Indirect transfer of pyriproxyfen to European honeybees via an autodissemination approach

The frequency of arboviral disease epidemics is increasing and vector control remains the primary mechanism to limit arboviral transmission. Container inhabiting mosquitoes such as Aedes albopictus and Aedes aegypti are the primary vectors of dengue, chikungunya, and Zika viruses. Current vector control methods for these species are often ineffective, suggesting the need for novel control approaches. A proposed novel approach is autodissemination of insect growth regulators (IGRs). The advantage of autodissemination approaches is small amounts of active ingredients compared to traditional insecticide applications are used to impact mosquito populations. While the direct targeting of cryptic locations via autodissemination seems like a significant advantage over large scale applications of insecticides, this approach could actually affect nontarget organisms by delivering these highly potent long lasting growth inhibitors such as pyriproxyfen (PPF) to the exact locations that other beneficial insects visit, such as a nectar source. Here we tested the hypothesis that PPF treated male Ae. albopictus will contaminate nectar sources, which results in the indirect transfer of PPF to European honey bees (Apis mellifera). We performed bioassays, fluorescent imaging, and mass spectrometry on insect and artificial nectar source materials to examine for intra- and interspecific transfer of PPF. Data suggests there is direct transfer of PPF from Ae. albopictus PPF treated males and indirect transfer of PPF to A. mellifera from artificial nectar sources. In addition, we show a reduction in fecundity in Ae. albopictus and Drosophila melanogaster when exposed to sublethal doses of PPF. The observed transfer of PPF to A. mellifera suggests the need for further investigation of autodissemination approaches in a more field like setting to examine for risks to insect pollinators.

The Genetic Basis for Salivary Gland Barriers to Arboviral Transmission

Arthropod-borne viruses (arboviruses) infect mosquito salivary glands and then escape to saliva prior to virus transmission. Arbovirus transmission from mosquitoes can be modulated by salivary gland infection barriers (SGIBs) and salivary gland escape barriers (SGEBs). We determined the influence of SGIBs and SGEBs by estimating the quantitative genetic contributions of Aedes aegypti half-sib families (Mapastepec, Mexico) infected with three dengue 2 (DENV2), two chikungunya (CHIKV), and two Zika (ZIKV) genotypes. We determined virus titer per salivary gland and saliva at seven days post-infection and virus prevalence in the half-sib population. CHIKV or ZIKV genotypes did not present SGIB, whereas DENV2 genotypes showed low rates of SGIB. However, virus titer and prevalence due to additive genetic factors in the half-sib family displayed a significant narrow-sense heritability (h2) for SGIB in two of the three DENV2 genotypes and one CHIKV and one ZIKV genotype. SGEBs were detected in all seven virus strains: 60–88% of DENV2 and 48–62% of CHIKV or ZIKV genotype infections. SGEB h2 was significant for all CHIKV or ZIKV genotypes but not for any of the DENV2 genotypes. SGIBs and SGEBs exhibited classical gene-by-gene interaction dynamics and are influenced by genetic factors in the mosquito and the virus.

Antiviral Compounds for Blocking Arboviral Transmission in Mosquitoes

Mosquito-borne arthropod-borne viruses (arboviruses) such as the dengue virus (DENV), Zika virus (ZIKV), and chikungunya virus (CHIKV) are important human pathogens that are responsible for significant global morbidity and mortality. The recent emergence and re-emergence of mosquito-borne viral diseases (MBVDs) highlight the urgent need for safe and effective vaccines, therapeutics, and vector-control approaches to prevent MBVD outbreaks. In nature, arboviruses circulate between vertebrate hosts and arthropod vectors; therefore, disrupting the virus lifecycle in mosquitoes is a major approach for combating MBVDs. Several strategies were proposed to render mosquitoes that are refractory to arboviral infection, for example, those involving the generation of genetically modified mosquitoes or infection with the symbiotic bacterium Wolbachia. Due to the recent development of high-throughput screening methods, an increasing number of drugs with inhibitory effects on mosquito-borne arboviruses in mammalian cells were identified. These antivirals are useful resources that can impede the circulation of arboviruses between arthropods and humans by either rendering viruses more vulnerable in humans or suppressing viral infection by reducing the expression of host factors in mosquitoes. In this review, we summarize recent advances in small-molecule antiarboviral drugs in mammalian and mosquito cells, and discuss how to use these antivirals to block the transmission of MBVDs.

Entomological Assessment of the Status and Risk of Mosquito-borne Arboviral Transmission in Ghana

Entomological surveillance is one of the tools used in monitoring and controlling vector-borne diseases. However, the use of entomological surveillance for arboviral infection vector control is often dependent on finding infected individuals. Although this method may suffice in highly endemic areas, it is not as effective in controlling the spread of diseases in low endemic and non-endemic areas. In this study, we examined the efficiency of using entomological markers to assess the status and risk of arbovirus infection in Ghana, which is considered a non-endemic country, by combining mosquito surveillance with virus isolation and detection. This study reports the presence of cryptic species of mosquitoes in Ghana, demonstrating the need to combine morphological identification and molecular techniques in mosquito surveillance. Furthermore, although no medically important viruses were detected, the importance of insect-specific viruses in understanding virus evolution and arbovirus transmission is discussed. This study reports the first mutualistic relationship between dengue virus and the double-stranded RNA Aedes aegypti totivirus. Finally, this study discusses the complexity of the virome of Aedes and Culex mosquitoes and its implication for arbovirus transmission.

Engineered resistance to Zika virus in transgenic Aedes aegypti expressing a polycistronic cluster of synthetic small RNAs

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive-based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered synthetic small RNAs targeting ZIKV is expressed and fully processed in Aedes aegypti, ensuring the formation of mature synthetic small RNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.

Concomitant Transmission of Dengue, Chikungunya, and Zika Viruses in Brazil: Clinical and Epidemiological Findings From Surveillance for Acute Febrile Illness

Background Since their emergence in the Americas, chikungunya (CHIKV) and Zika (ZIKV) viruses co-circulate with dengue virus (DENV), hampering clinical diagnosis. We investigated clinical and epidemiological characteristics of arboviral infections during the introduction and spread of CHIKV and ZIKV through northeastern Brazil. Methods Surveillance for arboviral diseases among febrile patients was performed at an emergency health unit of Salvador, Brazil, between September 2014 and July 2016. We interviewed patients to collect data on symptoms, reviewed medical records to obtain the presumptive diagnoses, and performed molecular and serological testing to confirm DENV, CHIKV, ZIKV, or nonspecific flavivirus (FLAV) diagnosis. Results Of 948 participants, 247 (26.1%) had an acute infection, of which 224 (23.6%) were single infections (DENV, 32 [3.4%]; CHIKV, 159 [16.7%]; ZIKV, 13 [1.4%]; and FLAV, 20 [2.1%]) and 23 (2.4%) coinfections (DENV/CHIKV, 13 [1.4%]; CHIKV/FLAV, 9 [0.9%]; and DENV/ZIKV, 1 [0.1%]). An additional 133 (14.0%) patients had serological evidence for a recent arboviral infection. Patients with ZIKV presented with rash and pruritus (69.2% each) more frequently than those with DENV (37.5% and 31.2%, respectively) and CHIKV (22.9% and 14.7%, respectively) (P < .001 for both comparisons). Conversely, arthralgia was more common in CHIKV (94.9%) and FLAV/CHIKV (100.0%) than in DENV (59.4%) and ZIKV (53.8%) (P < .001). A correct presumptive clinical diagnosis was made for 9%–23% of the confirmed patients. Conclusions Arboviral infections are frequent causes of febrile illness. Coinfections are not rare events during periods of intense, concomitant arboviral transmission. Given the challenge to clinically distinguish these infections, there is an urgent need for rapid, point-of-care, multiplex diagnostics.

Neighborhood Violence Impacts Disease Control and Surveillance: Case Study of Cali, Colombia from 2014 to 2016

Arboviruses are responsible for a large burden of disease globally and are thus subject to intense epidemiological scrutiny. However, a variable notably absent from most epidemiological analyses has been the impact of violence on arboviral transmission and surveillance. Violence impedes surveillance and delivery of health and preventative services and affects an individual’s health-related behaviors when survival takes priority. Moreover, low and middle-income countries bear a disproportionately high burden of violence and related health outcomes, including vector borne diseases. To better understand the epidemiology of arboviral outbreaks in Cali, Colombia, we georeferenced chikungunya (CHIKV), dengue (DENV), and Zika (ZIKV) viral cases from The National System of Surveillance in Public Health between October 2014 and April 2016. We extracted homicide data from the municipal monthly reports and kernel density of homicide distribution from IdeasPaz. Crucially, an overall higher risk of homicide is associated with increased risk of reported DENV, lower rates of acute testing, and higher rates of lab versus clinical discordance. In the context of high violence as a potential barrier to access to preventive health services, a community approach to improve health and peace should be considered.

Engineered resistance to Zika virus in transgenic Ae. aegypti expressing a polycistronic cluster of synthetic miRNAs

Recent Zika virus (ZIKV) outbreaks have highlighted the necessity for development of novel vector control strategies to combat arboviral transmission, including genetic versions of the sterile insect technique, artificial infection with Wolbachia to reduce population size and/or vectoring competency, and gene drive based methods. Here, we describe the development of mosquitoes synthetically engineered to impede vector competence to ZIKV. We demonstrate that a polycistronic cluster of engineered microRNAs (miRNAs) targeting ZIKV is expressed and fully processed following a blood meal in Ae. aegypti, ensuring the formation of mature synthetic miRNAs in the midgut where ZIKV resides in the early stages of infection. Critically, we demonstrate that engineered Ae. aegypti mosquitoes harboring the anti-ZIKV transgene have significantly reduced viral infection, dissemination, and transmission rates of ZIKV. Taken together, these compelling results provide a promising path forward for development of effective genetic-based ZIKV control strategies, which could potentially be extended to curtail other arboviruses.One Sentence SummaryHere we describe the generation of Ae. aegypti mosquitoes that are engineered to confer reduced vector competence to Zika virus (ZIKV) and we discuss how such engineering approach can be used to combat the major health burden of ZIKV and potentially other arboviruses in the future.

Diverse novel resident Wolbachia strains in Culicine mosquitoes from Madagascar

Wolbachia endosymbiotic bacteria are widespread throughout insect species and Wolbachia transinfected in Aedes mosquito species has formed the basis for biocontrol programs as Wolbachia strains inhibit arboviral replication and can spread through populations. Resident strains in wild Culicine mosquito populations (the vectors of most arboviruses) requires further investigation given resident strains can also affect arboviral transmission. As Madagascar has a large diversity of both Culicine species and has had recent arboviral outbreaks, an entomology survey was undertaken, in five ecologically diverse sites, to determine the Wolbachia prevalence. We detected diverse novel resident Wolbachia strains within the Aedeomyia, Culex, Ficalbia, Mansonia and Uranotaenia genera. Wolbachia prevalence rates and strain characterisation through Sanger sequencing with multilocus sequence typing (MLST) and phylogenetic analysis revealed significant diversity and we detected co-infections with the environmentally acquired bacterial endosymbiont Asaia. Mosquitoes were screened for major arboviruses to investigate if any evidence could be provided for their potential role in transmission and we report the presence of Rift Valley fever virus in three Culex species: Culex tritaeniorhynchus, Culex antennatus and Culex decens. The implications of the presence of resident Wolbachia strains are discussed and how the discovery of novel strains can be utilized for applications in the development of biocontrol strategies.