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Conceived and designed the experiments: ABF PM. Performed the experiments: LM KZ CAG VR. Analyzed the data: KZ CAG ABF. Contributed reagents/materials/analysis tools: LM KZ CAG VR. Wrote the paper: ABF KZ PM.
The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV).
To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal.
We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated.
Aedes albopictus is an invasive species that is expanding its natural range of geographic distribution. While it was previously considered a secondary vector of different arboviruses, this mosquito species is involved in the most recent outbreaks of chikungunya but contributes weakly to dengue outbreaks. Ae. albopictus naturally carries two strains of the bacterium Wolbachia, wAlbA and wAlbB. Present in 20% of insect species, Wolbachia is an obligate intracellular symbiont mainly transmitted through females. When inoculated into some mosquito hosts, Wolbachia is able to shorten the adult life span and to block arbovirus transmission. We have previously shown that Wolbachia is not capable of limiting chikungunya replication in the mosquito vector. In this study, we show that the native Wolbachia is able to limit dengue transmission by restricting the delivery of infectious viral particles from the mosquito saliva when biting. Therefore, our results might explain the low vector competence of Ae. albopictus for dengue, and thus its weak contribution as an epidemic dengue vector.
Dengue is the most important arboviral disease in tropical and subtropical areas where 2.5 billion people are at risk of infection. The four dengue virus serotypes (DENV-1, -2, -3, -4) are transmitted by Aedes genus mosquitoes, mainly Aedes aegypti and Aedes albopictus. Ae. aegypti presents feeding preferences restricted to humans and is considered the main epidemic vector of DENV. Ae. albopictus is rather regarded as a secondary vector, although it has been implicated in several dengue outbreaks, and its geographic expansion is proceeding through tropical and temperate regions . Currently, no specific antiviral therapy or vaccines are available against dengue. Thus, vector population control remains the principal tool for dengue prevention. Nevertheless, classical control methods such as the use of insecticides are failing to prevent a global increase in the incidence of dengue, and new approaches against this disease are needed.
Wolbachia can induce reproductive distortions in their hosts  increasing the reproductive success of infected females, thus enhancing their maternal transmission . The ability of some Wolbachia strains to reduce the Ae. aegypti mosquito lifespan and to interfere with the replication of several pathogens, including arboviruses in this mosquito, has promoted this symbiotic bacterium as one of the most promising tools to limit virus transmission , . To be transmitted successfully to the next host, DENV must replicate and disseminate in the mosquito's hemocoel before reaching the salivary glands. This period within the mosquito is called the extrinsic incubation period (EIP), and it determines how long a mosquito has to survive after having ingested the infectious blood-meal before becoming competent to transmit the virus. Only female mosquitoes that live longer than the EIP are of epidemiological interest. Therefore, a reduction in the number of old mosquitoes in a population may break disease transmission , .
Wolbachia strains can protect Drosophila flies from different RNA viruses resulting in infections showing lower titers and less associated pathologic effects , . Infection of the naturally Wolbachia-free Ae. aegypti with different Wolbachia strains generates mosquitoes with increased resistance to viral infection and a shortened lifespan –. However, Wolbachia-mediated antiviral protection is not ubiquitous . Natural Wolbachia strains that infect their native mosquito hosts have also been shown to induce resistance to viruses, although at a lower level than mosquito-transinfected strains .
Wolbachia is commonly found in mosquitoes including Ae. albopictus. This species is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB ,  and is able to transmit both DENV and chikungunya virus (CHIKV). On La Reunion Island, Ae. albopictus is the only species capable of sustaining an arboviral outbreak, because Ae. aegypti is scarcely present . The former is a very efficient vector of a particular variant of CHIKV ,  and, to a lesser extent, of DENV . Wolbachia is widely found in natural populations of Ae. albopictus ,  and is known to spread by inducing cytoplasmic incompatibility (CI) leading to successful increase of bacterial transmission through the female germline . Although it is still unclear how Wolbachia interferes with arboviruses in the different tissues of the mosquito where they coexist, mechanisms such as stimulation of the host immune responses or competition for resources have been proposed , , . Wolbachia is not able to limit DENV dissemination in Ae. albopictus, whereas a Wolbachia-mediated inhibition was observed in Ae. aegypti transfected with wAlbB . It has been demonstrated that the Wolbachia density in somatic tissues of Ae. albopictus, such as midgut and salivary glands, is too low to induce resistance to DENV. Indeed, a strong negative linear correlation was observed between Wolbachia density and DENV load . Nevertheless, when the wMel strain of Drosophila melanogaster was introduced into Ae. albopictus, a complete blockade of DENV transmission was observed suggesting that in addition to the host background, the Wolbachia strain plays a key role in viral inhibition .
Our previous studies have shown that Wolbachia does not suppress CHIKV replication in Ae. albopictus . Here, we examined whether Wolbachia is able to suppress DENV in Ae. albopictus, which is a poor DENV vector on La Reunion Island .
The Institut Pasteur animal facility has received accreditation from the French Ministry of Agriculture to perform experiments on live mice [see permit numbers at http://webcampus.pasteur.fr/jcms/c_97619/agrements-des-animaleries] in compliance with the French and European regulations on care and protection of the Laboratory Animals. This study was approved by the relative IACUC at the Institut Pasteur.
Two mosquito strains were used in experiments: the F2 generation of the wild-type superinfected Ae. albopictus ALPROV (La Reunion Island) with wAlbA and wAlbB strains and the aposymbiotic line generated by antibiotic treatments of ALPROV (tetracycline for larval stage and rifampicin for adult stage) . The ALPROV strain was generated from eggs collected in ovitraps placed in different houses in the “La Providence” neighborhood in the city of Saint-Denis. Obtained adults were maintained on a 10% sucrose at 28°C and 80% of relative humidity with a 16/8 light/dark cycle. Larvae were reared in pans with 200 larvae in 1 L of dechlorinated water, and fed with 1 yeast tablet every 2 days. The resulting adults were continuously provided with 10% sucrose. For routine colony maintenance, females were blood-fed three times a week on anesthetized mice.
The DENV-2 strain provided by Prof. Leon Rosen was isolated from a human serum collected in Bangkok (Thailand) in 1974. This virus had been passed in different mosquito species (Toxorhynchites amboinensis, Ae. albopictus and Ae. aegypti) by intrathoracic inoculation . Viral production and titration are described in . Blood-meals consisted of 1 mL of viral suspension in L-15 medium supplemented with 2% fetal bovine serum (FBS) and 2 mL of washed rabbit erythrocytes supplemented with ATP (5×10−3 M) as a phagostimulant. The infectious blood was transferred to a glass feeder maintained at 37°C and placed on top of plastic boxes containing 60 one-week-old mosquitoes that had been starved for 24 hours before the blood-meal. Mosquitoes from each box were allowed to feed for 20 min. Fully engorged females were transferred into cardboard containers and supplied with 10% sucrose at 28°C before being assayed.
Individual mosquitoes and dissected organs (midguts, wings, and salivary glands) were used to extract total nucleic acids. At different days post-infection (pi), 5–9 mosquitoes were washed in 70% ethanol, and then rinsed twice in sterile PBS. The midgut, wings, and salivary glands were dissected in sterile PBS. Each organ was rinsed individually in PBS before being transferred separately into tubes containing 350 µL RA1 solution (NucleoSpin RNA II kit, Macherey-Nagel), then homogenized with the mixer Mill MM 300 (Qiagen). Needles used for dissection were decontaminated after each set of mosquitoes. Total RNA/DNA was extracted using the NucleoSpin RNA/DNA buffer set (Macherey-Nagel) coupled to the NucleoSpin RNA II kit. RNA was used to determine viral load by qRT-PCR, and DNA to measure Wolbachia (wAlbA and wAlbB) density and actin gene content by qPCR.
To measure the number of DENV-2 genome copies, the total RNA extracted was eluted into 40 µL RNAse-free water and 5 µL were used in a one-step RT-PCR reaction performed with a Power SYBR Green RNA-to-CT one step kit (Applied Biosystem) in a volume of 25 µL. The reaction contained 5 µL RNA template, 12.5 µL 2× Power SYBR Green I RT-PCR Mix, 0.625 µL sense primer (10 µM), 0.625 µL anti-sense primer (10 µM), 0.2 µL RT enzyme mix and 6.02 µL ddH2O. Primers were selected in the capsid structural protein coding region and were: sense D1-Lm/C/153/+ (GAG AAA CCG CGT GTC AAC TG) and anti-sense TS2-Lm/C/219/- (GGA AAC GAA GGA ATG CCA CC). The PCR program was: 48°C for 30 min, 95°C for 10 min; 40 cycles of 95°C for 15 s, and 60°C for 1 min; 90°C for 15 s with a final ramping of 19 min 59 sec. The size of the PCR product was 113 bp. A standard curve was generated using duplicates of 102 to 108 synthetic RNA transcripts per reaction. Quantification of viral RNA was achieved by comparing the threshold cycle (Ct) values of samples to those of standards according to the ΔCt analysis. RNA viral copy number can give an overestimation of infectious viral particles and are more appropriate to describe viral dissemination than to estimate transmission probability.
To quantify Wolbachia (wAlbA and wAlbB) and actin genes, total DNA was extracted and used for q-PCR. The Wolbachia genome copy number was normalized using the nuclear actin gene. A plasmid (pQuantAlb), provided by Weill M  containing the three wAlbA, wAlbB wsp genes and the Ae. albopictus actin gene, was diluted from 102 to 108 copies/reaction and used to generate standard curves. Primers were: actAlb-dir (GCA AAC GTG GTA TCC TGA C) and actAlb-rev (GTC AGG AGA ACT GGG TGC T), QAdir1 (GGG TTG ATG TTG AAG GAG) and QArev2 (CAC CAG CTT TTA CTT GAC C), 183F (AAG GAA CCG AAG TTC ATG) and QBrev2 (AGT TGT GAG TAA AGT CCC), for wAlbA, wAlbB and actin, respectively. From 60 µL DNA solution extracted from one mosquito, 2 µL DNA was mixed with 0.3 µM of each primer and 12.5 µL FastStart Universal SYBR Green Master (Roche). PCR was run for 40 cycles (95°C for 10 min, 95°C for 15 sec, 60°C for 1 min). The mean number of wAlbA and wAlbB genomes was given per actin copy.
Mosquitoes exposed to a DENV-2 blood-meal were assayed for transmission at days 8 and 14 pi  by collecting saliva using the forced salivation technique . Briefly, mosquitoes were anesthetized on ice to remove their legs and wings. Then the proboscis was inserted into a capillary tube containing 5 µL of fetal bovine serum. After 45 min, saliva was transferred into 45 µL of L15 medium.
Saliva samples were titrated by fluorescent focus assay on C6/36 Ae. albopictus cell culture. Samples were serially diluted and inoculated into C6/36 cells in 96-well plates. After incubation at 28°C for 5 days, plates were stained using hyper-immune ascetic fluid specific to DENV-2 as the primary antibody and a goat anti-mouse conjugate as the second antibody.
Salivary glands were dissected from mosquitoes 14 days after exposure to an infectious blood-meal at a titer of 107 FFU (fluorescent focus units)/mL. Preparations of samples were as described . Briefly, organs were fixed for 20 min in freshly prepared 4% formaldehyde in PBS, and then washed once with PBS. Samples were incubated for 16 hours at 37°C in hybridization buffer [formamide 50%, SSC 5×, dextran sulfate 200 mg.l−1, poly(A) 250 mg.ml−1, salmon sperm DNA 250 mg.ml−1, tRNA 250 mg.ml−1, DTT 0.1 M, Denhartdt's solution 0.5×] containing 10 ng of two Wolbachia probes ,  and two DENV specific probes . Samples were washed twice in 1× SSC-10 mM DTT and twice in 0.5× SSC-10 mM DTT at 55°C for 15 min. Samples were then rinsed in PBS, mounted on a glass slide with glycerol and viewed under a confocal microscope (LSM510, Zeiss) at the Microscopy Centre of University Lyon I.
1-week-old Wolbachia-infected and Wolbachia-uninfected females were fed on a DENV-2 blood-meal as described previously . Engorged females were individually isolated in 50 mL tubes, maintained at 28°C and fed with 10% sucrose until death. Dead mosquitoes were scored daily to estimate the female life duration following exposure to an infectious blood-meal at a titer of 107 FFU/mL.
Statistical analyses were performed with Stata software (StataCorp LP, Texas, and USA). Means were compared using the Kruskal-Wallis test and the Khi2 test was used to compare rates.
To determine whether Wolbachia in Ae. albopictus affects DENV-2 replication, we fed Wolbachia-infected and Wolbachia-uninfected mosquitoes on a blood-meal proposed at two viral titers, 105 and 107 FFU/mL, and determined DENV-2 dynamics in mosquitoes. At a given viral titer of blood-meal, the number of DENV-2 genome copies in whole individuals was not significantly different between Wolbachia-infected and Wolbachia-uninfected strains (Mann-Whitney test: p>0.05) (Figure 1). Later, we compared DENV-2 loads in mosquitoes between Wolbachia-infected and Wolbachia-uninfected Ae. albopictus exposed to four viral titers, 104, 105, 106, and 107 FFU/mL. At days 2, 8, and 14 pi, the number of DENV-2 genome copies estimated by qRT-PCR was not significantly different between Wolbachia-infected and Wolbachia-uninfected mosquitoes at a given viral titer of blood-meal (Mann-Whitney test: p>0.05) (Figure 2). Concomitantly, at days 2, 8, and 14 pi, the loads of wAlbA (Figure 3A) and wAlbB (Figure 3B) remained roughly similar whatever the viral titer of blood-meal (Kruskall-Wallis test: p>0.05), albeit the Wolbachia densities were highly variable between mosquito individuals and day pi. Nevertheless, it was interesting to note that when a slight increase of wAlbA loads was observed between day 8 pi and day 14 pi, a concomitant decrease of wAlbB loads was detected. This result was not in line with previous findings with CHIKV .
Since the overall DENV-2 replication in Ae. albopictus did not differ between Wolbachia-infected and Wolbachia-uninfected mosquitoes, we compared the capacity of DENV to disseminate from the midgut to secondary organs by estimating viral loads in wings and salivary glands at days 2, 8, and 14 days after exposure of mosquitoes to blood-meals provided at four viral titers: 104, 105, 106, and 107 FFU/mL. The viral load in the midgut increased with the titer of the ingested blood-meal and the duration post-infection. In Wolbachia-infected mosquitoes, although virus dissemination to the wings was correlated with the blood-meal titer and day pi (Figure 4A). However, a strong reduction of viral load was observed in wings of Wolbachia-infected mosquitoes compared to Wolbachia-uninfected ones, suggesting that virions produced in the midgut epithelial cells were not massively released into the hemocoel. Thus, the occurrence of a midgut escape barrier in Wolbachia-infected Ae. albopictus can be suggested. Finally, salivary glands were only heavily infected at day 14 pi at the highest blood-meal titer assessed of 107 FFU/mL. Failure of DENV-2 to infect salivary glands might be due to low viral loads detected in the hemolymph (e.g. wings). On the other hand, in Wolbachia-uninfected mosquitoes, profiles observed in the different organs were significantly different with those observed in Wolbachia-infected mosquitoes (Kruskall-Wallis test: p<0.05). Indeed, DENV-2 loads in the midgut started to increase intensively from a blood-meal at 105 FFU/mL (Figure 4B). Dissemination from the midgut to wings and salivary glands remained relatively high at different titers in Wolbachia-uninfected individuals. Therefore, in Wolbachia-uninfected mosquitoes, DENV-2 did not seem to be restricted for dissemination to secondary organs suggesting the occurrence of a weak midgut escape barrier. Higher viral loads were found in Wolbachia-uninfected organs than in Wolbachia-infected ones, including salivary glands. Using specific FISH probes, we detected DENV-2 genomic RNA (green) (Figure 5, panels A, B, and C) and Wolbachia DNA (red) (Figure 5, panels D, E, and F). A co-localization of both Wolbachia and DENV-2 was detected in salivary glands (Figure 5, panel H). However, DENV-2 signals were relatively higher in Wolbachia-uninfected salivary glands (Figure 5, panel A) compared to a few dots in Wolbachia-infected ones (Figure 5, panel B), which is in agreement with PCR quantification data. As expected, no hybridization signal was observed in the salivary glands of DENV-uninfected and Wolbachia-uninfected individuals used as negative controls (Figure 5, panels C and F respectively).
30–40 mosquitoes that survived until day 14 pi were examined for transmission. DENV-2 particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after an infectious blood-meal at a titer of 107 FFU/mL; only 12.5% of mosquitoes (among 32 tested) were able to deliver viral particles (mean ± standard deviation: 18±28) (data not shown). This suggests that a salivary gland escape barrier preventing the release of virus from salivary glands operates in Wolbachia-uninfected Ae. albopictus. In addition, Wolbachia's effect depends on the viral titer of the blood-meal.
Removing Wolbachia did not affect Ae. albopictus survival when non-infected with DENV (data not shown). Then, to determine whether removing Wolbachia has an effect on DENV-2 infected mosquitoes, we compared the survival of Wolbachia-infected and Wolbachia-uninfected mosquitoes after infection with DENV-2 in two trials (Figure 6). Neither mosquito strains showed any difference in survival curves (logrank test: p>0.05). These results indicated that Wolbachia did not affect the longevity of Ae. albopictus when mosquitoes are infected with DENV-2.
Considering the overlapping distribution of Wolbachia and DENV in non-reproductive tissues of mosquitoes, such as midgut and salivary glands, we conducted experiments to characterize the role of Wolbachia in DENV-2 transmission by Ae. albopictus. Our results show that Wolbachia naturally infecting Ae. albopictus limits DENV-2 dissemination and the infection of salivary glands in the ALPROV strain, suggesting that Ae. albopictus is a poor epidemic vector of DENV in La Reunion Island .
Wolbachia is present in the midgut and salivary glands, two organs crucial for viral transmission . DENV penetrates into the midgut epithelial cells of mosquitoes following an infectious blood-meal. After replication and release of virions from the midgut, secondary organs such as salivary glands are subsequently infected. Infectious viral particles are then released from the salivary glands through saliva when the mosquito bites . Replication of DENV reaches a maximum in the mosquito at day 7–8 pi, as was observed for the principal DENV vector Ae. aegypti . Wolbachia densities in Ae. albopictus were not affected by the blood-meal titer; when increasing the blood-meal titer, wAlbA and wAlbB remained stable. This was consistently observed at different days pi (2, 8, and 14). Nevertheless, high levels of variation of Wolbachia densities were observed between individuals of the recently laboratory-established population of Ae. albopictus from La Reunion, as has been reported with mosquitoes from Thailand . When examining the midgut infection, we found that the viral loads in the midgut increased when the titer of the ingested blood-meal increased, as observed for other arboviruses , . In addition, virus dissemination to wings and salivary glands was correlated with the virus titer of blood-meals. Interestingly, we found that Wolbachia might have an inhibitory effect on DENV-2 dissemination. Indeed, the dissemination of infectious virions from the midgut to secondary organs, such as wings and salivary glands, was affected, suggesting the presence of a midgut escape barrier. Nevertheless, such a barrier could be overwhelmed when the blood-meal is provided at a titer higher than 106–107 FFU/mL. Later, the release of virus from salivary glands into the saliva of Wolbachia-infected mosquitoes at day 14 pi was completely inhibited suggesting the existence of a salivary gland escape barrier, possibly exerted by Wolbachia . Probably, at higher titers of blood-meals, this barrier may be less efficient. Thereby, a speculative suggestion can be made: if Ae. albopictus naturally carrying Wolbachia receives a high titer of blood-meal (>107 FFU/mL), the inhibition exerted by Wolbachia on DENV can be overcome. Thus viral transmission from a viremic patient to a mosquito vector is more likely to occur when high titers of blood-meals are provided. This can happen with the invasion and expansion of a new viral clade, resulting in high viremias in humans. Consequently, this leads to an enhanced mosquito transmission for the newly introduced viral lineage , . These invasive lineages causing higher viral titers are usually associated with more severe symptoms in humans . Conversely, in Wolbachia-uninfected mosquitoes, midgut infection was intensive and dissemination to wings and salivary glands was not strongly limited. Virus was only detected in saliva at day 14 pi in 12.5% of Wolbachia-uninfected mosquitoes. Contrary to Wolbachia-infected Ae. albopictus, Wolbachia-uninfected mosquitoes do not show the inhibition caused by a midgut escape barrier as well as a salivary gland escape barrier. Therefore, removing Wolbachia from Ae. albopictus also removes the inhibition exerted by Wolbachia on DENV-2 in Ae. albopictus. Low transmission to saliva might explain the low vector competence of Ae. albopictus for DENV. However, increasing the incubation period may favor transmission; in Ae. albopictus from Lebanon, virus was only detected at day 21 pi with 38% of mosquitoes delivering 174±455 (mean ± standard deviation) viral particles, whereas no viral particles were detected in the saliva at day 14 pi (Haddad N et al., unpublished data). Thus, the extended extrinsic incubation period of DENV in Ae. albopictus which requires that females live longer to be capable of transmitting, may contribute to reduce the potential for transmission of Ae. albopictus populations and weaken the role of the species as epidemic dengue vector.
The inhibitory effect of Wolbachia on different pathogens including DENV was demonstrated in the vector Ae. aegypti , . Similar interference was not found in Ae. albopictus naturally infected by Wolbachia. Both strains, wAlbA and wAlbB, are benign to Ae. albopictus  and contrary to reports on Drosophila , . It is not clear whether Wolbachia is able to remain a benign symbiont simply by maintaining a very low replication rate independent of the host cell or whether the bacterium actively coordinates its replication with the host through specific immune mechanisms. Immune responses to Wolbachia infection have only been observed in hosts artificially infected with new strains of Wolbachia , , . However, responses might not be solely be due to the Wolbachia infection but could reflect a consequence of the new host-symbiont combination such as elevated, unnatural Wolbachia densities . High densities of Wolbachia can provide high levels of protection whereas low densities provide limited protection , . Interference could also result from a direct competition between DENV and Wolbachia for the same resources or from an indirect disturbance by Wolbachia on the cellular environment required by DENV for its replication. Interestingly, replication of CHIKV in Ae. albopictus was concomitant with a decrease of Wolbachia densities , in contrast with the DENV replication pattern observed in the present study. The intensive replication of CHIKV in Ae. albopictus reaching up to 109 viral RNA from day 3 pi  induced negative effects on mosquito survival . Contrary to DENV, CHIKV has seemingly developed a subtle balance between intense replication and adverse effects in mosquitoes, though limited enough to maintain a high transmission capacity.
To conclude, our study showed that native Wolbachia was able to limit transmission of DENV in Ae. albopictus. Presumably, introducing an additional infection to the preexisting infection  or a new Wolbachia type in Wolbachia-free Ae. albopictus ,  might lead to a blockade of viral replication and dissemination making Ae. albopictus refractory to DENV.
We thank the ARS “Agence Régionale de Santé” in the Indian Ocean for providing mosquito strains. We also wish to thank Marie Vazeille for technical help. We are also grateful to Katherine Kean and Richard Paul for revision of our manuscript.