>Ae. aegypti</i> (AAPT) and <i>Ae. albopictus</i> (ALPROV).

<p><i>Ae. aegypti</i> (light-grey) and <i>Ae. albopictus</i> (dark-grey) were orally infected with a blood-meal containing both E1-226A and E1-226V provided at the same titer, 10^6.5 pfu/mL. Every day, 5 mosquitoes were sacrificed to isolate the midgut. The proportion of E1-226V compared to E1-226A (A) and the number of infectious particles (B) were determined. Error bars show the confidence intervals (95%) for % CHIKV E1-226V and the standard deviations for Log10 pfu/midgut.</p