DENV-1 Titer Impacts Viral Blocking in wMel Aedes aegypti with Brazilian Genetic Background

Several countries have been using Wolbachia deployments to replace highly competent native Aedes aegypti populations with Wolbachia-carrying mosquitoes with lower susceptibility to arboviruses such as dengue, Zika, and chikungunya. In Rio de Janeiro, Wolbachia deployments started in 2015 and still present a moderate introgression with a modest reduction in dengue cases in humans (38%). Here, we evaluated the vector competence of wild-type and wMel-infected Ae. aegypti with a Brazilian genetic background to investigate whether virus leakage could contribute to the observed outcomes in Brazil. We collected the specimens in three areas of Rio de Janeiro with distinct frequencies of mosquitoes with wMel strain and two areas with wild Ae. aegypti. The mosquitoes were orally exposed to two titers of DENV-1 and the saliva of DENV-1-infected Ae. aegypti was microinjected into wMel-free mosquitoes to check their infectivity. When infected with the high DENV-1 titer, the presence of wMel did not avoid viral infection in mosquitoes’ bodies and saliva but DENV-1-infected wMel mosquitoes produced lower viral loads than wMel-free mosquitoes. On the other hand, wMel mosquitoes infected with the low DENV-1 titer were less susceptible to virus infection than wMel-free mosquitoes, although once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and the saliva. Our results showed viral leakage in 60% of the saliva of wMel mosquitoes with Brazilian background; thus, sustained surveillance is imperative to monitor the presence of other circulating DENV-1 strains capable of overcoming the Wolbachia blocking phenotype, enabling timely implementation of action plans.


Introduction
Arthropod-borne viruses (arboviruses) such as dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) have spread in urbanized areas around the world, representing a significant public health burden, with 3.9 billion people at risk in 129 countries [1].Dengue is responsible for more than 90% of the arboviral cases reported in the Americas, reaching 2.8 million cases in 2022 [2,3].Aedes aegypti (Diptera: Culicidae) is the primary vector, and it is highly associated with anthropogenic environments, where female mosquitoes lay eggs mainly in artificial containers inside or near human habitations and feed preferentially on humans [4,5].
Due to a lack of a vaccine broadly available for many arboviruses, the most recommended way to mitigate transmission is through intensifying vector control.Traditional control methods, such as the search for and elimination of breeding sites and the use of insecticides, present limited effectiveness in avoiding outbreaks [6].Given this challenging scenario, designing alternative effective interventions is critical to reducing the disease burden.One of these strategies is the release of Ae. aegypti infected with the intracellular symbiont Wolbachia pipientis (Rickettsiales, Anaplasmataceae).This bacterium is naturally present in nearly 40% of arthropods but not in Ae. aegypti [7].Its use is based on at least three phenotypes to support introgression into native mosquito populations: reduction in arbovirus replication and transmission in mosquitoes [8][9][10], maternal transmission (MT), and capacity to induce cytoplasmic incompatibility (CI) [11].CI results in embryonic mortality in crosses between Wolbachia-free females and Wolbachia-infected males but not between females with Wolbachia and males with or without the bacteria [12,13].
Among the Wolbachia strains currently introgressed into Ae.aegypti, wMel (from Drosophila melanogaster) does not seem to influence mosquito fecundity and survival, but it reduces the oviposition success and the viability of eggs stored for many weeks under laboratory conditions [14][15][16].Regarding the refractoriness to arboviruses, the wMel strain inhibits the transmission of CHIKV [8], Yellow Fever [17], and Mayaro viruses [18], but its protective effect is not complete against DENV-1 [ [19][20][21] and ZIKV [22].Although the complete mechanism of Wolbachia-mediated blocking of arboviruses is not fully understood, there is evidence of mosquito immune priming, competition for lipids, and production of reactive oxygen species, among others (reviewed in [23,24]).The inhibition of arbovirus replication seems to also be bacterial density dependent, i.e., higher blocking is expected in individuals with higher Wolbachia density in mosquito tissues [25].
Wolbachia deployments in Ae. aegypti populations have been achieving promising outcomes around the globe.A randomized control trial in Yogyakarta, Indonesia, showed >70% wMel invasion after 4-7 months of release and a 77% reduction in dengue cases [26].In North Queensland, Australia, dengue incidence reduced nearly 65% during wMel releases for 28 months between 2014 and 2017 and >95% 24 months after releases [27].Another recent study in Colombia has shown that the Wolbachia deployments occurring since 2015 achieved an impressive dengue incidence decrease of 94-97%, with two out of the three cities that received the program being considered fully treated (60% of the local mosquitoes with Wolbachia [28,29]).
The first release of the wMel strain in Rio de Janeiro was conducted in 2015 at Tubiacanga, an isolated locality with around 750 premises.The release consisted of approximately 10,000 females/week for a period of 24 weeks.The wMel frequency in the field reached 80% in the 18th week of releases, and it remained between 85 and 95% one year after ceasing the releases [14].From 2017 to 2019, the release areas were expanded, covering a total area of 86.8 km 2 with around 890,000 inhabitants [30].Niterói (13 km away from Rio de Janeiro) was the first city in Brazil to adopt citywide deployment of Wolbachia [31].After a 2-year release program, the frequency of wMel-infected Ae. aegypti across the city varied from 33 to 90%, and an overall reduction of 69% in dengue cases was reported [32].A more modest outcome was obtained in the city of Rio de Janeiro.After releasing 67 million wMel Ae. aegypti mosquitoes between 2017 and 2019, a 32% wMel introgression into the wild population was achieved, and a reduction of 38% and 10% of dengue and chikungunya notifications were reported, respectively [30].The results of Rio de Janeiro are of interest, but a more in-depth investigation is warranted to assess the obstacles that limited wMel introgression and the reduction in arbovirus cases.
Several laboratory studies have been evaluating the effect of Wolbachia on the vector competence of Ae. aegypti with different genetic backgrounds to DENV strains, but most of them challenged mosquitoes with high doses of the virus (i.e., unrealistic viral loads) to ensure mosquito infection [19][20][21][33][34][35][36].For instance, wMel Ae. aegypti mosquitoes with a Brazilian genetic background showed refractoriness to high doses of DENV-1 (10 6 and 10 7 ) and ZIKV (10 6 and 10 8 ) viruses [32].In the field, Ae. aegypti, however, can also transmit arboviruses when fed the blood of infected vertebrates with low viremia [37].Moreover, different genetic backgrounds of arboviruses and mosquitoes circulate in the field, which ultimately can result in contrasting infection and transmission scenarios.Herein, we provide an independent dataset involving vector competence assays to investigate the capacity of wMel, wMel-free, and wild Ae.aegypti collected in five locations of Rio de Janeiro with different invasion patterns to become infected and transmit DENV-1 through experimental oral infections using low and high viral titers.
Two different sampling strategies were conducted simultaneously between September and November 2019.Using backpack aspirators, we captured 25 adult female Ae.aegypti in each of the study areas to check the prevalence and relative density of wMel in field mosquitoes.At the same time, 60 ovitraps were evenly distributed to obtain a minimum of 5000 eggs per area that best represents the genetic diversity of Ae. aegypti in the field.Five hundred eggs were hatched in plastic containers (45.5 cm × 28 cm × 7.7 cm) according to their area with 3 L of water and yeast, and larvae were fed daily with 4.5 mg of TetraMin ® fish food (Tetra, Melle, Germany) until the pupae stage.Adult mosquitoes were identified using taxonomic keys [43].Those identified as Ae.aegypti formed site-specific lab colonies that were kept under insectary conditions (80 ± 5% humidity and 25 ± 3 • C) with sugar solution (10%) ad libitum until 24 h before DENV infection.

Wolbachia DNA Detection and Quantification in Ae. aegypti
The DNA of Wolbachia was extracted as previously described [10] from both fieldcaught adults and lab-reared Ae. aegypti mosquitoes.wMel infection status was determined through a duplex PCR assay, which amplifies the Ae.aegypti ribosomal protein S17 (RpS17) as an internal mosquito control, and also the Wolbachia WD0513 gene to detect the wMel strain [19].Relative quantification was performed with TaqMan TM Fast 1-Step Master Mix (Applied Biosystems, Waltham, MA, USA), using the QuantStudio TM 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) as previously published [44].Five individuals from NR1 and NR2 were used as negative controls.

Wolbachia DNA Detection and Quantification in Ae. aegypti
The DNA of Wolbachia was extracted as previously described [10] from both fieldcaught adults and lab-reared Ae. aegypti mosquitoes.wMel infection status was determined through a duplex PCR assay, which amplifies the Ae.aegypti ribosomal protein S17 (RpS17) as an internal mosquito control, and also the Wolbachia WD0513 gene to detect the wMel strain [19].Relative quantification was performed with TaqMan TM Fast 1-Step Master Mix (Applied Biosystems, Waltham, MA, USA), using the QuantStudio TM 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, MA, USA) as previously published [44].Five individuals from NR1 and NR2 were used as negative controls.

Viral Strain and Oral Infections
Dengue-1 MV17 strain, isolated from a human case in 2015 at Minas Gerais (DENV1/Homo sapiens/Brasil/Contagem/MG/MV17/2015) [45], was obtained after four passages in C6/36 cell culture.The virus was inoculated in C6/36 cell culture 5 days before the experimental infections.F1 adult Ae. aegypti were kept in cages with access to sugar solution (10%) until they were 6-7 days old.They were deprived of sugar solution 24 h before blood feeding, which consisted of 1 mL of erythrocytes + 1 mL of fresh virus suspended in L15 medium for the infected group and 1 mL of erythrocytes + 1 mL of L15 medium for the uninfected groups.Female mosquitoes from the five populations were orally fed using an artificial feeder (Hemotek, Great Hardwood, UK) with human blood (approved by Fiocruz Ethics Committee-CAAE 53419815.9.0000.5248)at 37 °C for approximately 30 min.Only visually fully engorged females were selected for the analyses.Two viral titers of the same virus culture were offered: 6 × 10 8 FFU/mL (focusforming units/mL; hereafter called high titer') or 3 × 10 4 FFU/mL (hereafter called low

Viral Strain and Oral Infections
Dengue-1 MV17 strain, isolated from a human case in 2015 at Minas Gerais (DENV1/ Homo sapiens/Brasil/Contagem/MG/MV17/2015) [45], was obtained after four passages in C6/36 cell culture.The virus was inoculated in C6/36 cell culture 5 days before the experimental infections.F1 adult Ae. aegypti were kept in cages with access to sugar solution (10%) until they were 6-7 days old.They were deprived of sugar solution 24 h before blood feeding, which consisted of 1 mL of erythrocytes + 1 mL of fresh virus suspended in L15 medium for the infected group and 1 mL of erythrocytes + 1 mL of L15 medium for the uninfected groups.Female mosquitoes from the five populations were orally fed using an artificial feeder (Hemotek, Great Hardwood, UK) with human blood (approved by Fiocruz Ethics Committee-CAAE 53419815.9.0000.5248)at 37 • C for approximately 30 min.Only visually fully engorged females were selected for the analyses.Two viral titers of the same virus culture were offered: 6 × 10 8 FFU/mL (focus-forming units/mL; hereafter called 'high titer') or 3 × 10 4 FFU/mL (hereafter called 'low titer').The two viral titers were chosen as representative of natural viral loads from naturally infected human hosts [37].While the females were feeding, a virus aliquot was serially diluted and inoculated in C6/36 cells.Viral envelope protein E was detected by immunofluorescence in C6/36 cultures using DENV-specific monoclonal antibodies (purified from ascitic fluid anti-dengue virus 1; in-house lab LATAM production; product batch: 041118FDEN1P; technical expert: Tiago Pereira).The focus-forming units (FFUs) were counted in an EVOS ® FL Auto Imaging System (Life Technologies, Carlsbad, CA, USA).

Mosquito Saliva Collection and Intrathoracic Microinjection
Mosquito females were anesthetized on ice, and their wings and legs were removed to collect their saliva at 14 dpi.Their proboscises were individually inserted into sterile Viruses 2024, 16, 214 5 of 16 filtered 10 µL pipette tips containing 10 µL of sterile Fetal Bovine Serum (Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA) mixed with blue food coloring and allowed to salivate for 30 min.Only the saliva of mosquitoes with blueish abdomens was collected.The F1 generation of Ae. aegypti females from NR1 were intrathoracically injected with 69 nL of saliva collected from DENV-1-infected females of FI, PI1, PI2, NR1, and NR2.Uninfected saliva from NR1 females was also inoculated as negative controls.Injections were carried out with a Nanoject II (Drummond Scientific Company, Broomall, PA, USA), as previously described [8].
Each saliva sample was inoculated in 15 female mosquitoes.Nineteen saliva samples of wild mosquitoes (NR1), 26 from areas with recent releases of wMel (PI1 = 10, PI2 = 16), and 13 from an area with early wMel releases (FI) were injected into 870 NR1 7-dayold female mosquitoes susceptible to DENV-1 [45].We inoculated saliva only of those females that had their bodies positive for DENV-1 through RT-qPCR (excepting the negative controls).The injected female mosquitoes were killed seven days after saliva inoculations, and DENV-1 was screened through RT-qPCR (see next section for details).Saliva samples that produced at least one subsequent infection in the injected mosquitoes were classified as infective.In the case of females subjected to saliva microinjection, we assumed that susceptible mosquitoes would exhibit elevated viral loads in their bodies when exposed to saliva containing higher concentrations of infective virus.In essence, we postulated a positive correlation between the viral load in saliva-microinjected female bodies and the infective DENV load in the saliva.

DENV-1 RNA Detection and Quantification in Mosquitoes
The RNA of each sample was extracted individually from whole mosquitoes with a QlAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) 14 dpi and seven days after intrathoracic injections.Detection and quantification of viral RNA was performed with TaqMan TM Fast Virus 1-Step Master Mix (Applied Biosystems, Waltham, USA), using the QuantStudio TM 6 Flex Real-Time PCR System (Applied Biosystems, Waltham, USA).Each reaction was made with previously published primers and probes [46].Amplification conditions consisted of 12 nmoles of forward and reverse primers, 9 nmoles of probe, 5 µL of TaqMan TM Fast Virus 1-Step Master Mix (Applied Biosystems), and 5 µL of RNA.Cycling conditions were as follows: 45 • C for 15 min, 95 • C for 20 s, followed by 40 amplification cycles of 95 • C for 15 s, 58 • C for 5 s, and 60 • C for 30 s. Viral copy numbers were calculated by interpolation onto an internal standard curve made up of a six-point dilution series (10 1 -10 6 FFU/mL) of DENV-1.

Statistical Analysis
DENV-1 loads in whole mosquitoes (14 dpi) and in susceptible mosquitoes intrathoracically injected with mosquito saliva (7 dpi) were compared according to their collected area, Wolbachia presence and density, and to the two DENV-1 titers (3 × 10 4 FFU/mL and 6 × 10 8 FFU/mL).The distribution of DENV-1 load was not normally distributed (Shapiro-Wilk test = 0.85, p < 0.001) and, therefore, was compared through Wilcoxon-Mann-Whitney tests in the R environment [47].Moreover, logistic and linear regressions were used to estimate the effects of wMel and the viral titer on the infection status of mosquito bodies and saliva infectivity.The infection rate of mosquitoes was calculated as the number of mosquitoes infected with DENV-1 divided by the total number of mosquitoes tested.Linear regressions were also performed to infer the association between Wolbachia density and DENV-1 load.These analyses were performed with the 'glm' function using the binomial or Gaussian distributions in the R environment.

Prevalence and Density of wMel in Field and Lab Ae. aegypti Mosquitoes
We accessed the prevalence of Wolbachia in the five areas, three where wMel deployments took place before conducting the vector competence analyses (PI1, PI2, and FI; cf.Methods section for further details).The wMel strain was detected in 41 out of 75 mosquitoes (an overall frequency of 54.7%) of the field populations where wMel deployments occurred (Figure S1).All mosquitoes from FI (25/25) had the bacteria, while PI1 and PI2 had 20% (5/25) and 44% (11/25) of mosquitoes positive for wMel, respectively.Pairwise comparisons did not show significant differences between wMel densities in field-caught mosquitoes from the three deployed areas (W = 15; W = 107; W = 77 p > 0.05; Table S1).As expected, NR1 and NR2 mosquitoes were all wMel-free.

Vector Competence Assays
A total of 216 Ae. aegypti F1 from FI, PI1, and PI2 and 144 mosquitoes from NR1 and NR2 were exposed to DENV-1 oral infections with low and high titers, and their whole bodies were tested for infection via RT-qPCR.Ten wMel and wild mosquitoes (from FI and NR1, respectively) non-exposed to DENV-1 were used as negative controls for arbovirus detection.

Vector Competence Assays
A total of 216 Ae. aegypti F1 from FI, PI1, and PI2 and 144 mosquitoes from NR1 and NR2 were exposed to DENV-1 oral infections with low and high titers, and their whole bodies were tested for infection via RT-qPCR.Ten wMel and wild mosquitoes (from FI and NR1, respectively) non-exposed to DENV-1 were used as negative controls for arbovirus detection.

Saliva Infectivity for DENV-1
The saliva collected from mosquitoes with DENV-1 infectivity confirmed through RT-qPCR was injected into susceptible mosquitoes from NR1, and their whole bodies were analyzed seven days after injections.The DENV-1 infection rate of the injected saliva from the studied areas is shown in Table 1.Surprisingly, when infected with DENV-1 low titers, the viral load in wild Ae.aegypti who received infective saliva of wMel Ae. aegypti was similar to the viral load observed in the control group (W = 86.5, p > 0.05; W = 48, p > 0.05; W = 52, p = 0.02; Figure 4, Table S1).On the other hand, when infected with the higher DENV-1 titer, there was a decrease in the viral load in wMel Ae. aegypti saliva when compared with wild Ae.aegypti mosquitoes (W = 108, p < 0.05; W = 83, p < 0.05; W = 220.5,p < 0.0001; Figure 4, Table S1).The linear regression analyses did not detect a statistically significant correlation between DENV-1 copies in saliva-microinjected mosquitoes and the relative wMel density of the mosquitoes (F = 2.605, p > 0.05, R 2 = 0.063; Figures S2B and S3).

Interactions of wMel in DENV-1 Exposed Ae. aegypti
Generalized linear models revealed that when infected with low DENV-1 titer, wMel Ae. aegypti presented a lower infection rate in their whole bodies when compared to controls (wild Ae. aegypti; GLM: Wolbachia (Yes) estimate = −1.2,p = 0.0005; Figure S4, Table S2).Wild and wMel mosquitoes, however, had similar viral loads in their bodies (GLM: Wolbachia (Yes) estimate = 0.02, p = 0.885; Figure S4).When we analyzed the microinjected mosquitoes, both the rate of infection and the viral loads obtained seven days post injection of wMel mosquito saliva were similar to the controls (saliva of wild mosquitoes; GLM: Wolbachia (Yes) estimates = 0.6 and −0.4,p > 0.1, respectively; Table S2).

Interactions of wMel in DENV-1 Exposed Ae. aegypti
Generalized linear models revealed that when infected with low DENV-1 titer, wMel Ae. aegypti presented a lower infection rate in their whole bodies when compared to controls (wild Ae. aegypti; GLM: Wolbachia (Yes) estimate = −1.2,p = 0.0005; Figure S4, Table S2).Wild and wMel mosquitoes, however, had similar viral loads in their bodies (GLM: Wolbachia (Yes) estimate = 0.02, p = 0.885; Figure S4).When we analyzed the microinjected mosquitoes, both the rate of infection and the viral loads obtained seven days post injection of wMel mosquito saliva were similar to the controls (saliva of wild mosquitoes; GLM: Wolbachia (Yes) estimates = 0.6 and −0.4,p > 0.1, respectively; Table S2).

Discussion
In this study, the vector competence of Ae. aegypti was inferred in laboratory conditions by comparing the susceptibility and saliva infectivity of wMel and wMel-free mosquitoes collected in five localities of Rio de Janeiro, the first Brazilian city to receive Wolbachia deployment.We also tested if the relative density of wMel in mosquito bodies Forest plot showing the odds ratio (OR) and 95% confidence intervals for interactions of wMel in DENV-1-exposed Ae. aegypti mosquitoes."Infection" results are based on the number of infected and non-infected mosquitoes, and "Viral load" results are the absolute quantity of DENV-1 virus particles.Mosquito bodies were analyzed 14 days post infection ("Body"), and susceptible mosquitoes were analyzed 7 days after receiving an intrathoracic injection of saliva from a mosquito exposed to DENV-1 ("Saliva-microinjected").Mosquitoes were fed on blood infected with DENV-1 at 3 × 10 4 FFU/mL ("Low DENV-1 titer") or 6 × 10 8 FFU/mL ("High DENV-1 titer").The asterisks denote statistically significant negative association (i.e., OR < 1.0) between having wMel and body infection or viral loads.

Discussion
In this study, the vector competence of Ae. aegypti was inferred in laboratory conditions by comparing the susceptibility and saliva infectivity of wMel and wMel-free mosquitoes collected in five localities of Rio de Janeiro, the first Brazilian city to receive Wolbachia deployment.We also tested if the relative density of wMel in mosquito bodies influences DENV-1 infection.We subjected wMel and wMel-free Ae. aegypti to oral infections containing two DENV-1 titers (6 × 10 8 FFU/mL or 3 × 10 4 FFU/mL) and microinjected mosquito saliva into wMel-free mosquitoes.Our results highlighted that when mosquitoes were infected with the higher DENV-1 titer, wMel did not avoid viral infection in mosquitoes' bodies and saliva, but wMel mosquitoes produced lower DENV-1 loads than wMel-free mosquitoes (Figure 5).On the other hand, wMel mosquitoes subjected to a lower DENV-1 titer were less susceptible than wMel-free mosquitoes, but once infected, wMel and wMel-free mosquitoes exhibited similar viral loads in the body and to saliva-microinjected mosquitoes.
Several studies have been investigating the susceptibility of wMel and wMel-free Ae. aegypti to DENV [10,23,[48][49][50][51], but there are few regarding the DENV-1 serotype [19][20][21].In this study, we observed the superior DENV-1 infection rate (~69%) in wMel mosquitoes when infected with a higher viral titer, contrasting with those infected with a lower viral titer (~22%).Taking into consideration the saliva's infection rate, we observed that ~80% of wild Ae.aegypti were infective, whereas ~60% of wMel Ae. aegypti were positive for DENV-1.Ferguson and colleagues [19] observed similar results for wMel and wMel-free Ae. aegypti from Cairns, Australia, exposed to viremic blood from acute dengue cases in Vietnam.The infection rate of mosquito body was 100%, irrespective of whether the mosquito carried wMel or not for DENV-1 titers of ~10 8 at 14 dpi, but wMel mosquitoes had only ~40% of their saliva infected (lower than we observed in this study), whereas ~85% of infective saliva was observed for wMel-free Ae. aegypti.The authors, however, did not observe infected bodies of wMel and wMel-free mosquitoes exposed to DENV-1 titer of ~10 5 at 10 dpi.
Another study in Vietnam involving four DENV serotypes has shown that 71.5% of wMel-free Ae. aegypti and 58.7% of wMel Ae. aegypti had their abdomens infected with DENV.Furthermore, the same held true for infectious viruses in the saliva, in which 38.5% and 22.8% for wMel-free and wMel Ae. aegypti, respectively, were infected with DENV [20].In this study, the authors observed more mosquitoes with infective saliva when infected with DENV-1 than with DENV-4.
Flores and colleagues [21] also analyzed infection rates in wMel-Cairns Ae. aegypti (genetic background from Cairns, Australia), wMel-HCM Ae. aegypti (genetic background from Ho Chi Minh City, Vietnam) and wMel-free mosquitoes, respectively.They observed similar infection rates of abdomen/head-thorax/saliva to Carrington and colleagues [20] and did not find any difference in the prevalence of DENV in mosquitoes with different genetic backgrounds.The authors also described for DENV-1 intrathoracic microinjection a high infection rate (94%) and a ~10 3 viral load for wMel Ae. aegypti, while a 100% infection rate and ~10 5 TCID 50 /mL (for 1.2 × 10 5 TCID 50 /mL titer) viral load were observed for Cairns wMel-free Ae. aegypti.Concerning the Brazilian Ae. aegypti genetic background, Souto-Maior et al. [52] observed through DENV-1 microinjections of five viral titers (10 4 -10 8 TCID 50 /mL) that Wolbachia confers a slight protection against the virus.
Divergent results on studies regarding the vector competence to DENV of wMel Ae. aegypti might be related to the different virus genotypes [8,10,[19][20][21][48][49][50][51].In this context, the emergence of DENV variants able to replicate in wMel Ae. aegypti and surpass the viral blockage are of general concern.For example, an amino acid substitution (E203K) in the DENV-1 envelope protein has increased in frequency in virus populations following 20 passages in wMel Ae. aegypti compared to wMel-free Ae. aegypti cell cultures.Therefore, it is highly probable that wMel would exert selective pressure on dengue populations.It is still unknown, however, if these variants could efficiently replicate in the field where wMel mosquitoes have been released.Future studies regarding the genome sequence of DENV variants in the field may answer this question [53].
Wolbachia density has been positively linked to the strength of viral blocking to RNA viruses [9,[54][55][56].For instance, in Drosophila simulans, it was already observed that Wolbachia density is important for antiviral protection [57].Studies involving cell lines have shown almost complete DENV inhibition only if the cells have high Wolbachia density [55,56].Ae. albopictus is naturally infected with another Wolbachia strain, wAlbB, but is unable to block DENV, which has been linked to the lower bacterium density in this species.When wAlbB is transinfected into Ae.aegypti, it is present in higher densities in mosquito tissues, and a DENV-blocking phenotype is observed [56].In our study, wMel Ae. aegypti from the field exhibit a lower density of the bacterium than the F1 lab-reared mosquitoes, and it may be explained by several factors such as temperature [58,59], fitness cost [13,[60][61][62], and nutrition [63].High temperatures can impact CI and Wolbachia density, leading to a reduction in wMel frequencies and densities in Ae. aegypti [58,59].Although the fitness cost of having wMel is equal to both field and laboratory-reared mosquitoes [64,65], the latter are reared in optimal conditions of temperature, humidity, and nutrition, while field Ae. aegypti mosquitoes are subjected to adversities that may contribute to the lower wMel density.Regarding the lab-reared wMel Ae. aegypti exposed to DENV-1, there was no correlation between relative wMel density and virus infection of the mosquito's body or saliva (Figure 5), as already reported [66,67].Even though wMel density does not appear to influence DENV-1 infection, tissue-specific (ovaries, salivary glands, and saliva's infectivity) analysis could shed a brighter light on this issue.
We observed contrasting scenarios in Rio regarding the wMel geographical invasion and relative density in mosquitoes and the viral blocking phenotype of Ae. aegypti.FI is an isolated neighborhood with low human density where wMel deployment was completed in 2015 and had the highest wMel frequency and density.Moreover, its wMel mosquitoes had the lowest DENV-1 loads (Figures 1, 2 and 5).PI1, a highly urbanized and human-populated area surrounded by places with few or no Wolbachia deployments, where Wolbachia deployments occurred in early 2019, wMel is still in low prevalence, and mosquitoes had high viral loads in their bodies and saliva.In PI2, where wMel releases occurred in late 2019 and the bacterium is highly prevalent, the mosquitoes also had high viral loads in the saliva.Therefore, even though there is not a positive linear relationship between Wolbachia density and DENV-1 infection, the results observed in FI suggest there may exist a Wolbachia density threshold, which allows better protection for the arboviruses.Another hypothesis would be that the positive relationship between Wolbachia density and protection may occur in some strategic tissues for Wolbachia but not in others, and this outcome is masked in this study once we analyze the whole mosquito bodies.
The release of Ae. aegypti with Wolbachia has been a promising strategy to mitigate the transmission of arboviruses such as DENV, ZIKV, and CHIKV in urban settings.Nonetheless, the success of Wolbachia as a strategy to reduce arboviruses varies given the complexity of each city's environment and factors, such as the insecticide resistance of Ae. aegypti carrying Wolbachia, native field vector population sizes, and the maintenance of MT and CI in field conditions [30,36,[68][69][70][71].It is relevant to bear in mind that wMel deployment has a significant public health impact and likely achieves disease elimination in low/moderate arboviral transmission scenarios, but it seems to be less efficient to control the transmission in highly epidemic regions, particularly considering DENV-1 [19].Studies in Rio de Janeiro, Indonesia, and Australia have shown a reduction in dengue cases using the wMel strain.Australia had a great outcome with a reduction of >95% of DENV cases.In Indonesia, a better outcome was observed when compared to Rio's study, showing a reduction of 77% of the DENV cases against 38% [26,30].These differences may be associated not just with the moderate wMel introgression into wild Ae.aegypti population (average 40%) in Rio de Janeiro after 4-5 years of Wolbachia deployments across the city [30,42], but also to the potential DENV leakage in the saliva of Wolbachia-infected individuals that could reduce the epidemiological effectiveness of this strategy.It is also important to highlight that there are differences in the incidence rate (IR) of dengue cases in these countries.In 2022, Australia had an IR of 1.55 cases of dengue (per 100,000 habitants), while Indonesia showed an IR of 45.7 cases, and Brazil had an impressive IR of 1097 cases [72,73].Therefore, these distinct epidemiological scenarios may explain the different success rates in the reduction of dengue cases in these countries.Continuous monitoring of Wolbachia in the field and dengue cases in humans remains of utmost importance to identify any changes in the epidemiological scenario so action plans can be implemented in time.

Conclusions
This study provides insights into the tripartite interaction involving the wMel strain of Wolbachia, Ae. aegypti mosquitoes and DENV-1 in the highly epidemic scenario of Rio de Janeiro, Brazil.The presence of wMel did not avoid viral infection in mosquitoes' bodies and saliva when subjected to a higher DENV-1 titer, but wMel mosquitoes exhibited lower viral loads than wild mosquitoes.Regarding the lower DENV-1 titer, wMel mosquitoes were less susceptible than wMel-free mosquitoes, but once infected, wMel and wild mosquitoes exhibited similar viral loads in the body and the saliva-microinjected saliva.Furthermore, DENV-1 infection and viral titer in the body or in saliva-microinjected mosquitoes seem to not be related to wMel density.Future studies concerning the vector competence of wMel and wild Ae.aegypti with varied genetic backgrounds are necessary to check the impact on wMel's strategy to replace wild Ae.aegypti populations and diminish arbovirus transmission in the long term.

17 Figure 2 .
Figure 2. Relative density of wMel in Ae. aegypti mosquitoes infected with DENV-1 at low (3 × 10 4 FFU/mL) and high titers (6 × 10 8 FFU/mL).The numbers inside parentheses indicate the number of wMel-positive mosquitoes out of the total mosquitoes tested.Different letters (above the graph) indicate statistically significant differences in wMel density (Wilcoxon-Mann-Whitney test; p < 0.05).Horizontal red bars represent the medians.Yellow circles represent samples infected with wMel from PI1, pink circles from PI2, green circles from FI, and gray circles from wMel-positive control (FI).

Figure 2 .
Figure 2. Relative density of wMel in Ae. aegypti mosquitoes infected with DENV-1 at low (3 × 10 4 FFU/mL) and high titers (6 × 10 8 FFU/mL).The numbers inside parentheses indicate the number of wMel-positive mosquitoes out of the total mosquitoes tested.Different letters (above the graph) indicate statistically significant differences in wMel density (Wilcoxon-Mann-Whitney test; p < 0.05).Horizontal red bars represent the medians.Yellow circles represent samples infected with wMel from PI1, pink circles from PI2, green circles from FI, and gray circles from wMel-positive control (FI).

Figure 3 .
Figure 3. Number of DENV-1 copies in the whole body of wMel, wMel-free, and wild Ae.aegypti mosquitoes 14 days after the infection with the low titer (3 × 10 4 FFU/mL) (A) and the high titer (6 × 10 8 FFU/mL) (B).The numbers inside parentheses indicate the number of DENV-1-positive mosquitoes out of the total mosquitoes tested per area.Different letters (above the graph) indicate statistically significant differences (Wilcoxon-Mann-Whitney test; p < 0.05).Horizontal red bars represent the medians.Blue circles represent wMel-free mosquitoes, red circles represent mosquitoes with wMel, and brown circles represent wild mosquitoes.All negative controls for DENV-1 infection were negative.

Figure 3 .
Figure 3. Number of DENV-1 copies in the whole body of wMel, wMel-free, and wild Ae.aegypti mosquitoes 14 days after the infection with the low titer (3 × 10 4 FFU/mL) (A) and the high titer (6 × 10 8 FFU/mL) (B).The numbers inside parentheses indicate the number of DENV-1-positive mosquitoes out of the total mosquitoes tested per area.Different letters (above the graph) indicate statistically significant differences (Wilcoxon-Mann-Whitney test; p < 0.05).Horizontal red bars represent the medians.Blue circles represent wMel-free mosquitoes, red circles represent mosquitoes with wMel, and brown circles represent wild mosquitoes.All negative controls for DENV-1 infection were negative.

Figure 4 .
Figure 4. DENV-1 load in susceptible wild Ae.aegypti mosquitoes seven days after injecting the saliva of mosquitoes with confirmed infection of DENV-1 in the whole body.Each saliva was intrathoracically injected into 10 susceptible mosquitoes.The injected saliva was obtained from mosquitoes with and without wMel and subjected to DENV-1 oral infections with low (3 × 10 4 FFU/mL) and high titer (6 × 10 8 FFU/mL).The number of DENV-1 infective saliva out of the total number of mosquitoes tested is in parenthesis (below the neighborhood name).Horizontal red bars represent the medians.Different letters (above the graph) indicate statistically significant differences (Wilcoxon-Mann-Whitney test; p < 0.05).All mosquitoes that received saliva from DENV-1-negative mosquitoes were negative.

Figure 4 .
Figure 4. DENV-1 load in susceptible wild Ae.aegypti mosquitoes seven days after injecting the saliva of mosquitoes with confirmed infection of DENV-1 in the whole body.Each saliva was intrathoracically injected into 10 susceptible mosquitoes.The injected saliva was obtained from mosquitoes with and without wMel and subjected to DENV-1 oral infections with low (3 × 10 4 FFU/mL) and high titer (6 × 10 8 FFU/mL).The number of DENV-1 infective saliva out of the total number of mosquitoes tested is in parenthesis (below the neighborhood name).Horizontal red bars represent the medians.Different letters (above the graph) indicate statistically significant differences (Wilcoxon-Mann-Whitney test; p < 0.05).All mosquitoes that received saliva from DENV-1-negative mosquitoes were negative.Viruses 2024, 16, x FOR PEER REVIEW 10 of 17
2 , and it is spatially heterogeneous, with 163 neighborhoods where approximately 6.2 million people live [38].Wolbachia releases first started in 2015 and ceased in 2021, covering 29 neighborhoods that represent 17.8% of the city [39].Niterói is a city connected to Rio de Janeiro by a bridge across Guanabara Bay and has an area of 133 km 2 , with 482 thousand inhabitants and 52 neighborhoods [40].wMel releases first started in Niterói in late 2015, and in 2021 75% of the territory has already received Wolbachia [41].

Table 1 .
DENV-1 infection rate per area observed in wild Ae.aegypti mosquitoes 7 days post saliva microinjection with low titer (3 × 10 4 FFU/mL) and high titer (6 × 10 8 FFU/mL).The numbers inside parentheses indicate the number of DENV-1-positive mosquitoes out of the total mosquitoes tested per area.

Table 1 .
DENV-1 infection rate per area observed in wild Ae.aegypti mosquitoes 7 days post saliva microinjection with low titer (3 × 10 4 FFU/mL) and high titer (6 × 10 8 FFU/mL).The numbers inside parentheses indicate the number of DENV-1-positive mosquitoes out of the total mosquitoes tested per area.