Mosquito species (Diptera: culicidae) collected after tropical storm cristobal in Merida, Yucatan, South-east Mexico.

After the tropical storm Cristobal, we performed special adult entomological collections in the peri-domicile of 35 houses from 25 neighborhoods of Mérida, Yucatan, Mexico in response to complaints from the community about an increased nuisance due to an abundance of mosquitoes. A total of 1,275 specimens from four genera and 13 species were collected: Aedes taeniorhynchus (92%), Culex quinquefasciatus (72%), Aedes aegypti (72%), Psorophora mexicana (36%), Psorophora cyanescens (32%), Aedes scapularis (24%), Culex nigripalpus (24%), Aedes albopictus (8%), Psorophora ferox (4%), Haemagogus equinus (4%), Aedes trivittatus (4%), Culex coronator (4%), Culex iolambdis (4%). From these collections, the increased mosquito nuisance was mainly the result of invasive species such as Aedes taeniorhynchus and Psorophora. City wide, vehicle mounted ULV spraying was performed by the MoH and the municipality of Merida to control adult mosquito populations. We report Culex iolambdis for the first time in Merida and Psorophora mexicana for the state of Yucatan.

Endocytosis of flavivirus NS1 is required for NS1-mediated endothelial hyperpermeability and is abolished by a single N-glycosylation site mutation.

Arthropod-borne flaviviruses cause life-threatening diseases associated with endothelial hyperpermeability and vascular leak. We recently found that vascular leak can be triggered by dengue virus (DENV) non-structural protein 1 (NS1) via the disruption of the endothelial glycocalyx-like layer (EGL). However, the molecular determinants of NS1 required to trigger EGL disruption and the cellular pathway(s) involved remain unknown. Here we report that mutation of a single glycosylated residue of NS1 (N207Q) abolishes the ability of NS1 to trigger EGL disruption and induce endothelial hyperpermeability. Intriguingly, while this mutant bound to the surface of endothelial cells comparably to wild-type NS1, it was no longer internalized, suggesting that NS1 binding and internalization are distinct steps. Using endocytic pathway inhibitors and gene-specific siRNAs, we determined that NS1 was endocytosed into endothelial cells in a dynamin- and clathrin-dependent manner, which was required to trigger endothelial dysfunction in vitro and vascular leak in vivo. Finally, we found that the N207 glycosylation site is highly conserved among flaviviruses and is also essential for West Nile and Zika virus NS1 to trigger endothelial hyperpermeability via clathrin-mediated endocytosis. These data provide critical mechanistic insight into flavivirus NS1-induced pathogenesis, presenting novel therapeutic and vaccine targets for flaviviral diseases.

Protective effect of house screening against indoor Aedes aegypti in Mérida, Mexico: A cluster randomised controlled trial.

OBJECTIVE: To evaluate the protective effect of house screening (HS) on indoor Aedes aegypti infestation, abundance and arboviral infection in Merida, Mexico. METHODS: In 2019, we performed a cluster randomised controlled trial (6 control and 6 intervention areas: 100 households/area). Intervention clusters received permanently fixed fiberglass HS on all windows and doors. The study included two cross-sectional entomologic surveys, one baseline (dry season in May 2019) and one post-intervention (PI, rainy season between September and October 2019). The presence and number of indoor Aedes females and blood-fed females (indoor mosquito infestation) as well as arboviral infections with dengue (DENV) and Zika (ZIKV) viruses were evaluated in a subsample of 30 houses within each cluster. RESULTS: HS houses had significantly lower risk for having Aedes aegypti female mosquitoes (odds ratio [OR] = 0.56, 95% CI 0.33-0.97, p = 0.04) and blood-fed females (OR = 0.53, 95% CI 0.28-0.97, p = 0.04) than unscreened households from the control arm. Compared to control houses, HS houses had significantly lower indoor Ae. aegypti abundance (rate ratio [RR] = 0.50, 95% CI 0.30-0.83, p = 0.01), blood-fed Ae. aegypti females (RR = 0.48, 95% CI 0.27-0.85, p = 0.01) and female Ae. aegypti positive for arboviruses (OR = 0.29, 95% CI 0.10-0.86, p = 0.02). The estimated intervention efficacy in reducing Ae. aegypti arbovirus infection was 71%. CONCLUSIONS: These results provide evidence supporting the use of HS as an effective pesticide-free method to control house infestations with Aedes aegypti and reduce the transmission of Aedes-transmitted viruses such as DENV, chikungunya (CHIKV) and ZIKV.

Zika Virus Nonstructural Protein 1 Disrupts Glycosaminoglycans and Causes Permeability in Developing Human Placentas.

BACKGROUND: During pregnancy, the Zika flavivirus (ZIKV) infects human placentas, inducing defects in the developing fetus. The flavivirus nonstructural protein 1 (NS1) alters glycosaminoglycans on the endothelium, causing hyperpermeability in vitro and vascular leakage in vivo in a tissue-dependent manner. The contribution of ZIKV NS1 to placental dysfunction during ZIKV infection remains unknown. METHODS: We examined the effect of ZIKV NS1 on expression and release of heparan sulfate (HS), hyaluronic acid (HA), and sialic acid on human trophoblast cell lines and anchoring villous explants from first-trimester placentas infected with ZIKV ex vivo. We measured changes in permeability in trophoblasts and stromal cores using a dextran-based fluorescence assay and changes in HA receptor expression using immunofluorescent microscopy. RESULTS: ZIKV NS1 in the presence and absence of ZIKV increased the permeability of anchoring villous explants. ZIKV NS1 induced shedding of HA and HS and altered expression of CD44 and lymphatic endothelial cell HA receptor-1, HA receptors on stromal fibroblasts and Hofbauer macrophages in villous cores. Hyaluronidase was also stimulated in NS1-treated trophoblasts. CONCLUSIONS: These findings suggest that ZIKV NS1 contributes to placental dysfunction via modulation of glycosaminoglycans on trophoblasts and chorionic villi, resulting in increased permeability of human placentas.

Magnitude and Functionality of the NS1-Specific Antibody Response Elicited by a Live-Attenuated Tetravalent Dengue Vaccine Candidate.

BACKGROUND: Dengue virus (DENV) can cause life-threatening disease characterized by endothelial dysfunction and vascular leakage. DENV nonstructural protein 1 (NS1) induces human endothelial hyperpermeability and vascular leak in mice, and NS1 vaccination confers antibody-mediated protective immunity. We evaluated the magnitude, cross-reactivity, and functionality of NS1-specific IgG antibody responses in sera from a phase 2 clinical trial of Takeda's live-attenuated tetravalent dengue vaccine candidate (TAK-003). METHODS: We developed an enzyme-linked immunosorbent assay to measure anti-DENV NS1 IgG in sera from DENV-naive or preimmune subjects pre- and postvaccination with TAK-003 and evaluated the functionality of this response using in vitro models of endothelial permeability. RESULTS: TAK-003 significantly increased DENV-2 NS1-specific IgG in naive individuals, which cross-reacted with DENV-1, -3, and -4 NS1 to varying extents. NS1-induced endothelial hyperpermeability was unaffected by prevaccination serum from naive subjects but was variably inhibited by serum from preimmune subjects. After TAK-003 vaccination, all samples from naive and preimmune vaccinees completely abrogated DENV-2 NS1-induced hyperpermeability and cross-inhibited hyperpermeability induced by DENV-1, -3, and -4 NS1. Inhibition of NS1-induced hyperpermeability correlated with NS1-specific IgG concentrations. Postvaccination sera also prevented NS1-induced degradation of endothelial glycocalyx components. CONCLUSION: We provide evidence for functional NS1-specific IgG responses elicited by a candidate dengue vaccine. CLINICAL TRIALS REGISTRATION: NCT01511250.

Wolbachia in Native Populations of Aedes albopictus (Diptera: Culicidae) From Yucatan Peninsula, Mexico.

This study reports the results of a molecular screening for Wolbachia (Wb) infection in Aedes albopictus (Skuse) populations recently established in the Yucatan Peninsula, Mexico. To do so, collections of free-flying adults with BG traps and emerged adults from eggs after ovitrap field collections were performed in three suburban localities of the city of Merida, Yucatan. Overall, local populations of Ae. albopictus present a natural Wb infection rate of ~40% (18 of 45). Wb infection was detected in both field-collected adults (76.5%, 13 of 17) and eggs reared (17.8%, 5 of 28) and in 37.9% (11/29) of females and 43.7% (7/16) of male Ae. albopictus mosquitoes. An initial screening for Wolbachia strain typing showed that native Ae. albopictus were naturally coinfected with both wAlbA and wAlbB strains. The knowledge of the prevalence and diversity of Wolbachia strains in local populations of Aedes mosquitoes is part of the baseline information required for current and future Wolbachia-based vector control approaches to be conducted in Mexico.

Increased serum sialic acid is associated with morbidity and mortality in a murine model of dengue disease.

Dengue virus (DENV) causes the most prevalent arboviral infection of humans, resulting in a spectrum of outcomes, ranging from asymptomatic infection to dengue fever to severe dengue characterized by vascular leakage and shock. Previously, we determined that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability, disrupts the endothelial glycocalyx layer (EGL) in vitro and triggers shedding of structural components, including sialic acid (Sia) and heparan sulfate. Here, using a murine model of dengue disease disease, we found high levels of Sia and NS1 circulating in mice with DENV-induced morbidity and lethal DENV infection. Further, we developed a liquid chromatography/mass spectrometry-based method for quantifying free Sia in serum and determined that the levels of free N-glycolylneuraminic acid were significantly higher in DENV-infected mice than in uninfected controls. These data provide additional evidence that DENV infection disrupts EGL components in vivo and warrant further research assessing Sia as a biomarker of severe dengue disease.

Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components.

Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components.

Zika Virus Replicates in Proliferating Cells in Explants From First-Trimester Human Placentas, Potential Sites for Dissemination of Infection.

Background: Maternal Zika virus (ZIKV) infection with prolonged viremia leads to fetal infection and congenital Zika syndrome. Previously, we reported that ZIKV infects primary cells from human placentas and fetal membranes. Here, we studied viral replication in numerous explants of anchoring villi and basal decidua from first-trimester human placentas and midgestation amniotic epithelial cells (AmEpCs). Methods: Explants and AmEpCs were infected with American and African ZIKV strains at low multiplicities, and ZIKV proteins were visualized by immunofluorescence. Titers of infectious progeny, cell proliferation, and invasiveness were quantified. Results: In anchoring villus, ZIKV replicated reproducibly in proliferating cytotrophoblasts in proximal cell columns, dividing Hofbauer cells in villus cores, and invasive cytotrophoblasts, but frequencies differed. Cytotrophoblasts in explants infected by Nicaraguan strains were invasive, whereas those infected by prototype MR766 largely remained in cell columns, and titers varied by donor and strain. In basal decidua, ZIKV replicated in glandular epithelium, decidual cells, and immune cells. ZIKV-infected AmEpCs frequently occurred in pairs and expressed Ki67 and phosphohistone H3, indicating replication in dividing cells. Conclusions: ZIKV infection in early pregnancy could target proliferating cell column cytotrophoblasts and Hofbauer cells, amplifying infection in basal decidua and chorionic villi and enabling transplacental transmission.

Longitudinal Analysis of Antibody Cross-neutralization Following Zika Virus and Dengue Virus Infection in Asia and the Americas.

Background: The 4 dengue virus serotypes (DENV1-4) and Zika virus (ZIKV) are related mosquito-borne flaviviruses of major importance globally. While monoclonal antibodies and plasma from DENV-immune donors can neutralize or enhance ZIKV in vitro and in small-animal models, and vice versa, the extent, duration, and significance of cross-reactivity in humans remains unknown, particularly in flavivirus-endemic regions. Methods: We studied neutralizing antibodies to ZIKV and DENV1-4 in longitudinal serologic specimens collected through 3 years after infection from people in Latin America and Asia with laboratory-confirmed DENV infections. We also evaluated neutralizing antibodies to ZIKV and DENV1-4 in patients with Zika through 6 months after infection. Results: In patients with Zika, the highest neutralizing antibody titers were to ZIKV, with low-level cross-reactivity to DENV1-4 that was greater in DENV-immune individuals. We found that, in primary and secondary DENV infections, neutralizing antibody titers to ZIKV were markedly lower than to the infecting DENV and heterologous DENV serotypes. Cross-neutralization was greatest in early convalescence, then ZIKV neutralization decreased, remaining at low levels over time. Conclusions: Patterns of antibody cross-neutralization suggest that ZIKV lies outside the DENV serocomplex. Neutralizing antibody titers can distinguish ZIKV from DENV infections when all viruses are analyzed simultaneously. These findings have implications for understanding natural immunity and vaccines.

Dengue Virus NS1 Disrupts the Endothelial Glycocalyx, Leading to Hyperpermeability.

Dengue is the most prevalent arboviral disease in humans and a major public health problem worldwide. Systemic plasma leakage, leading to hypovolemic shock and potentially fatal complications, is a critical determinant of dengue severity. Recently, we and others described a novel pathogenic effect of secreted dengue virus (DENV) non-structural protein 1 (NS1) in triggering hyperpermeability of human endothelial cells in vitro and systemic vascular leakage in vivo. NS1 was shown to activate toll-like receptor 4 signaling in primary human myeloid cells, leading to secretion of pro-inflammatory cytokines and vascular leakage. However, distinct endothelial cell-intrinsic mechanisms of NS1-induced hyperpermeability remained to be defined. The endothelial glycocalyx layer (EGL) is a network of membrane-bound proteoglycans and glycoproteins lining the vascular endothelium that plays a key role in regulating endothelial barrier function. Here, we demonstrate that DENV NS1 disrupts the EGL on human pulmonary microvascular endothelial cells, inducing degradation of sialic acid and shedding of heparan sulfate proteoglycans. This effect is mediated by NS1-induced expression of sialidases and heparanase, respectively. NS1 also activates cathepsin L, a lysosomal cysteine proteinase, in endothelial cells, which activates heparanase via enzymatic cleavage. Specific inhibitors of sialidases, heparanase, and cathepsin L prevent DENV NS1-induced EGL disruption and endothelial hyperpermeability. All of these effects are specific to NS1 from DENV1-4 and are not induced by NS1 from West Nile virus, a related flavivirus. Together, our data suggest an important role for EGL disruption in DENV NS1-mediated endothelial dysfunction during severe dengue disease.

Primary dengue virus infections induce differential cytokine production in Mexican patients.

Severe dengue pathogenesis is not fully understood, but high levels of proinflammatory cytokines have been associated with dengue disease severity. In this study, the cytokine levels in 171 sera from Mexican patients with primary dengue fever (DF) and dengue haemorrhagic fever (DHF) from dengue virus (DENV) 1 (n = 116) or 2 (n = 55) were compared. DF and DHF were defined according to the patient's clinical condition, the primary infections as indicated by IgG enzymatic immunoassay negative results, and the infecting serotype as assessed by real-time reverse transcription-polymerase chain reaction. Samples were analysed for circulating levels of interleukin (IL)-12p70, interferon (IFN)-γ, tumour necrosis factor (TNF)-α, IL-6, and IL-8 using a commercial cytometric bead array. Significantly higher IFN-γ levels were found in patients with DHF than those with DF. However, significantly higher IL-12p70, TNF-α, and IL-6 levels were associated with DHF only in patients who were infected with DENV2 but not with DENV1. Moreover, patients with DF who were infected with DENV1 showed higher levels of IL-12p70, TNF-α, and IL-6 than patients with DHF early after-fever onset. The IL-8 levels were similar in all cases regardless of the clinical condition or infection serotype. These results suggest that the association between high proinflammatory cytokine levels and dengue disease severity does not always stand, and it once again highlights the complex nature of DHF pathogenesis.

The cytokine response of U937-derived macrophages infected through antibody-dependent enhancement of dengue virus disrupts cell apical-junction complexes and increases vascular permeability.

Severe dengue (SD) is a life-threatening complication of dengue that includes vascular permeability syndrome (VPS) and respiratory distress. Secondary infections are considered a risk factor for developing SD, presumably through a mechanism called antibody-dependent enhancement (ADE). Despite extensive studies, the molecular bases of how ADE contributes to SD and VPS are largely unknown. This work compares the cytokine responses of differentiated U937 human monocytic cells infected directly with dengue virus (DENV) or in the presence of enhancing concentrations of a humanized monoclonal antibody recognizing protein E (ADE-DENV infection). Using a cytometric bead assay, ADE-DENV-infected cells were found to produce significantly higher levels of the proinflammatory cytokines interleukin 6 (IL-6), IL-12p70, and tumor necrosis factor alpha (TNF-α), as well as prostaglandin E2 (PGE2), than cells directly infected. The capacity of conditioned supernatants (conditioned medium [CM]) to disrupt tight junctions (TJs) in MDCK cell cultures was evaluated. Exposure of MDCK cell monolayers to CM collected from ADE-DENV-infected cells (ADE-CM) but not from cells infected directly led to a rapid loss of transepithelial electrical resistance (TER) and to delocalization and degradation of apical-junction complex proteins. Depletion of either TNF-α, IL-6, or IL-12p70 from CM from ADE-DENV-infected cells fully reverted the disrupting effect on TJs. Remarkably, mice injected intraperitoneally with ADE-CM showed increased vascular permeability in sera and lungs, as indicated by an Evans blue quantification assay. These results indicate that the cytokine response of U937-derived macrophages to ADE-DENV infection shows an increased capacity to disturb TJs, while results obtained with the mouse model suggest that such a response may be related to the vascular plasma leakage characteristic of SD.

Prevalence of Flavivirus and A lphavirus in bats captured in the state of Yucatan, southeastern Mexico.

Flavivirus (family Flaviviridae) and Alphavirus (family Togaviridae) are mosquito-borne viruses that poses a significant risk to public health worldwide. Examples of these viruses include Dengue virus (DENV) and Zika virus (ZIKV) in the Flavivirus genus, and Chikungunya virus (CHIKV) in the Alphavirus genus. The potential contribution of bats in the mosquito-to-human transmission cycle of these viral genera in the tropics has not been studied. Here, a total of 144 bats belonging to three families (Emballonuridae, Phyllostomidae, and Molossidae) and six species were captured for one year using mist nets in sites with different landscapes (forest and grassland) in the state of Yucatan, southeastern Mexico. Blood samples and rectal and oral swabs were collected to detect Flavivirus and Alphavirus RNA genomes through RT-PCR. Flavivirus RNA was detected in 53 individuals (36.8%; 95% CI: 29.4%-44.9%), and Alphavirus RNA was detected in 59 individuals (40.1%; 95% CI: 33.2%-49.2%). The sequences obtained were consistent with ZIKV and DENV, into the Flavivirus, and CHIKV into the Alphavirus positive samples. The prevalence of both Flavivirus and Alphavirus was higher during the dry season compared with the rainy season. This high positivity rate, highlighted in both Flavivirus and Alphavirus, suggests a potential contribution of bats in the circulation of these viral genera in sylvatic environments. Seasonal variation in viral genera prevalence, with higher prevalence during dry seasons than rainy seasons, may suggest specific viral activity patterns in response to climatic conditions.

Wolbachia pipientis (Rickettsiales: Rickettsiaceae) mediated effects on the fitness and performance of Aedes aegypti (Diptera: Culicidae) under variable temperatures and initial larval densities.

Wolbachia pipientis (Hertig, 1936), also referred as Wolbachia, is a bacterium present across insect taxa, certain strains of which have been demonstrated to impact the fitness and capacity to transmit viruses in mosquitoes, particularly Aedes aegypti (Linnaeus, 1762). Most studies examine these impacts in limited sets of environmental regimes. Here we seek to understand the impacts of environmentally relevant conditions such as larval density, temperature, and their interaction on wAlbB-infected A. aegypti. Using a factorial design, we measured wAlbB stability (relative density, post-emergence in females, and in progeny), the ability for wAlbB to induce cytoplasmic incompatibility, and bacterial effects on mosquito fitness (fecundity, fertility, and body mass) and performance (adult survival and time to pupation) across 2 temperature regimes (fluctuating and constant) and 2 initial larval densities (low and high). Fluctuating daily regimes of temperature (27 to 40 °C) led to decreased post-emergence wAlbB density and increased wAlbB density in eggs compared to constant temperature (27 °C). An increased fecundity was found in wAlbB-carrying females reared at fluctuating temperatures compared to uninfected wild-type females. wAlbB-carrying adult females showed significantly increased survival than wild-type females. Contrarily, wAlbB-carrying adult males exhibited a significantly lower survival than wild-type males. We found differential effects of assessed treatments (Wolbachia infection status, temperature, and larval density) across mosquito sexes and life stages. Taken together, our results indicate that realistic conditions may not impact dramatically the stability of wAlbB infection in A. aegypti. Nonetheless, understanding the ecological consequence of A. aegypti-wAlbB interaction is complex due to life history tradeoffs under conditions faced by natural populations.

Evidence of Ongoing Transmission of Zika Virus in Mérida, Mexico.

Since the Zika virus (ZIKV) pandemic in 2015-2017, there has been a near absence of reported cases in the Americas outside of Brazil. However, the conditions for Aedes-borne transmission persist in Latin America, and the threat of ZIKV transmission is increasing as population immunity wanes. Mexico has reported only 70 cases of laboratory-confirmed ZIKV infection since 2020, with no cases recorded in the Yucatán peninsula. Here, we provide evidence of active ZIKV transmission, despite the absence of official case reports, in the city of Mérida, Mexico, the capital of the state of Yucatán. Capitalizing on an existing cohort, we detected cases in participants with symptoms consistent with flavivirus infection from 2021 to 2022. Serum samples from suspected cases were tested for ZIKV RNA by polymerase chain reaction or ZIKV-reactive IgM by ELISA. To provide more specific evidence of exposure, focus reduction neutralization tests were performed on ELISA-positive samples. Overall, we observed 25 suspected ZIKV infections for an estimated incidence of 2.8 symptomatic cases per 1,000 persons per year. Our findings emphasize the continuing threat of ZIKV transmission in the setting of decreased surveillance and reporting.

The TIRS trial: Enrollment procedures and baseline characterization of a pediatric cohort to quantify the epidemiologic impact of targeted indoor residual spraying on Aedes-borne viruses in Merida, Mexico.

Aedes mosquito-borne viruses (ABVs) place a substantial strain on public health resources in the Americas. Vector control of Aedes mosquitoes is an important public health strategy to decrease or prevent spread of ABVs. The ongoing Targeted Indoor Residual Spraying (TIRS) trial is an NIH-sponsored clinical trial to study the efficacy of a novel, proactive vector control technique to prevent dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) infections in the endemic city of Merida, Yucatan, Mexico. The primary outcome of the trial is laboratory-confirmed ABV infections in neighborhood clusters. Despite the difficulties caused by the COVID-19 pandemic, by early 2021 the TIRS trial completed enrollment of 4,792 children aged 2-15 years in 50 neighborhood clusters which were allocated to control or intervention arms via a covariate-constrained randomization algorithm. Here, we describe the makeup and ABV seroprevalence of participants and mosquito population characteristics in both arms before TIRS administration. Baseline surveys showed similar distribution of age, sex, and socio-economic factors between the arms. Serum samples from 1,399 children were tested by commercially available ELISAs for presence of anti-ABV antibodies. We found that 45.1% of children were seropositive for one or more flaviviruses and 24.0% were seropositive for CHIKV. Of the flavivirus-positive participants, most were positive for ZIKV-neutralizing antibodies by focus reduction neutralization testing which indicated a higher proportion of participants with previous ZIKV than DENV infections within the cohort. Both study arms had statistically similar seroprevalence for all viruses tested, similar socio-demographic compositions, similar levels of Ae. aegypti infestation, and similar observed mosquito susceptibility to insecticides. These findings describe a population with a high rate of previous exposure to ZIKV and lower titers of neutralizing antibodies against DENV serotypes, suggesting susceptibility to future outbreaks of flaviviruses is possible, but proactive vector control may mitigate these risks.

Targeted indoor residual insecticide applications shift Aedes aegypti age structure and arbovirus transmission potential.

While residual insecticide applications have the potential to decrease pathogen transmission by reducing the density of vectors and shifting the age structure of the adult mosquito population towards younger stages of development, this double entomological impact has not been documented for Aedes aegypti. Aedes collected from households enrolled in a cluster-randomized trial evaluating the epidemiological impact of targeted indoor residual spraying (TIRS) in Merida, Mexico, were dissected and their age structure characterized by the Polovodova combined with Christopher's ovariole growth methods. In total, 813 females were dissected to characterize age structure at 1, 3, 6, and 9 months post-TIRS. Significant differences in the proportion of nulliparous Ae. aegypti females between the treatment groups was found at one-month post-TIRS (control: 35% vs. intervention: 59%), three months (20% vs. 49%) but not at six or nine months post-TIRS. TIRS significantly shiftted Ae. aegypti age structure towards younger stages and led to a non-linear reduction in survivorship compared to the control arm. Reduced survivorship also reduced the number of arbovirus transmitting females (those who survived the extrinsic incubation period). Our findings provide strong evidence of the full entomological impact of TIRS, with important implications for quantifying the epidemiological impact of vector control methods.

Flavivirus NS1 Triggers Tissue-Specific Disassembly of Intercellular Junctions Leading to Barrier Dysfunction and Vascular Leak in a GSK-3ß-Dependent Manner.

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins ß-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of ß-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of ß-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3ß (GSK-3ß). Supporting this model, we found that a chemical inhibitor of GSK-3ß reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3ß as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.

Wolbachia wAlbB remains stable in Aedes aegypti over 15 years but exhibits genetic background-dependent variation in virus blocking.

The ability of the maternally transmitted endosymbiotic bacterium Wolbachia to induce cytoplasmic incompatibility (CI) and virus blocking makes it a promising weapon for combatting mosquito-borne diseases through either suppression or replacement of wild-type populations. Recent field trials show that both approaches significantly reduce the incidence of dengue fever in humans. However, new questions emerge about how Wolbachia-mosquito associations will co-evolve over time and whether Wolbachia-mediated virus blocking will be affected by the genetic diversity of mosquitoes and arboviruses in the real world. Here, we have compared the Wolbachia density and CI expression of two wAlbB-infected Aedes aegypti lines transinfected 15 years apart. We have also assessed wAlbB-mediated virus blocking against dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) viruses and examined whether host genetic backgrounds modulate viral blocking effects by comparing ZIKV infection in mosquitoes with a Mexican genetic background to those with a Singaporean background. Our results show that over 15 years, wAlbB maintained the capacity to form a stable association with Ae. aegypti in terms of both density and CI expression. There were variations in wAlbB-induced virus blocking against CHIKV, DENV, and ZIKV, and higher inhibitory effects on ZIKV in mosquitoes on the Singaporean genetic background than on the Mexican background. These results provide important information concerning the robustness and long-term stability of Wolbachia as a biocontrol agent for arbovirus disease control.