The recent association of Zika virus with cases of microcephaly has sparked a global health crisis and highlighted the need for mechanisms to combat the Zika vector, Aedes aegypti mosquitoes. Wolbachia pipientis, a bacterial endosymbiont of insect, has recently garnered attention as a mechanism for arbovirus control. Here we report that Aedes aegypti harboring Wolbachia are highly resistant to infection with two currently circulating Zika virus isolates from the recent Brazilian epidemic. Wolbachia-harboring mosquitoes displayed lower viral prevalence and intensity and decreased disseminated infection and, critically, did not carry infectious virus in the saliva, suggesting that viral transmission was blocked. Our data indicate that the use of Wolbachia-harboring mosquitoes could represent an effective mechanism to reduce Zika virus transmission and should be included as part of Zika control strategies.

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As many different strains of the bacterium cause this inhibition, we hypothesized that the wMel Wolbachia strain (wMel_Br), currently being utilized as part of dengue control efforts in Brazil, might be able to restrict ZIKV infection and transmission in Ae. aegypti. To that end, we performed experimental infections with two currently circulating ZIKV isolates and used a qRT-PCR-based assay to a quantify ZIKV levels in mosquito tissues and saliva, in order to assess whether Wolbachia could potentially be used to combat the emerging Zika pandemic.

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et al. Zika virus in the Americas: Early epidemiological and genetic findings. 1 Br males (the eggs of field-collected Br mosquitoes) in wMel_Br colony cages over 2 years, both lines had a similar genetic background (see Through experimental infection and transmission assays using two currently circulating Brazilian ZIKV isolates (BRPE243/2015 [BRPE] and SPH/2015 [SPH]) (), we compared ZIKV infection in wMel-infected mosquitoes (wMel_Br) with Wolbachia-uninfected mosquitoes collected in Urca, Rio de Janeiro, Brazil in early 2016 (Br). Due to the regular introduction of FBr males (the eggs of field-collected Br mosquitoes) in wMel_Br colony cages over 2 years, both lines had a similar genetic background (see Supplemental Experimental Procedures ).

Table 1 Effects of Wolbachia on ZIKV Prevalence Isolate ZIKV Titer (PFU/mL) Days Post-infection wMel_Br Br wMel_Br Br wMel_Br Br Head/Thorax Infection Rate Abdomen Infection Rate Saliva Infection Rate BRPE 5.0 × 106 7 0 65 55 85 – – 14 10 100 35 100 45 100 SPH 8.7 × 103 7 5 95 30 90 – – 14 25 95 30 95 – – Ae. aegypti were orally infected with fresh, low-passage ZIKV. Initial viral titer was determined by plaque-forming assay. Saliva infection was only examined for mosquitoes at 14 days post-infection with the BRPE isolate. Infection rates are given as percentages. n = 20 per group unless specified. ZIKV, Zika virus; PFU, plaque-forming units; BRPE, ZIKV/H. sapiens/Brazil/BRPE243/2015; SPH, ZIKV/H. sapiens/Brazil/SPH/2015; wMel_Br, Wolbachia-infected; Br, Wolbachia-uninfected. Figure 1 Wolbachia Infection Restricts ZIKV Infection in Ae. aegypti Mosquitoes Show full caption (A–C) Wolbachia-infected (green circles) and -uninfected (black circles) mosquitoes were orally challenged with either (A) the BRPE or (B) the SPH ZIKV isolates. Wolbachia infection reduced both prevalence and intensity of ZIKV infection in mosquito heads/thoraces and abdomens at 7 and 14 dpi. Saliva was then collected for mosquitoes infected with the BRPE ZIKV isolate at 14 dpi infection (C), and we observed that saliva from Wolbachia-infected mosquitoes had a significantly lower rate of saliva infection and median viral load. (D) When these saliva samples were injected into ZIKV-uninfected Br mosquitoes, all of the Br saliva samples contained infectious virus, while no wMel_Br saliva produced a subsequent infection (columns: black, percentage infected; white, percentage uninfected; +, saliva contained infectious virus, −, saliva did not contain infectious virus). Absolute ZIKV copy numbers were quantified via qRT-PCR. In (A)–(C), each circle represents tissue or saliva from a single adult female (n = 20 per group). Red lines indicate the median ZIKV copies. ∗∗∗, p < 0.0001; analysis by Mann-Whitney U test. In (D), each column represents mosquitoes injected with a single saliva sample. The ZIKVs were isolated in the field in late 2015 and maintained in cell culture, and viral titers were quantified via plaque-forming assay prior to experimental infection ( Table 1 ). In two separate experiments, fresh ZIKV-infected supernatant was harvested from culture, mixed with human blood, and used to orally infect wMel_Br and Br mosquitoes. ZIKV levels were quantified in mosquito heads/thoraces and in abdomens at 7 and 14 days post-infection (dpi) using a TaqMan-based qRT-PCR assay ( Figure 1 ).

The prevalence of ZIKV infection was significantly reduced among Wolbachia-infected mosquitoes ( Table 1 , analysis via Fisher’s exact test, p < 0.0001 unless stated). For the BRPE isolate ( Figure 1 A), Wolbachia decreased ZIKV prevalence by 35% in abdomens, although there was no significant difference for this tissue (p > 0.05), by 100% in head/thoraces at 7 dpi, and by 65% and 90% at 14 dpi, respectively. For the SPH isolate ( Figure 1 B), Wolbachia reduced prevalence by 95% and 67% in head/thoraces and abdomens (p = 0.0002), respectively, at 7 dpi, and by 74% and 68% in head/thoraces and abdomens, respectively, at 14 dpi.

Likewise, the intensity of ZIKV infection was greatly reduced in wMel_Br mosquitoes for both tissues and time points (Mann-Whitney U tests, p < 0.0001). Additionally, we observed that median ZIKV titers in the head/thoraces of Br mosquitoes increased over time for both isolates (Mann-Whitney U test; BRPE, p < 0.0001; SPH, p = 0.0094), while there was no such effect in wMel_Br mosquitoes.

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Tan C.H. Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika virus. Saliva was collected from Br and wMel_Br mosquitoes at 14 dpi, after the 5- to 10-day ZIKV extrinsic incubation period was likely completed (), in order to determine if Wolbachia infection also inhibited ZIKV transmission ( Figure 1 C). We used mosquitoes infected with the BRPE isolate as it had a higher titer in culture ( Table 1 ). ZIKV levels were quantified directly for individual saliva samples using the same qRT-PCR assay. We observed that Wolbachia infection reduced ZIKV prevalence in individual saliva samples by 55% (Fisher’s exact test, p < 0.0001) and median ZIKV copies by approximately 5 logs (Mann-Whitney U test, p < 0.0001).

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et al. Modeling the impact on virus transmission of Wolbachia-mediated blocking of dengue virus infection of Aedes aegypti. To determine if the virus in these samples was infectious, a further ten wMel_Br and ten Br saliva samples, from the samples described above, were intrathoracically injected into 8–14 naive Br mosquitoes each ( Figure 1 D), using a previously described method (). The overall mortality rate among injected mosquitoes was 11.93%. The presence or absence of ZIKV infection was determined at 5 dpi in eight mosquitoes injected with each saliva, amounting to a mean proportion sampled of 0.68. Of the 80 mosquitoes injected with Br saliva, 68 (85%) became infected with ZIKV, with all Br saliva samples producing at least one infected mosquito. In contrast, none of the 80 mosquitoes injected with wMel_Br saliva became infected (Fisher’s exact test, p < 0.0001; odds ratio 882.3, 95% CI, 51.3–15187), indicating that while some of the wMel_Br saliva samples did contain detectable ZIKV, we saw no evidence that the saliva contained infectious virus.

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et al. The wMel Wolbachia strain blocks dengue and invades caged Aedes aegypti populations. Our results indicate that the ability of Wolbachia infection to greatly reduce the capacity of mosquitoes to harbor and transmit a range of medically important pathogens, including the dengue and chikungunya viruses () also extends to ZIKV. While wMel did not completely inhibit ZIKV infection, we observed a similar decrease in prevalence and intensity of infection to that of wMel-infected Ae. aegypti challenged with viremic blood from dengue patients, which was considered sufficient to drastically decrease viral transmission (). Additionally, the fact that we did not observe an increase in disseminated ZIKV infection over time, and that ZIKV prevalence and infectivity in wMel_Br mosquito saliva was significantly decreased, may indicate that, as for dengue, wMel extends the ZIKV extrinsic incubation period (). This in turn would likely further decrease overall ZIKV transmission rates, given the small decrease in lifespan associated with wMel infection ().

We observed that the wMel Wolbachia infection in Ae. aegypti greatly inhibited ZIKV infection in mosquito abdomens, and it reduced disseminated infection in heads and thoraces and ZIKV prevalence in mosquito saliva. Most critically, our results suggest that saliva from wMel-infected mosquitoes did not contain infectious virus. That this inhibition occurred for two ZIKV isolates that circulated in Brazil during the 2015 epidemic, and for mosquitoes with a wild-type genetic background, suggests that wMel could greatly reduce ZIKV transmission in field populations of Ae. aegypti, which in turn would likely reduce the frequency of Zika-associated pathology in humans.

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O’Neill S.L. Stability of the wMel Wolbachia Infection following invasion into Aedes aegypti populations. Wolbachia can invade and persist in wild mosquito populations () and represents a relatively low-cost, self-sustaining form of mosquito control that is already being trialed in countries where ZIKV outbreaks have been reported and has recently been recommended by the World Health Organization as a suitable tool to control ZIKV transmission ( http://migre.me/tDWVe ). It is important to point out that extensive public engagement will be required before releases of Wolbachia-infected mosquitoes can be scaled up for use in other areas. However, the results presented here indicate that wMel-infected Ae. aegypti represent a realistic and effective option to combat the ZIKV burden in Brazil and potentially in other countries and should be considered as an integral part of future control efforts.

The work reported in this paper was performed under the oversight of the Committee for Ethics in Research (CEP)/FIOCRUZ (License CEP 732.621).