Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE: ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

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.

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.

Evaluation of a novel West Nile virus transmission control strategy that targets Culex tarsalis with endectocide-containing blood meals.

Control of arbovirus transmission remains focused on vector control through application of insecticides directly to the environment. However, these insecticide applications are often reactive interventions that can be poorly-targeted, inadequate for localized control during outbreaks, and opposed due to environmental and toxicity concerns. In this study, we developed endectocide-treated feed as a systemic endectocide for birds to target blood feeding Culex tarsalis, the primary West Nile virus (WNV) bridge vector in the western United States, and conducted preliminary tests on the effects of deploying this feed in the field. In lab tests, ivermectin (IVM) was the most effective endectocide tested against Cx. tarsalis and WNV-infection did not influence mosquito mortality from IVM. Chickens and wild Eurasian collared doves exhibited no signs of toxicity when fed solely on bird feed treated with concentrations up to 200 mg IVM/kg of diet, and significantly more Cx. tarsalis that blood fed on these birds died (greater than 80% mortality) compared to controls (less than 25% mortality). Mosquito mortality following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. Preliminary field testing over one WNV season in Fort Collins, Colorado demonstrated that nearly all birds captured around treated bird feeders had detectable levels of IVM in their blood. However, entomological data showed that WNV transmission was non-significantly reduced around treated bird feeders. With further development, deployment of ivermectin-treated bird feed might be an effective, localized WNV transmission control tool.

American Aedes vexans Mosquitoes are Competent Vectors of Zika Virus.

AbstractStarting in 2013-2014, the Americas have experienced a massive outbreak of Zika virus (ZIKV) which has now reached at least 49 countries. Although most cases have occurred in South America and the Caribbean, imported and autochthonous cases have occurred in the United States. Aedes aegypti and Aedes albopictus mosquitoes are known vectors of ZIKV. Little is known about the potential for temperate Aedes mosquitoes to transmit ZIKV. Aedes vexans has a worldwide distribution, is highly abundant in particular localities, aggressively bites humans, and is a competent vector of several arboviruses. However, it is not clear whether Ae. vexans mosquitoes are competent to transmit ZIKV. To determine the vector competence of Ae. vexans for ZIKV, wild-caught mosquitoes were exposed to an infectious bloodmeal containing a ZIKV strain isolated during the current outbreak. Approximately 80% of 148 mosquitoes tested became infected by ZIKV, and approximately 5% transmitted infectious virus after 14 days of extrinsic incubation. These results establish that Ae. vexans are competent ZIKV vectors. Their relative importance as vectors (i.e., their vectorial capacity) depends on feeding behavior, longevity, and other factors that are likely to vary in ecologically distinct environments.

Rapid and specific detection of Asian- and African-lineage Zika viruses.

Understanding the dynamics of Zika virus transmission and formulating rational strategies for its control require precise diagnostic tools that are also appropriate for resource-poor environments. We have developed a rapid and sensitive loop-mediated isothermal amplification (LAMP) assay that distinguishes Zika viruses of Asian and African lineages. The assay does not detect chikungunya virus or flaviviruses such as dengue, yellow fever, or West Nile viruses. The assay conditions allowed direct detection of Zika virus RNA in cultured infected cells; in mosquitoes; in virus-spiked samples of human blood, plasma, saliva, urine, and semen; and in infected patient serum, plasma, and semen samples without the need for RNA isolation or reverse transcription. The assay offers rapid, specific, sensitive, and inexpensive detection of the Asian-lineage Zika virus strain that is currently circulating in the Western hemisphere, and can also detect the African-lineage Zika virus strain using separate, specific primers.

Vector Competence of American Mosquitoes for Three Strains of Zika Virus.

In 2015, Zika virus (ZIKV; Flaviviridae; Flavivirus) emerged in the Americas, causing millions of infections in dozens of countries. The rapid spread of the virus and the association with disease outcomes such as Guillain-Barré syndrome and microcephaly make understanding transmission dynamics essential. Currently, there are no reports of vector competence (VC) of American mosquitoes for ZIKV isolates from the Americas. Further, it is not clear whether ZIKV strains from other genetic lineages can be transmitted by American Aedes aegypti populations, and whether the scope of the current epidemic is in part facilitated by viral factors such as enhanced replicative fitness or increased vector competence. Therefore, we characterized replication of three ZIKV strains, one from each of the three phylogenetic clades in several cell lines and assessed their abilities to be transmitted by Ae. aegypti mosquitoes. Additionally, laboratory colonies of different Culex spp. were infected with an American outbreak strain of ZIKV to assess VC. Replication rates were variable and depended on virus strain, cell line and MOI. African strains used in this study outcompeted the American strain in vitro in both mammalian and mosquito cell culture. West and East African strains of ZIKV tested here were more efficiently transmitted by Ae. aegypti from Mexico than was the currently circulating American strain of the Asian lineage. Long-established laboratory colonies of Culex mosquitoes were not efficient ZIKV vectors. These data demonstrate the capacity for additional ZIKV strains to infect and replicate in American Aedes mosquitoes and suggest that neither enhanced virus replicative fitness nor virus adaptation to local vector mosquitoes seems likely to explain the extent and intensity of ZIKV transmission in the Americas.

The selective in vitro cytotoxicity of carcinoma cells by AZT is enhanced by concurrent treatment with delocalized lipophilic cations.

This study assessed the selective growth inhibitory effect on cultured carcinoma cells by 3'-azido-3'-deoxythymidine (AZT), as a single agent, and in combination with delocalized lipophilic cations (DLCs) that are known to inhibit mitochondrial function. In cytotoxicity assays, treatment of cells with varying concentrations of AZT induced a dose-dependent inhibition of cell growth of the human carcinoma lines DU-145 (prostate; IC50 at 24 microM), MCF-7 (breast; IC50 at 22 microM), and CX-1 (colon; IC50 at 23 microM), yet caused no significant effect on the growth of the control epithelial cell line CV-1 (monkey kidney) at a concentration as high as 50 microM AZT. Combination treatment employing a constant concentration (1.25 microM) of the DLC dequalinium chloride (DECA) plus varying concentrations of AZT (0-50 microM) enhanced the AZT-induced cytotoxicity of carcinoma cells at least fourfold for MCF-7 and CX-1 cells (IC50 at 5 microM AZT), and twofold for DU-145 cells (IC50 at 11 microM AZT). Similar results were obtained in DU-145 cells using a constant concentration of the DLC MKT-077 (1.0 microM) and varying concentrations of AZT (IC50 at 12.5 microM). As expected, the drug combination of constant DLC and varying AZT had no significant effect on the growth of CV-1 cells. Clonogenic assays demonstrated up to 20-fold enhancement of selective carcinoma cell killing by combination vs. single agent treatment, depending on the specific drug combination and concentrations used. It is hypothesized that the efficacy of the AZT/DLC drug combination in carcinoma cell killing may be based on a dual selectivity involving inhibition of mitochondrial energy metabolism and inhibition of DNA synthesis due to limited deoxythymidine monophosphate availability.

Functional genetic characterization of salivary gland development in Aedes aegypti.

BACKGROUND: Despite the devastating global impact of mosquito-borne illnesses on human health, very little is known about mosquito developmental biology. In this investigation, functional genetic analysis of embryonic salivary gland development was performed in Aedes aegypti, the dengue and yellow fever vector and an emerging model for vector mosquito development. Although embryonic salivary gland development has been well studied in Drosophila melanogaster, little is known about this process in mosquitoes or other arthropods. RESULTS: Mosquitoes possess orthologs of many genes that regulate Drosophila melanogaster embryonic salivary gland development. The expression patterns of a large subset of these genes were assessed during Ae. aegypti development. These studies identified a set of molecular genetic markers for the developing mosquito salivary gland. Analysis of marker expression allowed for tracking of the progression of Ae. aegypti salivary gland development in embryos. In Drosophila, the salivary glands develop from placodes located in the ventral neuroectoderm. However, in Ae. aegypti, salivary marker genes are not expressed in placode-like patterns in the ventral neuroectoderm. Instead, marker gene expression is detected in salivary gland rudiments adjacent to the proventriculus. These observations highlighted the need for functional genetic characterization of mosquito salivary gland development. An siRNA- mediated knockdown strategy was therefore employed to investigate the role of one of the marker genes, cyclic-AMP response element binding protein A (Aae crebA), during Ae. aegypti salivary gland development. These experiments revealed that Aae crebA encodes a key transcriptional regulator of the secretory pathway in the developing Ae. aegypti salivary gland. CONCLUSIONS: The results of this investigation indicated that the initiation of salivary gland development in Ae. aegypti significantly differs from that of D. melanogaster. Despite these differences, some elements of salivary gland development, including the ability of CrebA to regulate secretory gene expression, are conserved between the two species. These studies underscore the need for further analysis of mosquito developmental genetics and may foster comparative studies of salivary gland development in additional insect species.