Abstract
First isolated from a forest in East Africa in the mid-20th century, Zika virus (ZIKV) has now emerged worldwide in urbanized areas where its mosquito vectors, mainly Aedes aegypti and Ae. albopictus, are present. Europe and French overseas territories in the Indian Ocean have been so far spared despite the presence of Ae. albopictus, the Asian tiger mosquito. However, because they have strong economic and touristic links with regions affected by ZIKV, French territories in the Indian Ocean have a high risk of introduction. Here, we assess the susceptibility of two Ae. albopictus populations from Metropolitan France and the Reunion island (a French oversea territory in the Indian Ocean) for a ZIKV isolate from the Asian genotype at a titer ranging from 3 to 7.5 × 106 focus-forming units per milliliter. High infection rates and unpreceded levels of systemic infection rates were observed in both Metropolitan France and the Reunion island populations, without differences in infection rates or intra-mosquito systemic infection dynamics between the two mosquito populations. Ten and 20-days were needed by the virus to disseminate in 50% and 100% of the exposed mosquitoes respectively. Such slow intra-mosquito viral dynamics, in addition to repeatedly reported high transmission barrier in the literature, can impact ZIKV transmission when potentially vectored by Ae. albopictus. However, because mosquito-borne virus intra-host transmission dynamics can be influenced by numerous factors, including virus dose dynamics inside infectious humans or viral evolution towards shorter extrinsic incubation periods (EIP), our results highlight that Ae. albopictus populations present in Metropolitan France and the French territoires in the Indian Ocean might become potential vector for autochthonous ZIKV transmissions.
Introduction
Several mosquito-borne viruses have left their original enzootic cycles in tropical primary forests to emerge worldwide in transmission cycles involving humans and mosquitoes highly adapted to urban environments. While Aedes aegypti is considered as the main vector species of viruses affecting human health, the Asian tiger mosquito Aedes albopictus is trailing second in the list and might soon move ahead because of its outstanding invasive capacity. Originating from South-East Asia, Ae. albopictus has invaded the world and is now present in all continents, including temperate Europe, due to its potential of enduring harsh winter conditions through the induction of diapause in eggs1,2. The species is also displacing Ae. aegypti populations in areas where both species co-localize due to competitive advantages, notably at the larval stage3–5.
Zika virus (ZIKV) is the lastest mosquito-borne virus that has emerged on a global scale and that can be sustained in urban cycles involving solely human-mosquito transmissions. ZIKV is a RNA virus from the Flavivirus genus, Flaviviridae family, which includes other human pathogens such as yellow fever virus (YFV) or dengue viruses (DENV). First isolated from a rhesus monkey in the Zika forest in Uganda in 1947, ZIKV has emerged concomitantly in 2007 in Gabon6 and in the Federated States of Micronesia where it triggered a major outbreak in Yap Island7. The virus then spread through the South Pacific islands from 2013 to 20148–10 before its emergence in north-eastern Brazil in 201511, the starting point toward a large outbreak that hit a total of 50 territories and countries in the Americas12. Usually relatively mild, ZIKV infection can impact human health by leading to Guillain-Barré syndromes and cases of microcephaly in newborns, as well as other neurological impairments13.
Aedes albopictus mosquitoes are known to vector several major arthropod-borne viruses (arboviruses)14 and have been incriminated in the transmission of chikungunya (CHIKV) and DENV viruses in major recent outbreaks in the Reunion island, a French oversea territory in the Indian Ocean15,16. This species was also responsible for the transmission of these viruses in Europe (e.g. autochthonous cases in South France17–19) and a CHIKV outbreak in north-eastern Italy with over 200 confirmed cases20. European French populations of Ae. albopictus mosquitoes were shown to experimentally transmit DENV and CHIKV as efficiently as the typical tropical vector Ae. aegypti21. On the other hand, ZIKV has exhibited lower dissemination and transmission rates in experimental exposure assays in both Ae. albopictus and Ae. aegypti when compared to other RNA mosquito-borne viruses such as DENV, YFV or CHIKV 22–24. Such barriers preventing systemic infection (virus dissemination from midgut to secondary tissues) or transmission22,24–27 suggest limited risk of Zika virus transmission in Europe or the Reunion island22,25. To our knowledge, no ZIKV outbreak or autochthonous transmission was yet reported in Metropolitan France or French overseas territories where this species is established, but the implication of Ae. albopictus was strongly suspected in West Africa6. French overseas territories in the Indian Ocean have repeatedly been hit by mosquito-borne viruses and a large amount of goods and travelers are transiting every year between Metropolitan France and these tropical overseas territories, putting these regions at risk of ZIKV emergence.
In the present studies, we aim to assess the susceptibility of Aedes albopictus populations from Metropolitan France and Oversea France (the Reunion island, Indian Ocean) for ZIKV infection and compare their intra-host systemic infection dynamics. Two Ae. albopictus mosquito populations were orally exposed to a 3 to 7.5 ×106 focus-forming units per mL (FFU/mL) infectious blood meal of a ZIKV isolate from the Asian genotype. These virus titers lie between median infection doses that were reported for Ae. albopictus27. Mosquitoes from each population were sampled at five different times post virus exposure to assess intra-mosquito systemic infection dynamics. This assessement of intra-mosquito systemic infection dynamics of ZIKV for both tropical and temperate Ae. albopictus mosquito populations shed light on the risk of ZIKV emergence in both temperate and tropical areas.
Materials and Methods
Zika virus isolate
In this study, we used the ZIKV isolate SL1602 that was isolated from the plasma of a traveller returning from Suriname in 201628. The full-length consensus genome sequence of the isolate is available from GenBank under accession number KY348640. This isolate belongs to the Asian lineage and is phylogenetically close to other viruses that have recently been isolated in the Americas28. The virus was passaged four times in C6/36 cells (Aedes albopictus) prior use for mosquito infections. To prepare virus stock, sub-confluent C6/36 cells were infected using a viral multiplicity of infection (MOI) of 0.1 (one infectious viral particle for 10 cells) in 25-cm2 culture flasks and incubated for 7 days at 28 °C with 5 mL of Leibovitz’s L-15 medium supplemented with 0.1% penicillin (10,000 U/mL)/streptomycin (10,000 μg/mL) (Life Technologies, Grand Island, NY, USA), 1X non-essential amino acids (Life Technologies) and 2% fetal bovine serum (FBS, Life Technologies). At the end of the incubation, the cell culture medium was harvested, adjusted to 10% FBS and pH ∼8 with sodium bicarbonate, aliquoted and stored at −80 °C. An additional 5 mL of Leibovitz’s L-15 medium prepared as described above were added to infected confluent C6/36 cells and harvested 3 days later. This procedure increases virus titres in the stock solution.
Virus titration was performed by focus-forming assay (FFA) on one aliquot that has been stored at −80 °C as previously described with minor modifications29. This assay relies on inoculation of 10-fold dilutions of a sample onto a sub-confluent culture of C6/36 cells, followed by incubation and subsequent visualization of infectious foci by indirect immunofluorescence. Modifications to the previously published protocol include an 1 hour incubation step at 37 °C with 40 μL/well of mouse anti-Flavivirus group antigen antibody clone D1-4G2-4-15 (Merck Millipore, Molsheim, France) diluted 1:200 in PBS + 1% bovine serum albumin (BSA) (Interchim, Montluçon, France). After another three washes in PBS, cells were incubated at 37 °C for 30 min with 40 μL/well of a goat anti-mouse IgG (H+L), FITC-conjugated antibody (Merck Millipore). After three more washes in PBS and a final wash in ultrapure water, infectious foci were counted under a fluorescent microscope and converted into focus-forming units/mL (FFU/mL). The titre of the frozen virus stock was estimated at 6.35 × 105 focus-forming units per mL (FFU/mL) for the first medium culture harvest and 2.75 × 108 FFU/mL for the second medium culture harvest. All infectious experiments were conducted in a Biosafety Level-3 (BSL-3) insectary (IHU Méditerranée Infection, Marseille).
Mosquito populations
Two mosquito populations of Aedes albopictus species were used in this study, one from Metropolitan France and the other from the Réunion island, a French overseas territory in the Indian Ocean. Ae. albopictus mosquitoes from Metropolitan France were collected both as eggs using ovitraps and human landing catches in three different locations in Marseille city (5th, 12th and 13th districts) in June 2018. Ae. albopictus mosquitoes from French overseas territory were all collected as eggs in June 2018 from 6 different localities of the Réunion island named Sainte Marie Duparc, Saint André centre-ville, Saint Paul, Le Port Rivière des Galets, Saint Leu Pointe des châteaux, Saint Pierre Bois d’olive (Figure 1). An first generation population was obtained from the eggs of adults originating from ovitraps or captured by human landing catches. For each population, larvae from different collection sites were combined and adult mosquitoes were maintained in standardized insectary condition (28°C, 75 ± 5% relative humidity, 16:8 hours light-dark cycle) until the 4th generation by mass sib-mating and collective oviposition. Adult females from each population were blood-fed on human blood obtained from the Etablissement Français du Sang (EFS) through a membrane feeding system (Hemotek Ltd, Blackburn, UK) using pig intestine as membrane. Access to human blood was based upon an agreement with the EFS. Eggs were hatched in reverse osmosis water and larvae were reared with a standardized diet of fish food in 24 × 34 × 9 cm plastic trays at a density of about 400 larvae per tray. A maximum of 800 male and female adults were maintained in 24 × 24 × 24 cm screened cages with permanent access to a 10% sucrose solution.
Experimental mosquito infections
Nine to 13 days old females were transferred to the biosafety level 3 laboratory and deprived of sucrose solution 24 hours before experimental virus exposure. Sixty to 80 females were confined into 80-mm high and 80/84 mm in (inside/outside) diameter cardboard containers. Containers were sealed on the top with mosquito mesh and with a 65-mm high polystyrene piston covered up with a plasticized fabric that match the inside diameter of the container at the bottom. Pistons of each container were rose before to infectious blood meal exposure to contain all mosquitoes in a tight space below the infectious blood meal. This procedure increases the yield of engorged mosquitoes. Females were allowed to feed for 20 min from an artificial feeding system (Hemotek) covered by a pig intestine membrane that contained the infectious blood meal maintained at 37 °C. Feeders were placed on top of the mesh that sealed the containers. The infectious blood meal consisted of two volumes of washed human erythrocytes and one volume of viral suspension. Human erythrocytes were collected and washed one day before the experimental infection. An aliquot of the artificial blood meal was collected immediately prior to blood feeding and titred by FFA as described above without a freezing step. Final ZIKV virus titres in blood meals were 7.5 × 106 FFU/mL and 3 × 106 FFU/mL for experiment 1 and 2, respectively. After virus exposure, fully engorged females were cold anesthetized and sorted on ice before being individually transferred to new cardboard containers and maintained under controlled conditions (28°C, 75 ± 5% relative humidity, 16:8 hours light-dark cycle with permanent access to a 10% sucrose solution).
ZIKV RNA detection
ZIKV RNA was detected in mosquito bodies and heads after a fresh dissection from freeze-killed mosquitoes at 5,10, 14, 17 and 21-days post virus exposure (DPE) for both Ae. albopictus populations. Presence of ZIKV RNA in bodies indicates a midgut infection, while presence of the virus RNA in mosquito heads indicates a systemic (disseminated) infection30. These two vector competence indices were determined qualitatively (i.e. presence or absence of virus in mosquito bodies and heads, respectively). Dissected mosquito heads and bodies were homogenized individually in 400 μL of lysis buffer (NucleoSpin® 96 Virus Core Kit, Macherey-Nagel, Hoerdt, France) during three rounds of 20 sec at 5,000 rpm in a mixer mill (Precellys 24, Bertin Technologies, Montigny le Bretonneux, France). Viral RNA from individual organs was extracted using the NucleoSpin® 96 Virus Core Kit (Macherey-Nagel) according to the manufacturer’s instructions. At the final step, viral RNA from each sample was eluted in 100 μL of RNase-free elution buffer.
Detection of ZIKV RNA was performed with an end-point reverse transcription polymerase chain reaction (RT-PCR) assay. ZIKV genomic RNA was first reverse transcribed to complementary DNA (cDNA) with random hexamers using M-MLV Reverse Transcriptase (Life Technologies) according to the manufacturer’s instructions. cDNA was amplified by 35 cycles of PCR using the set of primers targeting the RNA-dependent RNA polymerase NS5 gene: ZIKV-F: 5’-GCCATCTGGTATATGTGG-3’ and ZIKV-R: 5’-CAAGACCAAAGGGGGAGCGGA-3’. Amplicons (393 bp) were visualized by electrophoresis on 1.5% agarose gels.
Statistical analysis
The time-dependent effect of the mosquito population on mosquito body infection and systemic infection was analysed by Firth’s penalized likelihood logistic regression by considering each phenotype as a binary response variable. Penalized logistic regression, implemented through the logistf R package31 was used to solve problem of separation that can occur in logistic regression when all observations have the same event status for a combination of predictors or when a continuous covariate predict the outcome too perfectly. A full-factorial generalized linear model that included the time post-virus exposure and the mosquito population was fitted to the data with a binomial error structure and a logit link function. Statistical significance of the effects was assessed by an analysis of deviance.
The intra-host dynamic of systemic infection was assessed by a global likelihood function for each mosquito species as described by Fontaine et al, 201830. Probabilities of systemic infection at each time point post virus exposure were estimated with a 3-parameter logistic model. The probability of systemic infection at a given time point (t) is governed by K: the saturation level (the maximum proportion of mosquitoes with a systemic infection), B: the slope factor (the maximum value of the slope during the exponential phase of the cumulative function, scaled by K) and M: the lag time (the time at which the absolute increase in cumulative proportion is maximal). For easier biological interpretation, B was transformed into a rising time Δt, which correspond to the time required to rise from 10% to 90% of the saturation level with the formula: Δt = ln (81) / B. For each mosquito population, the subplex32 R function was used to provide the best estimates of the three parameters to maximize the global likelihood function (i.e., the sum of binomial probabilities at each time point post virus exposure). This method accounts for differences in sample size when estimating parameters values.
All statistical analyses were performed in the statistical environment R v3.5.233. Figures were prepared using the package ggplot234.
Results
A total of 227 and 175 engorged female mosquitoes from the Metropolitan France and Overseas France populations, respectively, were analyzed in this study. Two ZIKV experimental exposure assays were performed with infectious blood meals titred at 7.5 × 106 FFU/mL (98 and 120 engorged mosquitoes for Metropolitan France and Overseas France populations, respectively) and 3 × 106 FFU/mL (129 and 55 engorged mosquitoes for Metropolitan France and Overseas France populations, respectively). Because each assay was performed with a single dose, the virus dose effect was here confounded with the experiment effect. High body infection prevalences were obtained for both populations, independently of the time post exposure (Figure 2). Averaged over time, mean body infection prevalences were 84% for the Metropolitan France population for each experiment and over 87% for the Overseas France population. A full factorial logistic regression model revealed no statistically significant difference for body infection prevalences across mosquito populations and experiment replicates. However, there was a statistically significant interaction effect between time post-virus exposure and the experiment replicate (analysis of deviance, p-value = 0.048) which was driven by a relatively low body prevalence at 5 days post-virus exposure for mosquitoes from the Metropolitan France population at 7.5 × 106 FFU/mL.
Systemic infection prevalences were measured by counting the number of mosquitoes with infected heads over the number of mosquitoes with an infected body. Systemic infection prevalences were significantly influenced by the time post-exposure (analysis of deviance, p-value < 2 ×10−16) but not by mosquito population or experiment replicate. Intra-host dynamic of systemic ZIKV infection was inferred in all combinations of mosquito population and virus doses by fitting a 3-parameters logistic model that assumes a sigmoidal distribution of the cumulative proportion of mosquitoes with a systemic infection over time. The model failed to provide relevant estimates for the Overseas France population at the lowest virus dose due to a lack of samples at early times post-virus exposure. However, systemic infection prevalences saturated at 100% for both mosquito populations and experiment replicates. In addition, the model inferred a lag time (M, the inflexion point of the sigmoid which represents the time needed to reach 50% of the saturation level) and a rising time (Δt, time to go from 10% to 90% of the saturation level). The estimated time required to reach 50% of systemic infections was 10 and 11 days for the Metropolitan France and the Overseas France populations, respectively, independently of the experiment replicate for the Metropolitan France population. In the first experiment replicate (highest virus titer at 7.5 × 106 FFU/mL), an estimated 9 and 13 days were needed to go from 10% to 90% of systemic infections for the Overseas France and Metropolitan France populations, respectively. This rising time (Δt) was estimated to 9 days for the Metropolitan France population in the second experiment replicate (virus titer at 3 × 106 FFU/mL).
Discussion
The French overseas territories in the Indian Ocean have housed several major mosquito-borne virus outbreaks over the last fifteen years. From 2005 to 2006, chikungunya virus has emerged in the Indian Ocean, causing 244,000 cases in the Reunion island, which represented 40% of the island population. Fifteen years later, this region was affected by a dengue virus outbreak that caused more than 6,500 cumulated cases from austral summer 2017 to winter 201816. Neighbouring islands, such as the Seychelles, Mayotte and Mauritius, were also affected16,35. These regions are popular destinations for tourists, especially those coming from Metropolitan France. In 2018, the Reunion island hosted more than 400,000 visitors from Metropolitan France, which represents around 80% of the island global tourism flow of the year36. This region also has historic, touristic and economic links with countries in the Indian Ocean basin, including India and south-eastern African countries. Chikungunya viruses that were responsible for the 2005-2006 outbreak in the Reunion island were tracked back to Kenya37 and then propagated to India38. The DENV epidemic that occurred in both the Reunion island and the Seychelles in 2018 was suspected to involve virus strains originating from India or China16. To our knowledge, no Zika virus outbreak has been documented in the Reunion island or surrounding islands. Yet, Zika viruses from the Asian lineage were circulating in India from 2016 to 201839, transmitted by the mosquito Ae. aegypti40. The risk of ZIKV introduction in the Reunion island and surrounding islands is therefore significant.
ZIKV was isolated from Aedes africanus mosquitoes in 1948 in the Zika Forest, Uganda, one year after its first isolation from a sentinel rhesus monkey in the same area41. While Aedes hensilli was strongly suspected to transmit ZIKV during the Yap island outbreak7, the species Aedes aegypti was incriminated in all major outbreaks that have occurred in the Pacific Ocean, Brazil or the Caribbean, especially in urban transmissions42. Ae. albopictus was found to be competent for ZIKV experimentally24,27,43–46 and strongly suspected to sustain a ZIKV epidemic activity (African lineage) in Gabon in 20076. Ae. albopictus has been implicated in all arbovirus transmissions occurring in the Reunion island and could thus vector ZIKV after an introduction in the island. Southern Europe is also at risk in areas where Ae. albopictus is implanted, which represents 42 (43.75%) over 96 Metropolitan French departments in 201847. To our knowledge, no autochthonous cases of ZIKV has occurred in Metropolitan France despite the presence of imported cases48, while several cases of autochthonous CHIKV and DENV were repeatedly reported during the last fifteen years17,18,49.
Here we described high infection rates and unpreceded levels of systemic infection rates that reach 100% in both Metropolitan France and Indian Ocean populations. Importantly, we considered the dynamic nature of vector competence by assessing systemic infection (dissemination) rate over time post-virus exposure. No differences in infection rates or intra-mosquito systemic infection dynamics were revealed between Ae. albopictus populations originating from Metropolitan France and the Indian Ocean (the Reunion island). Vector competence studies performed on Ae. albopictus often revealed a low systemic infection rate for ZIKV24,25,43,44,50. All these studies used a ZIKV isolate from the Asian genotype and a virus dose in the same range than our study (from 3 × 106 FFU/mL to 107 FFU/mL; or 107 TCID50/mL) and some have used mosquito populations that can be assumed to be closely related to our Ae. albopictus populations (Indian Ocean or European origins). In addition, three of these works assessed systemic infection prevalence at a time that exceed our estimated saturation level (i.e. 21 days post virus exposure). Mosquito vector competence for viruses were reported to differ according to mosquito populations51,52, virus isolates30, their combinations53 or the virus dose27. More complex interactions between intrinsic (eg. mosquito virome54, endogenous non-retroviral elements55 or bacterial microbiome56) or environmental (eg. temperature57–59, rearing or experimental settings) conditions might further complicate comparisons across vector competence studies. An almost unanimous consensus is however prevailing on the existence of a low transmission efficiency22,24,25,27,43,44,50,60 in the Ae. albopictus/ZIKV couple. Forced salivation assays allow to determine viable infectious particles in single mosquito saliva, avoiding the use of animal experiments. In these assays, mosquito wings and legs are removed prior to collect the saliva from the remaining portion of live mosquito by inserting the proboscis into a capillary filled with different types of media, including defibrinated blood, fetal bovine serum or mineral oil61,62. Transmission dynamic estimates must be considered with caution because the method to detect viruses in mosquito saliva might not distinguish true negatives from negatives resulting from mosquito that did not expectorate saliva. This can be however improved by the visualization of the saliva droplet with the use of a hydrophobic medium. Further experiments are needed to assess the presence of a transmission barrier in our settings. It is worth noting that on the contrary, two studies have revealed high transmission rates of ZIKV in Ae. albopictus45,46.
Interestingly, our results show that ZIKV has slower intra-host systemic infection dynamics than other mosquito-borne viruses: by comparison, 100% systemic infections of Aedes aegypti can be observed for several dengue isolates around 10 days (2 folds faster)30. Combined with vector longevity, intra-host mosquito dynamic is the most powerful contributor to vectorial capacity, a restatement of the basic reproductive rate (R0) of a pathogen 30,63. In presence of a ZIKV outbreak sustained solely by Ae. albopictus mosquitoes, older individuals (>20 days post their first infectious blood meal) would ultimately be implied in virus transmission and any anti-vector measures designed to reduce mosquito life span should thus have significant effect to control the outbreak.
The efficient infection of Ae. albopictus however leaves the opportunity for ZIKV to evolve towards shorter intra-mosquito dynamics, as had already occurred in other systems, enhancing the epidemic potential of the Ae. albopictus/ZIKV couple64,65. In addition, intra-mosquito systemic infection dynamics and the duration of the extrinsic incubation periods (EIP) depend on the amount of virus ingested during the blood meal29,66. ZIKV viremia was shown to peak during the first days of infection and to decline quickly in the next few days when assessed on nonhuman primate models67,68. In humans, the amount of ZIKV genomic RNA was only assessed post-symptom onsets: ZIKV genomic RNA copies per millilitre were at their highest level at the first sampling time post-symptom onsets but rarely exceeded 6 logs69–71, which would translate into a much lower concentration of infectious particles due to a genomic RNA/infectious particles ratio > 172. At these dose levels the probability to infect Ae. albopictus mosquitoes would be minimal, and long EIP values distribution would probably arise. We currently lack information about the distribution of infectious ZIKV concentration in human before symptom onsets. Here, we estimated that around 10 days are required to reach 50% of systemic infections in Ae. albopictus at a dose ranging from 3 to 7.5 × 106 FFU/mL. We can hypothesize that faster intra-mosquito virus dynamic could be achieved at a higher virus doses and that infected humans would better contribute to ZIKV transmission in their early days’ pre-symptom onsets. Further works are needed to assess the ZIKV dose impact on intra-mosquito systemic and transmission dynamics.
Taken together, our results suggests that despite being reported as a relatively poor vector of ZIKV, Ae. albopictus mosquitoes present in Metropolitan France and French territories in the Indian Ocean basin cannot be considered per se as an obstacle to autochthonous ZIKV transmissions. Further works on the assessment of a transmission barrier for ZIKV and the influence of the virus dose on intra-host dynamics are clearly needed to explore the full epidemic potential of the ZIKV/Ae. albopictus couple.
Conflict of interests
The authors declare that there is no conflict of interest regarding the publication of this article.
Funding statement and acknowledgments
This study was funded by the Direction Générale de l’Armement (grant no PDH-2-NRBC-2-B-2113) and supported by the European Virus Archive goes Global (EVAg) project that has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement N° 653316. The contents of this publication are the sole responsibility of the authors. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. Sebastian Lequime is funded by a postdoctoral grant of the Fonds Wetenschappelijk Onderzoek – Vlaanderen (FWO). We thank Nathalie Wurtz, Muriel Militello and Jean-Marc Feuerstein for their help and contributions concerning the development of the biological safety level 3 procedure settings concerning experimental mosquito exposure to ZIKV. We also thank Joël Mosnier and Isabelle Fonta for their help with the seeting up of BSL3 experiments.