Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Artificial Feeding Systems for Vector-Borne Disease Studies.

This review examines the advancements and methodologies of artificial feeding systems for the study of vector-borne diseases, offering a critical assessment of their development, advantages, and limitations relative to traditional live host models. It underscores the ethical considerations and practical benefits of such systems, including minimizing the use of live animals and enhancing experimental consistency. Various artificial feeding techniques are detailed, including membrane feeding, capillary feeding, and the utilization of engineered biocompatible materials, with their respective applications, efficacy, and the challenges encountered with their use also being outlined. This review also forecasts the integration of cutting-edge technologies like biomimicry, microfluidics, nanotechnology, and artificial intelligence to refine and expand the capabilities of artificial feeding systems. These innovations aim to more accurately simulate natural feeding conditions, thereby improving the reliability of studies on the transmission dynamics of vector-borne diseases. This comprehensive review serves as a foundational reference for researchers in the field, proposing a forward-looking perspective on the potential of artificial feeding systems to revolutionize vector-borne disease research.

Erratum: AC-DC Electropenetrography as a Tool to Quantify Probing and Ingestion Behaviors of the Yellow Fever Mosquito (Aedes aegypti) on Mice in Biocontainment.

This corrects the article DOI: 10.30802/AALAS-CM-23-000037
When the above article was first published in the Vol 3 No 6 (December 2023) issue of Comparative Medicine, figure images were incorrectly associated with the figure legends. The correct version of this article has been reprinted in full in volume 74, issue 1 of the February issue of Comparative Medicine.
The publisher apologizes for this error and any inconvenience caused.

Neurodevelopmental assessment of normocephalic children born to Zika virus exposed and unexposed pregnant people.

BACKGROUND: Studies examining the association between in utero Zika virus (ZIKV) exposure and child neurodevelopmental outcomes have produced varied results. METHODS: We aimed to assess neurodevelopmental outcomes among normocephalic children born from pregnant people enrolled in the Zika in Pregnancy in Honduras (ZIPH) cohort study, July-December 2016. Enrollment occurred during the first prenatal visit. Exposure was defined as prenatal ZIKV IgM and/or ZIKV RNA result at enrollment. Normocephalic children, >6 months old, were selected for longitudinal follow-up using the Bayley Scales of Infant and Toddler Development (BSID-III) and the Ages & Stages Questionnaires: Social-Emotional (ASQ:SE-2). RESULTS: One hundred fifty-two children were assessed; after exclusion, 60 were exposed and 72 were unexposed to ZIKV during pregnancy. Twenty children in the exposed group and 21 children in the unexposed group had a composite score <85 in any of the BSID-III domains. Although exposed children had lower cognitive and language scores, differences were not statistically significant. For ASQ:SE-2 assessment, there were not statistically significant differences between groups. CONCLUSIONS: This study found no statistically significant differences in the neurodevelopment of normocephalic children between in utero ZIKV exposed and unexposed. Nevertheless, long-term monitoring of children with in utero ZIKV exposure is warranted. IMPACT: This study found no statistically significant differences in the neurodevelopment in normocephalic children with in utero Zika virus exposure compared to unexposed children, although the exposed group showed lower cognitive and language scores that persisted after adjustment by maternal age and education and after excluding children born preterm and low birth weight from the analysis. Children with prenatal Zika virus exposure, including those normocephalic and have no evidence of abnormalities at birth, should be monitored for neurodevelopmental delays. Follow-up is important to be able to detect developmental abnormalities that might not be detected earlier in life.

Development of an automated biomaterial platform to study mosquito feeding behavior.

Mosquitoes carry a number of deadly pathogens that are transmitted while feeding on blood through the skin, and studying mosquito feeding behavior could elucidate countermeasures to mitigate biting. Although this type of research has existed for decades, there has yet to be a compelling example of a controlled environment to test the impact of multiple variables on mosquito feeding behavior. In this study, we leveraged uniformly bioprinted vascularized skin mimics to create a mosquito feeding platform with independently tunable feeding sites. Our platform allows us to observe mosquito feeding behavior and collect video data for 30-45 min. We maximized throughput by developing a highly accurate computer vision model (mean average precision: 92.5%) that automatically processes videos and increases measurement objectivity. This model enables assessment of critical factors such as feeding and activity around feeding sites, and we used it to evaluate the repellent effect of DEET and oil of lemon eucalyptus-based repellents. We validated that both repellents effectively repel mosquitoes in laboratory settings (0% feeding in experimental groups, 13.8% feeding in control group, p < 0.0001), suggesting our platform's use as a repellent screening assay in the future. The platform is scalable, compact, and reduces dependence on vertebrate hosts in mosquito research.

Aedes aegypti and Aedes albopictus (Diptera: Culicidae) Oviposition Activity and the Associated Socio-environmental Factors in the New Orleans Area.

The transmission of Aedes-borne viruses is on the rise globally. Their mosquito vectors, Aedes aegypti (Linnaeus, Diptera: Culicidae) and Ae. albopictus (Skuse, Diptera: Culicidae), are focally abundant in the Southern United States. Mosquito surveillance is an important component of a mosquito control program. However, there is a lack of long-term surveillance data and an incomplete understanding of the factors influencing vector populations in the Southern United States. Our surveillance program monitored Ae. aegypti and Ae. albopictus oviposition intensity in the New Orleans area using ovicups in a total of 75 sites from 2009 to 2016. We found both Aedes spp. throughout the study period and sites. The average number of Ae. aegypti and Ae. albopictus hatched from collected eggs per site per week was 34.1 (SD = 57.7) and 29.0 (SD = 46.5), respectively. Based on current literature, we formed multiple hypotheses on how environmental variables influence Aedes oviposition intensity, and constructed Generalized Linear Mixed Effect models with a negative binomial distribution and an autocorrelation structure to test these hypotheses. We found significant associations between housing unit density and Ae. aegypti and Ae. albopictus oviposition intensity, and between median household income and Ae. albopictus oviposition intensity. Temperature, relative humidity, and accumulated rainfall had either a lagged or an immediate significant association with oviposition. This study provides the first long-term record of Aedes spp. distribution in the New Orleans area, and sheds light on factors associated with their oviposition activity. This information is vital for the control of potential Aedes-borne virus transmission in this area.

Microcephaly Outcomes among Zika Virus-Infected Pregnant Women in Honduras.

The impact of Zika virus (ZIKV) infection on pregnancies shows regional variation emphasizing the importance of studies in different geographical areas. We conducted a prospective study in Tegucigalpa, Honduras, recruiting 668 pregnant women between July 20, 2016, and December 31, 2016. We performed Trioplex real-time reverse transcriptase-PCR (rRT-PCR) in 357 serum samples taken at the first prenatal visit. The presence of ZIKV was confirmed in seven pregnancies (7/357, 2.0%). Nine babies (1.6%) had microcephaly (head circumference more than two SDs below the mean), including two (0.3%) with severe microcephaly (head circumference [HC] more than three SDs below the mean). The mothers of both babies with severe microcephaly had evidence of ZIKV infection. A positive ZIKV Trioplex rRT-PCR was associated with a 33.3% (95% CI: 4.3-77.7%) risk of HC more than three SDs below the mean.

Factors associated with Chikungunya infection in a cohort of women aged 15-39 y in Fortaleza, Brazil.

BACKGROUND: Outbreaks of Chikungunya virus (CHIKV) occurred in Brazil during 2015-2017. Fortaleza was the city that reported the most cases. METHODS: The first round of a cohort study was conducted among women aged 15-39 y in Fortaleza, Brazil, in 2018 (Zika in Fortaleza). We collected sera to detect CHIKV IgG and IgM antibodies. Factors for CHIKV infection were identified using a Poisson regression model. RESULTS: We evaluated 1466 serum samples and 13.8% and 37.2% of women were found positive for CHIKV IgM and IgG antibodies, respectively. Living with more than four others in the same house and having an abandoned house nearby were associated with CHIKV infection. Being currently pregnant was associated with a decreased probability of CHIKV infection, which was also associated with pregnant women reporting using more repellent, both inside and outside the house, than non-pregnant women. CONCLUSIONS: Crowding in households and abandoned houses nearby can increase potential transmission. Policies providing better living conditions and regulation of abandoned sites and buildings are necessary to control the mosquito population. Programmes providing repellant at low or no cost to pregnant women should be implemented in the neighbourhoods where arbovirus infections are endemic.

Effects of Ascogregarina culicis (Eugregarinorida: Lecudinidae) on Mosquito Size and Dengue Virus Infection in Aedes aegypti (Diptera: Culicidae).

Dengue virus infection, transmitted via mosquito bites, poses a substantial risk to global public health. Studies suggest that the mosquito's microbial community can profoundly influence vector-borne pathogen transmissions, including dengue virus. Ascogregarina culicis (Ross) of the phylum Apicomplexa is among the most common parasites of Aedes aegypti (Linnaeus), the principal vector of dengue. Despite a high prevalence worldwide, including in the areas where dengue is endemic, the impact of A. culicis on Ae. aegypti vector competence for dengue virus is unknown. This study aimed to investigate the effects of A. culicis infection on mosquito size and fitness, as measured by wing length, and the susceptibility to dengue virus infection in Ae. aegypti. Our results showed that there was no statistically significant difference in wing lengths between Ae. aegypti infected and not infected with A. culicis. Furthermore, A. culicis infection did not significantly affect dengue virus infection or disseminated infection rate. However, there was a significant association between shorter wings and higher dengue virus infection rate, whereby a 0.1-mm increase in wing length decreased the odds of the mosquito being infected by 32%. Thus, based on our result, A. culicis infection does not influence the body size and dengue virus infection in Ae. aegypti. This study helps to shed light on a common but neglected eukaryotic mosquito parasite.

Immune outcomes of Zika virus infection in nonhuman primates.

Although the Zika virus (ZIKV) epidemic is subsiding, immune responses that are important for controlling acute infection have not been definitively characterized. Nonhuman primate (NHP) models were rapidly developed to understand the disease and to test vaccines, and these models have since provided an understanding of the immune responses that correlate with protection during natural infection and vaccination. Here, we infected a small group of male rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques with a minimally passaged Brazilian ZIKV isolate and used multicolor flow cytometry and transcriptional profiling to describe early immune patterns following infection. We found evidence of strong innate antiviral responses together with induction of neutralizing antibodies and T cell responses. We also assessed the relative importance of CD8 T cells in controlling infection by carrying out CD8 T cell depletion in an additional two animals of each species. CD8 depletion appeared to dysregulate early antiviral responses and possibly increase viral persistence, but the absence of CD8 T cells ultimately did not impair control of the virus. Together, these data describe immunological trends in two NHP species during acute ZIKV infection, providing an account of early responses that may be important in controlling infection.

An Investigation of Human-Mosquito Contact Using Surveys and Its Application in Assessing Dengue Viral Transmission Risk.

Aedes-borne viral diseases such as dengue fever are surging in incidence in recent years. To investigate viral transmission risks, the availability of local transmission parameters is essential. One of the most important factors directly determining infection risk is human-mosquito contact. Yet the contact rate is not often characterized, compared with other risk metrics such as vector density, because of the limited research tool options. In this study, human-mosquito contact was assessed in two study sites in the Southern United States using self-administered standardized survey instruments. The fraction of mosquito bites attributed to important vector species was estimated by human landing sampling. The survey participants reported a significantly higher outdoor mosquito bite exposure than indoor. The reported bite number was positively correlated with outdoor time during at-risk periods. There was also a significant effect of the study site on outdoor bite exposure, possibly due to the differing vector density. Thus, the levels of human-mosquito contact in this study were influenced both by the mosquito density and human behaviors. A dengue virus transmission model demonstrated that the observed difference in the contact rates results in differential virus transmission risks. Our findings highlight the practicality of using surveys to investigate human-mosquito contact in a setting where bite exposure levels differ substantially, and serve as a basis for further evaluations. This study underscores a new avenue that can be used in combination with other field methods to understand how changes in human behavior may influence mosquito bite exposure which drives mosquito-borne virus transmission.

Short Report: Asymptomatic Zika virus infections with low viral loads not likely to establish transmission in New Orleans Aedes populations.

Aedes aegypti and Aedes albopictus are both vectors of Zika virus and both are endemic to the New Orleans Metropolitan area. Fortunately, to date there has been no known autochthonous transmission of Zika virus in New Orleans. No studies of the vector competence of local populations of Ae. aegypti and Ae. albopictus for Zika virus transmission have been conducted. To determine if New Orleans Ae. aegypti and Ae. albopictus mosquitoes are competent for Zika virus, mosquitoes were reared to generation F3 from eggs collected in New Orleans during the 2018 mosquito season. Adults were fed an infectious blood meal and kept for 15 days in an environmental chamber. Transmission assays were conducted at 4, 10, and 15 days post exposure and RT-PCR was run on bodies and saliva to detect the presence of Zika virus RNA. We observed remarkably low susceptibility of both Ae. aegypti and Ae. albopictus from New Orleans to a Zika strain from Panama after oral challenge. These results suggest a limited risk of Zika virus transmission should it be introduced to the New Orleans area, and may partially explain why no transmission was detected in Louisiana during the 2016 epidemic in the Americas, despite multiple known travel associated introductions to New Orleans. Despite these results these mosquito populations are known to be competent vectors for some other mosquito-borne viruses and control measures should not be relaxed.

Zika Virus and the World Health Organization Criteria for Determining Recent Infection Using Plaque Reduction Neutralization Testing.

The recent Zika virus (ZIKV) epidemic swept across Latin America and the Caribbean, where dengue virus (DENV) is endemic. The antigenic similarities of these closely related flaviviruses left researchers and clinicians with challenges to interpret serological tests. Thirty-six women attending a prenatal clinic in Honduras and with positive DENV IgM enzyme-linked immunoabsorbent assays (ELISAs) were screened with a ZIKV immunoglobulin M ELISA, reverse transcription polymerase chain reaction for ZIKV and DENV 1-4, and plaque reduction neutralization tests (PRNTs) for ZIKV and DENV-2. Plaque reduction neutralization test results were interpreted using the World Health Organization (WHO) and Centers for Disease Control and Prevention (CDC) criteria. Using the WHO criteria of a PRNT90 titer ≥ 20 and a 4-fold difference between ZIKV and DENV titers, we determined that 69.4% of samples had a recent ZIKV infection, compared with 5.6% using CDC criteria. The interpretation of ZIKV PRNTs in a DENV-endemic region is highly dependent on the choice of interpretation criteria.

Congenital Transmission of Trypanosoma cruzi in Argentina, Honduras, and Mexico: An Observational Prospective Study.

Compared with South America, there is a lack of epidemiologic studies about the risk of congenital transmission of Trypanosoma cruzi in Central America and Mexico. It has been suggested that T. cruzi genotypes might differ by region and that congenital transmission might vary according to the parasite's genotype. Our objective was to compare T. cruzi congenital transmission rates in three countries. We performed an observational prospective study in 2011-2014 enrolling women at delivery in one hospital in Argentina, two hospitals in Honduras, and two hospitals in Mexico. Congenital T. cruzi infection was defined as the presence of one or more of the following criteria: presence of parasites in cord blood (direct parasitological microscopic examination) with positive polymerase chain reaction (PCR) in cord blood, presence of parasites in infant's blood at 4-8 weeks (direct parasitological microscopic examination), and persistence of T. cruzi-specific antibodies at 10 months, as measured by at least two tests. Among 28,145 enrolled women, 347 had at least one antibody rapid test positive in cord blood and a positive enzyme-linked immunosorbent assay in maternal blood. PCR in maternal blood was positive in 73.2% of the cases, and genotyping identified a majority of non-TcI in the three countries. We found no (0.0%; 95% confidence interval [CI]: 0.0, 2.0) confirmed congenital case in Honduras. Congenital transmission was 6.6% (95% CI: 3.1, 12.2) in Argentina and 6.3% (95% CI: 0.8, 20.8) in Mexico. Trypanosoma cruzi non-TcI predominated and risks of congenital transmission were similar in Argentina and Mexico.

Effective population sizes of a major vector of human diseases, Aedes aegypti.

The effective population size (Ne ) is a fundamental parameter in population genetics that determines the relative strength of selection and random genetic drift, the effect of migration, levels of inbreeding, and linkage disequilibrium. In many cases where it has been estimated in animals, Ne is on the order of 10%-20% of the census size. In this study, we use 12 microsatellite markers and 14,888 single nucleotide polymorphisms (SNPs) to empirically estimate Ne in Aedes aegypti, the major vector of yellow fever, dengue, chikungunya, and Zika viruses. We used the method of temporal sampling to estimate Ne on a global dataset made up of 46 samples of Ae. aegypti that included multiple time points from 17 widely distributed geographic localities. Our Ne estimates for Ae. aegypti fell within a broad range (~25-3,000) and averaged between 400 and 600 across all localities and time points sampled. Adult census size (Nc) estimates for this species range between one and five thousand, so the Ne /Nc ratio is about the same as for most animals. These Ne values are lower than estimates available for other insects and have important implications for the design of genetic control strategies to reduce the impact of this species of mosquito on human health.

Design and Testing of Novel Lethal Ovitrap to Reduce Populations of Aedes Mosquitoes: Community-Based Participatory Research between Industry, Academia and Communities in Peru and Thailand.

BACKGROUND: Dengue virus (and Chikungunya and Zika viruses) is transmitted by Aedes aegypti and Aedes albopictus mosquitoes and causes considerable human morbidity and mortality. As there is currently no vaccine or chemoprophylaxis to protect people from dengue virus infection, vector control is the only viable option for disease prevention. The purpose of this paper is to illustrate the design and placement process for an attractive lethal ovitrap to reduce vector populations and to describe lessons learned in the development of the trap. METHODS: This study was conducted in 2010 in Iquitos, Peru and Lopburi Province, Thailand and used an iterative community-based participatory approach to adjust design specifications of the trap, based on community members' perceptions and feedback, entomological findings in the lab, and design and research team observations. Multiple focus group discussions (FGD) were held over a 6 month period, stratified by age, sex and motherhood status, to inform the design process. Trap testing transitioned from the lab to within households. RESULTS: Through an iterative process of working with specifications from the research team, findings from the laboratory testing, and feedback from FGD, the design team narrowed trap design options from 22 to 6. Comments from the FGD centered on safety for children and pets interacting with traps, durability, maintenance issues, and aesthetics. Testing in the laboratory involved releasing groups of 50 gravid Ae. aegypti in walk-in rooms and assessing what percentage were caught in traps of different colors, with different trap cover sizes, and placed under lighter or darker locations. Two final trap models were mocked up and tested in homes for a week; one model was the top choice in both Iquitos and Lopburi. DISCUSSION: The community-based participatory process was essential for the development of novel traps that provided effective vector control, but also met the needs and concerns of community members.

Effect of Spatial Repellent Exposure on Dengue Vector Attraction to Oviposition Sites.

BACKGROUND: Aedes aegypti is a primary vector of dengue virus (DENV), the causative agent of dengue fever, an arthropod-borne disease of global importance. Although a vaccine has been recommended for prevention, current dengue prevention strategies rely on vector control. Recently, volatile pyrethroids-spatial repellents-have received interest as a novel delivery system for adult Ae. aegypti control. Understanding the full range of behavioral effects spatial repellents elicit in mosquito species will be critical to understanding the overall impact these products have on vector populations and will guide expectations of efficacy against DENV transmission. METHODOLOGY/PRINCIPAL FINDINGS: The current study quantified changes in attraction of gravid Ae. aegypti to experimental oviposition sites following exposure to the spatial repellent transfluthrin. Responses were measured with two-choice olfaction bioassays using 'sticky-screens' covering cups to prevent contact with the oviposition substrate. Two cups contained a bacterial attractant composed of four species of bacteria in calcium alginate beads in water and two cups contained only deionized water. Results from 40 replicates (n = 780 females total per treatment) indicated an estimated difference in attraction of 9.35% ± 0.18 (p ≤ 0.003), implying that the transfluthrin-exposed mosquitoes were more attracted to the experimental oviposition sites than the non-exposed mosquitoes. CONCLUSIONS/SIGNIFICANCE: Findings from this study will further characterize the role of spatial repellents to modify Ae. aegypti behavior related to dengue prevention specifically, and encourage innovation in vector control product development more broadly.

Zika virus infection in pregnant women in Honduras: study protocol.

BACKGROUND: Although there is increasing evidence for a relationship between symptomatic Zika virus (ZIKV) maternal infection, and microcephaly, a firm causal relation has yet to be established by epidemiologic studies. Studies also need to be conducted in recently infected settings. Our objectives are to assess the frequency of ZIKV infection during pregnancy in Honduras and the association of microcephaly with ZIKV infection. METHODS/DESIGN: We will perform a prospective study enrolling pregnant women at their first antenatal visit and following them up until delivery. At the time of enrollment, women will be interviewed to collect socio-demographic data, data needed to locate them for potential additional follow-up, and data about ZIKV symptoms during pregnancy. We will also collect maternal blood as soon as possible after enrollment. A probable maternal ZIKV infection will be defined as positive for maternal ZIKV IgM. A confirmed maternal ZIKV infection will be defined as positive for ZIKV IgM confirmed by plaque reduction neutralization test. Microcephaly at birth will be defined as an occipito-frontal circumference <2SD for sex and gestational age. Our objective is to enroll 2000 pregnant women. In a first step, we will follow a case cohort design and only analyze blood samples for cases and a sub-cohort of 200 women randomly selected. Blood samples for the entire population will be analyzed at a later stage if funds are available. DISCUSSION: This protocol was designed to be implemented with minimal resources. It allows a cohort to be built, which could be a foundation for future in-depth and follow-up studies.

Prospective study of pregnancy and newborn outcomes in mothers with West nile illness during pregnancy.

BACKGROUND: A previous case report of West Nile virus (WNV) illness during pregnancy suggested that WNV could be a cause of congenital defects. We performed a prospective, longitudinal cohort study of pregnant women with WNV illness to increase our knowledge of the effects of WNV illness during pregnancy. METHODS: Participants were enrolled in 2005 to 2008 from pregnant women with serologically confirmed WNV illness reported to the Centers for Disease Control and Prevention. Comparison was made to WNV-uninfected women, matched on maternal age and enrollment month. Pregnancy and newborn data were collected; cord blood WNV serology was obtained. Pediatric exams and the Bayley Scales of Infant and Toddler Development-Third Edition (Bayley-III) were performed. RESULTS: Twenty-eight WNV-infected mothers and 25 WNV-uninfected mothers participated. Maternal demographics were similar except for a higher rate of planned pregnancies, education, and household income in the WNV-uninfected mothers. There were no differences in pregnancy and delivery characteristics except that infected mothers had a higher incidence of febrile illnesses and used more medications. Birth weight, length, head circumference, and rate of congenital malformations were similar in babies born to WNV-infected and -uninfected mothers. Follow-up physical exams were generally normal. The Bayley-III assessments, available for 17 children born to mothers with WNV illness, showed performance at or above age level across domains. CONCLUSION: The risk for adverse pregnancy and newborn outcomes in women experiencing WNV illness in pregnancy appears to be low, but future studies with larger numbers are needed to rule out a small risk. Birth Defects Research (Part A) 106:716-723, 2016. © 2016 Wiley Periodicals, Inc.