Corrigendum: Intrinsic factors driving mosquito vector competence and viral evolution: a review.

[This corrects the article DOI: 10.3389/fcimb.2023.1330600.].

Sibling species of the major malaria vector Anopheles gambiae display divergent preferences for aquatic breeding sites in southern Nigeria.

BACKGROUND: When integrated with insecticide-treated bed nets, larval control of Anopheles mosquitoes could fast-track reductions in the incidence of human malaria. However, larval control interventions may deliver suboptimal outcomes where the preferred breeding places of mosquito vectors are not well known. This study investigated the breeding habitat choices of Anopheles mosquitoes in southern Nigeria. The objective was to identify priority sites for mosquito larval management in selected urban and periurban locations where malaria remains a public health burden.  METHODS: Mosquito larvae were collected in urban and periurban water bodies during the wet-dry season interface in Edo, Delta, and Anambra States. Field-collected larvae were identified based on PCR gel-electrophoresis and amplicon sequencing, while the associations between Anopheles larvae and the properties and locations of water bodies were assessed using a range of statistical methods. RESULTS: Mosquito breeding sites were either man-made (72.09%) or natural (27.91%) and mostly drainages (48.84%) and puddles (25.58%). Anopheles larvae occurred in drainages, puddles, stream margins, and a concrete well, and were absent in drums, buckets, car tires, and a water-holding iron pan, all of which contained culicine larvae. Wild-caught Anopheles larvae comprised Anopheles coluzzii (80.51%), Anopheles gambiae sensu stricto (s.s.) (11.54%), and Anopheles arabiensis (7.95%); a species-specific PCR confirmed the absence of the invasive urban malaria vector Anopheles stephensi among field-collected larvae. Anopheles arabiensis, An. coluzzii, and An. gambiae s.s. displayed preferences for turbid, lowland, and partially sunlit water bodies, respectively. Furthermore, An. arabiensis preferred breeding sites located outside 500 m of households, whereas An. gambiae s.s. and An. coluzzii had increased detection odds in sites within 500 m of households. Anopheles gambiae s.s. and An. coluzzii were also more likely to be present in natural water bodies; meanwhile, 96.77% of An. arabiensis were in man-made water bodies. Intraspecific genetic variations were little in the dominant vector An. coluzzii, while breeding habitat choices of populations made no statistically significant contributions to these variations. CONCLUSION: Sibling malaria vectors in the An. gambiae complex display divergent preferences for aquatic breeding habitats in southern Nigeria. The findings are relevant for planning targeted larval control of An. coluzzii whose increasing evolutionary adaptations to urban ecologies are driving the proliferation of the mosquito, and An. arabiensis whose adults typically evade the effects of treated bed nets due to exophilic tendencies.

Evaluation of Vector-Enabled Xenosurveillance in Rural Guatemala.

Surveillance methods that permit rapid detection of circulating pathogens in low-resource settings are desperately needed. In this study, we evaluated a mosquito bloodmeal-based surveillance method ("xenosurveillance") in rural Guatemala. Twenty households from two villages (Los Encuentros and Chiquirines) in rural southwest Guatemala were enrolled and underwent weekly prospective surveillance from August 2019 to December 2019 (16 weeks). When febrile illness was reported in a household, recently blood-fed mosquitoes were collected from within dwellings and blood samples taken from each member of the household. Mosquitoes were identified to species and blood sources identified by sequencing. Shotgun metagenomic sequencing was used to identify circulating viruses. Culex pipiens (60.9%) and Aedes aegypti (18.6%) were the most abundant mosquitoes collected. Bloodmeal sources were most commonly human (32.6%) and chicken (31.6%), with various other mammal and avian hosts detected. Several mosquito-specific viruses were detected, including Culex orthophasma virus. Human pathogens were not detected. Therefore, xenosurveillance may require more intensive sampling to detect human pathogens in Guatemala and ecologically similar localities in Central America.

Colorado tick fever virus: a review of historical literature and research emphasis for a modern era.

Colorado tick fever virus is an understudied tick-borne virus of medical importance that is primarily transmitted in the western United States and southwestern Canada. The virus is the type species of the genus Coltivirus (Spinareoviridae) and consists of 12 segments that remain largely uncharacterized. Patterns of viral distribution are driven by the presence of the primary vector, the Rocky Mountain wood tick, Dermacentor andersoni. Infection prevalence in D. andersoni can range from 3% to 58% across the geographic distribution of the tick. Infection in humans can be severe and often presents with fever relapses but is rarely fatal. Here, we review the literature from primary characterizations in the early 20th century to current virus/vector research being conducted and identify vacancies in current research.

Predicted reduction in transmission from deployment of ivermectin-treated birdfeeders for local control of West Nile virus.

Ivermectin (IVM)-treated birds provide the potential for targeted control of Culex mosquitoes to reduce West Nile virus (WNV) transmission. Ingestion of IVM increases mosquito mortality, which could reduce WNV transmission from birds to humans and in enzootic maintenance cycles affecting predominantly bird-feeding mosquitoes and from birds to humans. This strategy might also provide an alternative method for WNV control that is less hampered by insecticide resistance and the logistics of large-scale pesticide applications. Through a combination of field studies and modeling, we assessed the feasibility and impact of deploying IVM-treated birdfeed in residential neighborhoods to reduce WNV transmission. We first tracked 105 birds using radio telemetry and radio frequency identification to monitor their feeder usage and locations of nocturnal roosts in relation to five feeder sites in a neighborhood in Fort Collins, Colorado. Using these results, we then modified a compartmental model of WNV transmission to account for the impact of IVM on mosquito mortality and spatial movement of birds and mosquitoes on the neighborhood level. We found that, while the number of treated lots in a neighborhood strongly influenced the total transmission potential, the arrangement of treated lots in a neighborhood had little effect. Increasing the proportion of treated birds, regardless of the WNV competency status, resulted in a larger reduction in infection dynamics than only treating competent birds. Taken together, model results indicate that deployment of IVM-treated feeders could reduce local transmission throughout the WNV season, including reducing the enzootic transmission prior to the onset of human infections, with high spatial coverage and rates of IVM-induced mortality in mosquitoes. To improve predictions, more work is needed to refine estimates of daily mosquito movement in urban areas and rates of IVM-induced mortality. Our results can guide future field trials of this control strategy.

Tracking antimalarial drug resistance using mosquito blood meals: a cross-sectional study.

BACKGROUND: Strong surveillance systems with wide geographic coverage are needed to detect and respond to reports of antimalarial drug resistance on the African continent. We aimed to assess the utility and feasibility of using blood-fed mosquitos (xenomonitoring) to conduct rapid surveillance of molecular markers associated with resistance in human populations. METHODS: We conducted three cross-sectional surveys in two rainy seasons and the interim dry season in southwest Burkina Faso between Oct 10, 2018, and Sept 17, 2019. We collected human blood samples and blood-fed mosquitos residing in household clusters across seven village sectors. Samples were assessed for Plasmodium falciparum with ultrasensitive quantitative PCR, genotyped for two markers of reduced drug susceptibility, pfmdr1 256A>T (Asn86Tyr) and pfcrt 227A>C (Lys76Thr), and sequenced for four markers of clonality. We assessed statistical equivalence using a 10% margin of equivalence. FINDINGS: We identified 551 infections in 1483 human blood samples (mean multiplicity of infection [MOI] 1·94, SD 1·47) and 346 infections in 2151 mosquito blood meals (mean MOI 2·2, SD 1·67). The frequency of pfmdr1 Asn86Tyr was 4% in survey 1, 2% in survey 2, and 12% in survey 3 in human samples, and 3% in survey 1, 0% in survey 2, and 8% in survey 3 in mosquito blood meals, and inter-host frequencies were statistically equivalent in surveys 1 and 2 (p<0·0001) but not Survey 3 (p=0·062) within a tolerability of 0·10. The frequency of pfcrt Lys76Thr was 16% in survey 1, 55% in survey 2, and 11% in survey 3 in humans and 40% in survey 1, 72% in survey 2, and 13% in survey 3 in mosquitos, and inter-host frequencies were equivalent in survey 3 only (p=0·032) within a tolerability of 0·10. In simulations, multiple but not preferential feeding behaviour in mosquitos reduced the accuracy of frequency estimates between hosts, particularly for markers circulating at higher frequencies. INTERPRETATION: Molecular markers in mosquito blood meals and in humans exhibited similar temporal trends but frequencies were not statistically equivalent in all scenarios. More work is needed to determine empirical and pragmatic thresholds of difference. Xenomonitoring might be an efficient tool to provide rapid information on emerging antimalarial resistance in regions with insufficient surveillance. FUNDING: National Institute of Allergy and Infectious Diseases. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

Repeat Ivermectin Mass Drug Administrations for Malaria Control II: Protocol for a Double-blind, Cluster-Randomized, Placebo-Controlled Trial for the Integrated Control of Malaria.

BACKGROUND: The gains made against malaria have stagnated since 2015, threatened further by increasing resistance to insecticides and antimalarials. Improvement in malaria control necessitates a multipronged strategy, which includes the development of novel tools. One such tool is mass drug administration (MDA) with endectocides, primarily ivermectin, which has shown promise in reducing malaria transmission through lethal and sublethal impacts on the mosquito vector. OBJECTIVE: The primary objective of the study is to assess the impact of repeated ivermectin MDA on malaria incidence in children aged ≤10 years. METHODS: Repeat Ivermectin MDA for Malaria Control II is a double-blind, placebo-controlled, cluster-randomized, and parallel-group trial conducted in a setting with intense seasonal malaria transmission in Southwest Burkina Faso. The study included 14 discrete villages: 7 (50%) randomized to receive standard measures (seasonal malaria chemoprevention [SMC] and bed net use for children aged 3 to 59 months) and placebo, and 7 (50%) randomized to receive standard measures and monthly ivermectin MDA at 300 µg/kg for 3 consecutive days, provided under supervision to all eligible village inhabitants, over 2 successive rainy seasons. Nonpregnant individuals >90 cm in height were eligible for ivermectin MDA, and cotreatment with ivermectin and SMC was not permitted. The primary outcome is malaria incidence in children aged ≤10 years, as assessed by active case surveillance. The secondary safety outcome of repeated ivermectin MDA was assessed through active and passive adverse event monitoring. RESULTS: The trial intervention was conducted from July to November in 2019 and 2020, with additional sampling of humans and mosquitoes occurring through February 2022 to assess postintervention changes in transmission patterns. Additional human and entomological assessments were performed over the 2 years in a subset of households from 6 cross-sectional villages. A subset of individuals underwent additional sampling in 2020 to characterize ivermectin pharmacokinetics and pharmacodynamics. Analysis and unblinding will commence once the database has been completed, cleaned, and locked. CONCLUSIONS: Our trial represents the first study to directly assess the impact of a novel approach for malaria control, ivermectin MDA as a mosquitocidal agent, layered into existing standard-of-care interventions. The study was designed to leverage the current SMC deployment infrastructure and will provide evidence regarding the additional benefit of ivermectin MDA in reducing malaria incidence in children. TRIAL REGISTRATIONS: ClinicalTrials.gov NCT03967054; https://clinicaltrials.gov/ct2/show/NCT03967054 and Pan African Clinical Trials Registry PACT201907479787308; https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=8219. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/41197.

Intrinsic factors driving mosquito vector competence and viral evolution: a review.

Mosquitoes are responsible for the transmission of numerous viruses of global health significance. The term "vector competence" describes the intrinsic ability of an arthropod vector to transmit an infectious agent. Prior to transmission, the mosquito itself presents a complex and hostile environment through which a virus must transit to ensure propagation and transmission to the next host. Viruses imbibed in an infectious blood meal must pass in and out of the mosquito midgut, traffic through the body cavity or hemocoel, invade the salivary glands, and be expelled with the saliva when the vector takes a subsequent blood meal. Viruses encounter physical, cellular, microbial, and immunological barriers, which are influenced by the genetic background of the mosquito vector as well as environmental conditions. Collectively, these factors place significant selective pressure on the virus that impact its evolution and transmission. Here, we provide an overview of the current state of the field in understanding the mosquito-specific factors that underpin vector competence and how each of these mechanisms may influence virus evolution.

Back to the Future: Quantifying Wing Wear as a Method to Measure Mosquito Age.

Vector biologists have long sought the ability to accurately quantify the age of wild mosquito populations, a metric used to measure vector control efficiency. This has proven difficult due to the difficulties of working in the field and the biological complexities of wild mosquitoes. Ideal age grading techniques must overcome both challenges while also providing epidemiologically relevant age measurements. Given these requirements, the Detinova parity technique, which estimates age from the mosquito ovary and tracheole skein morphology, has been most often used for mosquito age grading despite significant limitations, including being based solely on the physiology of ovarian development. Here, we have developed a modernized version of the original mosquito aging method that evaluated wing wear, expanding it to estimate mosquito chronological age from wing scale loss. We conducted laboratory experiments using adult Anopheles gambiae held in insectary cages or mesocosms, the latter of which also featured ivermectin bloodmeal treatments to change the population age structure. Mosquitoes were age graded by parity assessments and both human- and computational-based wing evaluations. Although the Detinova technique was not able to detect differences in age population structure between treated and control mesocosms, significant differences were apparent using the wing scale technique. Analysis of wing images using averaged left- and right-wing pixel intensity scores predicted mosquito age at high accuracy (overall test accuracy: 83.4%, average training accuracy: 89.7%). This suggests that this technique could be an accurate and practical tool for mosquito age grading though further evaluation in wild mosquito populations is required.

Two-year Decay of Zika Virus Neutralizing Antibodies in People Living in an Endemic Region in Brazil.

It is currently not clear whether humoral immunity to Zika virus (ZIKV) elicited upon natural ZIKV infection is long-lasting. In addition, cross-reactivity of anti-ZIKV antibodies with antigenically related dengue viruses (DENV) may have biological implications in nonnaive individuals who subsequently acquire a heterotypic infection. Cross-reactive humoral immunity between ZIKV and DENV also complicates the interpretation of serological tests to evaluate previous exposure to either virus. Here, we have measured the 2-year decay of ZIKV neutralizing antibodies in people living in a ZIKV/DENV endemic area in Brazil who were identified as having an acute infection (group 1) or past (but recent) infection (group 2) with ZIKV in 2015-16. The titers of neutralizing antibodies to ZIKV decreased 9.1 and 2.3 times in groups 1 and 2, respectively. We also show that the plaque reduction neutralization assay (PRNT) is a reliable method to measure past exposure to ZIKV in coendemic areas.

Effects of ivermectin treatment of backyard chickens on mosquito dynamics and West Nile virus transmission.

BACKGROUND: Vector control strategies typically rely on pesticides to target mosquitoes involved in enzootic and zoonotic transmission of West Nile virus (WNV). Nevertheless, increasing insecticide resistance and a desire to reduce pesticide usage provide the impetus for developing alternative strategies. Ivermectin (IVM), an antiparasitic drug which is widely used in human and veterinary medicine, is a potential alternative for targeted control because Culex mosquitoes experience increased mortality following ingestion of IVM in bloodmeals. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a randomized field trial to investigate the impact of treating backyard chicken flocks with IVM in urban neighborhoods across Davis, California on mosquito populations and WNV transmission dynamics. We observed a significant reduction in WNV seroconversions in treated vs. untreated chickens, suggesting a reduction in WNV transmission intensity around treated flocks. We also detected a reduction in parity rates of Cx. tarsalis near treated vs. untreated flocks and increased mortality in wild mosquitoes following a bloodmeal on treated chickens (IVM serum concentration > 5ng/mL) vs. chickens with IVM serum concentrations < 5 ng/mL. However, we did not find a significant difference in abundance or infection prevalence in mosquitoes between treatment groups associated with the reductions in seroconversions. Mosquito immigration from surrounding larval habitat, relatively low WNV activity in the study area, and variable IVM serum concentrations likely contributed to uncertainty about the impact. CONCLUSIONS/SIGNIFICANCE: Taken together, our results point to a reduction in WNV transmission due to the impact of IVM on Culex mosquito populations and support the ongoing investigation of oral administration of IVM to wild birds for local control of WNV transmission, although further work is needed to optimize dosing and understand effects on entomological endpoints.

Characterization of subclinical ZIKV infection in immune-competent guinea pigs and mice.

An infectious agent's pathogenic and transmission potential is heavily influenced by early events during the asymptomatic or subclinical phase of disease. During this phase, the presence of infectious agent may be relatively low. An important example of this is Zika virus (ZIKV), which can cross the placenta and infect the foetus, even in mothers with subclinical infections. These subclinical infections represent roughly 80 % of all human infections. Initial ZIKV pathogenesis studies were performed in type I interferon receptor (IFNAR) knockout mice. Blunting the interferon response resulted in robust infectivity, and increased the utility of mice to model ZIKV infections. However, due to the removal of the interferon response, the use of these models impedes full characterization of immune responses to ZIKV-related pathologies. Moreover, IFNAR-deficient models represent severe disease whereas less is known regarding subclinical infections. Investigation of the anti-viral immune response elicited at the maternal-foetal interface is critical to fully understand mechanisms involved in foetal infection, foetal development, and disease processes recognized to occur during subclinical maternal infections. Thus, immunocompetent experimental models that recapitulate natural infections are needed. We have established subclinical intravaginal ZIKV infections in mice and guinea pigs. We found that these infections resulted in: the presence of both ZIKV RNA transcripts and infectious virus in maternal and placental tissues, establishment of foetal infections and ZIKV-mediated CXCL10 expression. These models will aid in discerning the mechanisms of subclinical ZIKV mother-to-offspring transmission, and by extension can be used to investigate other maternal infections that impact foetal development.

Three Immunocompetent Small Animal Models That Do Not Support Zika Virus Infection.

Zika virus (ZIKV) is a mosquito-borne flavivirus that is primarily transmitted to humans through the bite of an infected mosquito. ZIKV causes disease in infected humans with added complications of Guillain-Barré syndrome and birth defects in infants born to mothers infected during pregnancy. There are several large immunocompetent animal models for ZIKV including non-human primates (NHPs). NHP models closely reflect human infection; however, due to sample size restrictions, investigations into the effects of transmission route and the impacts on disease dynamics have been understudied. Mice have been widely used for modeling ZIKV infection, yet there are few ZIKV-susceptible immunocompetent mouse models and none of these have been used to investigate sexual transmission. In an effort to identify a small immunocompetent animal model to characterize sexual transmission of ZIKV, we attempt experimental infection of multimammate mice, New Zealand white rabbits, and Hartley guinea pigs. The multimammate mouse is the natural reservoir of Lassa fever virus and has been identified to harbor other human pathogens. Likewise, while NZW rabbits are susceptible to West Nile virus, they have not yet been examined for their susceptibility to infection with ZIKV. Guinea pigs have been successfully used as models for ZIKV infection, but only in immunocompromised life stages (young or pregnant). Here, it was found that the multimammate mouse and New Zealand White (NZW) rabbits are not susceptible ZIKV infection as determined by a lack viral RNA in tissues and fluids collected. Sexually mature male Hartley guinea pigs were inoculated subcutaneously and by mosquito bite, but found to be refractory to ZIKV infection, contrary to findings of other studies in young and pregnant guinea pigs. Interestingly, here it is shown that adult male guinea pigs are not susceptible to ZIKV infection, even when infected by natural route (e.g., mosquito bite). Although a new small animal model for the sexual transmission for ZIKV was not established through this study, these findings provide information on outbred animal species that are not permissive to infection (NZW rabbits and multimammate mice) and new information surrounding limitations of a previously established animal model (guinea pigs).

Design and analysis of a 2-year parallel follow-up of repeated ivermectin mass drug administrations for control of malaria: Small sample considerations for cluster-randomized trials with count data.

BACKGROUND: Cluster-randomized trials allow for the evaluation of a community-level or group-/cluster-level intervention. For studies that require a cluster-randomized trial design to evaluate cluster-level interventions aimed at controlling vector-borne diseases, it may be difficult to assess a large number of clusters while performing the additional work needed to monitor participants, vectors, and environmental factors associated with the disease. One such example of a cluster-randomized trial with few clusters was the "efficacy and risk of harms of repeated ivermectin mass drug administrations for control of malaria" trial. Although previous work has provided recommendations for analyzing trials like repeated ivermectin mass drug administrations for control of malaria, additional evaluation of the multiple approaches for analysis is needed for study designs with count outcomes. METHODS: Using a simulation study, we applied three analysis frameworks to three cluster-randomized trial designs (single-year, 2-year parallel, and 2-year crossover) in the context of a 2-year parallel follow-up of repeated ivermectin mass drug administrations for control of malaria. Mixed-effects models, generalized estimating equations, and cluster-level analyses were evaluated. Additional 2-year parallel designs with different numbers of clusters and different cluster correlations were also explored. RESULTS: Mixed-effects models with a small sample correction and unweighted cluster-level summaries yielded both high power and control of the Type I error rate. Generalized estimating equation approaches that utilized small sample corrections controlled the Type I error rate but did not confer greater power when compared to a mixed model approach with small sample correction. The crossover design generally yielded higher power relative to the parallel equivalent. Differences in power between analysis methods became less pronounced as the number of clusters increased. The strength of within-cluster correlation impacted the relative differences in power. CONCLUSION: Regardless of study design, cluster-level analyses as well as individual-level analyses like mixed-effects models or generalized estimating equations with small sample size corrections can both provide reliable results in small cluster settings. For 2-year parallel follow-up of repeated ivermectin mass drug administrations for control of malaria, we recommend a mixed-effects model with a pseudo-likelihood approximation method and Kenward-Roger correction. Similarly designed studies with small sample sizes and count outcomes should consider adjustments for small sample sizes when using a mixed-effects model or generalized estimating equation for analysis. Although the 2-year parallel follow-up of repeated ivermectin mass drug administrations for control of malaria is already underway as a parallel trial, applying the simulation parameters to a crossover design yielded improved power, suggesting that crossover designs may be valuable in settings where the number of available clusters is limited. Finally, the sensitivity of the analysis approach to the strength of within-cluster correlation should be carefully considered when selecting the primary analysis for a cluster-randomized trial.

Nootkatone Is an Effective Repellent against Aedes aegypti and Aedes albopictus.

We tested a nootkatone product for insecticide activity against the most prominent vectors of Zika virus (ZIKV), Aedes aegypti, and Aedes albopictus. We tested the permethrin-resistant (PERM-R) Vergel strain of A. aegypti and the permethrin-susceptible (PERM-S) New Orleans strain of A. aegypti to determine if insecticide resistance affected their susceptibility to nootkatone. Bottle bioassays showed that the PERM-S strain (New Orleans) was more susceptible to nootkatone than the confirmed A. aegypti permethrin-resistant (PERM-R) strain, Vergel. The A. albopictus strain ATM-NJ95 was a known PERM-S strain and Coatzacoalcos permethrin susceptibility was unknown but proved to be similar to the ATM-NJ95 PERM-S phenotype. The A. albopictus strains (ATM-NJ95 and Coatzacoalcos) were as susceptible to nootkatone as the New Orleans strain. Bottle bioassays conducted with ZIKV-infected mosquitoes showed that the New Orleans (PERM-S) strain was as susceptible to nootkatone as the mock-infected controls, but the PERM-R strain was less susceptible to nootkatone than the mock-infected controls. Repellency/irritancy and biting inhibition bioassays (RIBB) of A. aegypti determined whether the nootkatone-treated arms of three human subjects prevented uninfected A. aegypti mosquitoes from being attracted to the test subjects and blood-feeding on them. The RIBB analyses data calculated the spatial activity index (SAI) and biting inhibition factor (BI) of A. aegypti at different nootkatone concentrations and then compared the SAI and BI of existing repellency products. We concluded that nootkatone repelled mosquitoes at a rate comparable to 7% DEET or 5% picaridin and has the potential to be an efficacious repellent against adult A. aegypti mosquitoes.

Characterizing and Quantifying Arbovirus Transmission by Aedes aegypti Using Forced Salivation and Analysis of Bloodmeals.

Arbovirus transmission studies are dependent on the ability to estimate the titer of virus transmitted from infectious mosquitoes to a host. There are several methods for estimating virus titer in mosquito saliva, including (1) using forced salivation (FS) whereby the infectious mosquito's proboscis is forced into a capillary tube containing media to collect and test their saliva for virus, and (2) by quantifying virus expectorated into host tissues or into the blood contained in an artificial feeder immediately after blood feeding. We studied FS and bloodmeals to estimate and compare titers of Zika virus and chikungunya virus transmitted by the mosquito vector Aedes aegypti. Infectious virus and viral genomes of both viruses were detected more often from individual mosquitoes using immersion oil for the FS media compared to fetal bovine serum (FBS) plus glycerol, but the FS media had no influence on virus quantification from positive samples. FS virus titers were equivalent when comparing individuals or groups of mosquitoes that never received a blood meal compared to those that were blood fed immediately prior, showing that blood feeding does not influence FS. This suggested that performing FS on mosquitoes after blood feeding might be an efficient way to estimate virus transmitted during blood feeding. However, detecting virus from the blood remaining in an artificial feeder post-blood feeding was mostly unsuccessful relative to quantifying virus from FS of the post-blood fed mosquitoes. In contrast, immunocompromised mice always became infected after being fed on by Zika-infected mosquitoes, even when no infectious virus was detected in their saliva by FS post-blood feed. Due to this discrepancy, we tested the ingested bloodmeals of individual mosquitoes that fed on artificial blood feeders for virus, and compared these to virus in their saliva harvested from FS and to virus in their bodies. These experiments revealed ~50-100 times higher virus titers in the dissected bloodmeals compared to those detected in the same mosquitoes' saliva, demonstrating how mosquitoes re-ingest much of their saliva during artificial blood feeding, and highlighting a large increase in virus transmission during Aedes aegypti blood feeding. Both FS and the dissected bloodmeals of artificially blood-fed mosquitoes showed that the quantity of viral RNA expectorated by mosquitoes was 2-5 logs more than the quantity of infectious virus. The results from this study add critical information to understanding and quantifying the transmission of Aedes aegypti arboviruses.

Follow-Up Household Serosurvey in Northeast Brazil for Zika Virus: Sexual Contacts of Index Patients Have the Highest Risk for Seropositivity.

BACKGROUND: Zika virus (ZIKV) is a mosquito-borne virus that is also transmitted sexually; however, the epidemiological relevance of ZIKV sexual transmission in endemic regions is unclear. METHODS: We performed a household-based serosurvey in Northeast Brazil to evaluate the differential exposure to ZIKV and chikungunya virus (CHIKV) among households. Individuals who participated in our previous arboviral disease cohort (indexes) were recontacted and enrolled, and their household members were newly enrolled. RESULTS: The relative risk of sexual partners being ZIKV-seropositive when living with a ZIKV-seropositive index participant was significantly higher, whereas this was not observed among nonsexual partners of the index. For CHIKV, both sexual and nonsexual partner household members living with a CHIKV-seropositive index had a significantly higher risk of being seropositive. In the nonindex-based dyadic and generalized linear mixed model analyses, the odds of sexual dyads having a concordant ZIKV plaque reduction neutralization test result was significantly higher. We have also analyzed retrospective clinical data according to the participants' exposure to ZIKV and CHIKV. CONCLUSIONS: Our data suggest that ZIKV sexual transmission may be a key factor for the high ZIKV seroprevalence among households in endemic areas and raises important questions about differential disease from the 2 modes of transmission.

Considerations for Human Blood-Feeding and Arthropod Exposure in Vector Biology Research: An Essential Tool for Investigations and Disease Control.

Eventually there may be a broadly acceptable, even perfected, substitute for the human host requirement for direct feeding experiments by arthropods, most notably mosquitoes. However, for now, direct and indirect feeding on human volunteers is an important, if not essential, tool in vector biology research (VBR). This article builds on the foundational publication by Achee et al. (2015) covering considerations for the use of human participants in VBR pursuits. The authors introduced methods involving human participation in VBR, while detailing human-landing collections (catches) as a prime example. Benedict et al. (2018) continued this theme with an overview of human participation and considerations for research that involves release of mosquito vectors into the environment. In this study, we discuss another important aspect of human use in VBR activities: considerations addressing studies that require an arthropod to feed on a live human host. Using mosquito studies as our principal example, in this study, we discuss the tremendous importance and value of this approach to support and allow study of a wide variety of factors and interactions related to our understanding of vector-borne diseases and their control. This includes establishment of laboratory colonies for test populations, characterization of essential nutrients that contribute to mosquito fitness, characterization of blood-feeding (biting) behavior and pathogen transmission, parameterization for modeling transmission dynamics, evaluation of human host attraction and/or agents that repel, and the effectiveness of antivector or parasite therapeutic drug studies.

Partitiviruses Infecting Drosophila melanogaster and Aedes aegypti Exhibit Efficient Biparental Vertical Transmission.

Partitiviruses are segmented, multipartite double-stranded RNA (dsRNA) viruses that until recently were only known to infect fungi, plants, and protozoans. Metagenomic surveys have revealed that partitivirus-like sequences are also commonly associated with arthropods. One arthropod-associated partitivirus, galbut virus, is common in wild populations of Drosophila melanogaster To begin to understand the processes that underlie this virus's high global prevalence, we established colonies of wild-caught infected flies. Infection remained at stably high levels over 3 years, with between 63 and 100% of individual flies infected. Galbut virus infects fly cells and replicates in tissues throughout infected adults, including reproductive tissues and the gut epithelium. We detected no evidence of horizontal transmission via ingestion, but vertical transmission from either infected females or infected males was ∼100% efficient. Vertical transmission of a related partitivirus, verdadero virus, that we discovered in a laboratory colony of Aedes aegypti mosquitoes was similarly efficient. This suggests that efficient biparental vertical transmission may be a feature of at least a subset of insect-infecting partitiviruses. To study the impact of galbut virus infection free from the confounding effect of other viruses, we generated an inbred line of flies with galbut virus as the only detectable virus infection. We were able to transmit infection experimentally via microinjection of homogenate from these galbut-only flies. This sets the stage for experiments to understand the biological impact and possible utility of partitiviruses infecting model organisms and disease vectors.IMPORTANCE Galbut virus is a recently discovered partitivirus that is extraordinarily common in wild populations of the model organism Drosophila melanogaster Like for most viruses discovered through metagenomics, most of the basic biological questions about this virus remain unanswered. We found that galbut virus, along with a closely related partitivirus found in Aedes aegypti mosquitoes, is transmitted from infected females or males to offspring with ∼100% efficiency and can be maintained in laboratory colonies over years. This efficient transmission mechanism likely underlies the successful spread of these viruses through insect populations. We created Drosophila lines that contained galbut virus as the only virus infection and showed that these flies can be used as a source for experimental infections. This provides insight into how arthropod-infecting partitiviruses may be maintained in nature and sets the stage for exploration of their biology and potential utility.