Brian D. Foy

Probable Non–Vector-borne Transmission of Zika Virus, Colorado, USA

Clinical and serologic evidence indicate that 2 American scientists contracted Zika virus infections while working in Senegal in 2008. One of the scientists transmitted this arbovirus to his wife after his return home. Direct contact is implicated as the transmission route, most likely as a sexually transmitted infection.

Aedes aegypti uses RNA interference in defense against Sindbis virus infection

BackgroundRNA interference (RNAi) is an important anti-viral defense mechanism. The Aedes aegypti genome encodes RNAi component orthologs, however, most populations of this mosquito are readily infected by, and subsequently transmit flaviviruses and alphaviruses. The goal of this study was to use Ae. aegypti as a model system to determine how the mosquitos anti-viral RNAi pathway interacts with recombinant Sindbis virus (SINV; family Togaviridae, genus Alphavirus).ResultsSINV (TR339-eGFP) (+) strand RNA, infectious virus titers and infection rates transiently increased in mosquitoes following dsRNA injection to cognate Ago2, Dcr2, or TSN mRNAs. Detection of SINV RNA-derived small RNAs at 2 and 7 days post-infection in non-silenced mosquitoes provided important confirmation of RNAi pathway activity. Two different recombinant SINV viruses (MRE16-eGFP and TR339-eGFP) with significant differences in infection kinetics were used to delineate vector/virus interactions in the midgut. We show virus-dependent effects on RNAi component transcript and protein levels during infection. Monitoring midgut Ago2, Dcr2, and TSN transcript levels during infection revealed that only TSN transcripts were significantly increased in midguts over blood-fed controls. Ago2 protein levels were depleted immediately following a non-infectious bloodmeal and varied during SINV infection in a virus-dependent manner.ConclusionWe show that silencing RNAi components in Ae. aegypti results in transient increases in SINV replication. Furthermore, Ae. aegypti RNAi is active during SINV infection as indicated by production of virus-specific siRNAs. Lastly, the RNAi response varies in a virus-dependent manner. These data define important features of RNAi anti-viral defense in Ae. aegypti.

Use of Sindbis virus-mediated RNA interference to demonstrate a conserved role of Broad-Complex in insect metamorphosis

The transcription factor Broad-Complex (BR-C) is required for differentiation of adult structures as well as for the programmed death of obsolete larval organs during metamorphosis of the fruit fly Drosophila melanogaster. Whether BR-C has a similar role in other holometabolous insects could not be proven without a loss-of-function genetic test, performed in a non-drosophilid species. Here we use a recombinant Sindbis virus as a tool to silence BR-C expression in the silkmoth Bombyx mori. The virus expressing a BR-C antisense RNA fragment reduced endogenous BR-C mRNA levels in infected tissues (adult wing and leg primordia) via RNA interference (RNAi). The RNAi knock-down of BR-C resulted in the failure of animals to complete the larval–pupal transition or in later morphogenetic defects, including differentiation of adult compound eyes, legs, and wings from their larval progenitors. BR-C RNAi also perturbed the programmed cell death of larval silk glands. These developmental defects correspond to loss-of-function phenotypes of BR-C Drosophila mutants in both the morphogenetic and degenerative aspects, suggesting that the critical role of BR-C in metamorphosis is evolutionarily conserved. We also demonstrate that the Sindbis virus is a useful vehicle for silencing of developmental genes in new insect models.

Comparative genomics of small RNA regulatory pathway components in vector mosquitoes

BackgroundSmall RNA regulatory pathways (SRRPs) control key aspects of development and anti-viral defense in metazoans. Members of the Argonaute family of catalytic enzymes degrade target RNAs in each of these pathways. SRRPs include the microRNA, small interfering RNA (siRNA) and PIWI-type gene silencing pathways. Mosquitoes generate viral siRNAs when infected with RNA arboviruses. However, in some mosquitoes, arboviruses survive antiviral RNA interference (RNAi) and are transmitted via mosquito bite to a subsequent host. Increased knowledge of these pathways and functional components should increase understanding of the limitations of anti-viral defense in vector mosquitoes. To do this, we compared the genomic structure of SRRP components across three mosquito species and three major small RNA pathways.ResultsThe Ae. aegypti, An. gambiae and Cx. pipiens genomes encode putative orthologs for all major components of the miRNA, siRNA, and piRNA pathways. Ae. aegypti and Cx. pipiens have undergone expansion of Argonaute and PIWI subfamily genes. Phylogenetic analyses were performed for these protein families. In addition, sequence pattern recognition algorithms MEME, MDScan and Weeder were used to identify upstream regulatory motifs for all SRRP components. Statistical analyses confirmed enrichment of species-specific and pathway-specific cis-elements over the rest of the genome.ConclusionAnalysis of Argonaute and PIWI subfamily genes suggests that the small regulatory RNA pathways of the major arbovirus vectors, Ae. aegypti and Cx. pipiens, are evolving faster than those of the malaria vector An. gambiae and D. melanogaster. Further, protein and genomic features suggest functional differences between subclasses of PIWI proteins and provide a basis for future analyses. Common UCR elements among SRRP components indicate that 1) key components from the miRNA, siRNA, and piRNA pathways contain NF-kappaB-related and Broad complex transcription factor binding sites, 2) purifying selection has occurred to maintain common pathway-specific elements across mosquito species and 3) species-specific differences in upstream elements suggest that there may be differences in regulatory control among mosquito species. Implications for arbovirus vector competence in mosquitoes are discussed.

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.

Ivermectin to reduce malaria transmission: a research agenda for a promising new tool for elimination

BackgroundThe heterogeneity of malaria transmission makes widespread elimination a difficult goal to achieve. Most of the current vector control measures insufficiently target outdoor transmission. Also, insecticide resistance threatens to diminish the efficacy of the most prevalent measures, indoor residual spray and insecticide treated nets. Innovative approaches are needed. The use of endectocides, such as ivermectin, could be an important new addition to the toolbox of anti-malarial measures. Ivermectin effectively targets outdoor transmission, has a novel mechanism of action that could circumvent resistance and might be distributed over the channels already in place for the control of onchocerciasis and lymphatic filariasis.MethodsThe previous works involving ivermectin and Anopheles vectors are reviewed and summarized. A review of ivermectin’s safety profile is also provided. Finally three definitive clinical trials are described in detail and proposed as the evidence needed for implementation. Several smaller and specific supportive studies are also proposed.ConclusionsThe use of ivermectin solves many challenges identified for future vector control strategies. It is an effective and safe endectocide that was approved for human use more than 25 years ago. Recent studies suggest it might become an effective and complementary strategy in malaria elimination and eradication efforts; however, intensive research will be needed to make this a reality.

The effect of oral anthelmintics on the survivorship and re-feeding frequency of anthropophilic mosquito disease vectors.

In the Tropics, there is substantial temporal and spatial overlap of diseases propagated by anthropophilic mosquito vectors (such as malaria and dengue) and human helminth diseases (such as onchocerciasis and lymphatic filariasis) that are treated though mass drug administrations (MDA). This overlap will result in mosquito vectors imbibing significant quantities of these drugs when they blood feed on humans. Since many anthelmintic drugs have broad anti-invertebrate effects, the possibility of combined helminth control and mosquito-borne disease control through MDA is apparent. It has been previously shown that ivermectin can reduce mosquito survivorship when administered in a blood meal, but more detailed examinations are needed if MDA is to ever be developed into a tool for malaria or dengue control. We examined concentrations of drugs that follow human pharmacokinetics after MDA and that matched with mosquito feeding times, for effects against the anthropophilic mosquito vectors Anopheles gambiae s.s. and Aedes aegypti. Ivermectin was the only human-approved MDA drug we tested that affected mosquito survivorship, and only An. gambiae s.s. were affected at concentrations respecting human pharmacokinetics at indicated doses. Ivermectin also delayed An. gambiae s.s. re-feeding frequency and defecation rates, and two successive ivermectin-spiked blood meals following human pharmacokinetic concentrations compounded mortality effects compared to controls. These findings suggest that ivermectin MDA in Africa may be used to decrease malaria transmission if MDAs were administered more frequently. Such a strategy would broaden the current scope of polyparasitism control already afforded by MDAs, and which is needed in many African villages simultaneously burdened by many parasitic diseases.

Mass drug administration of ivermectin in south-eastern Senegal reduces the survivorship of wild-caught, blood fed malaria vectors

BackgroundIn south-eastern Senegal, malaria and onchocerciasis are co-endemic. Onchocerciasis in this region has been controlled by once or twice yearly mass drug administration (MDA) with ivermectin (IVM) for over fifteen years. Since laboratory-raised Anopheles gambiae s.s. are susceptible to ivermectin at concentrations found in human blood post-ingestion of IVM, it is plausible that a similar effect could be quantified in the field, and that IVM might have benefits as a malaria control tool.MethodsIn 2008 and 2009, wild-caught blood fed An. gambiae s.l. mosquitoes were collected from huts of three pairs of Senegalese villages before and after IVM MDAs. Mosquitoes were held in an insectary to assess their survival rate, subsequently identified to species, and their blood meals were identified. Differences in mosquito survival were statistically analysed using a Glimmix model. Lastly, changes in the daily probability of mosquito survivorship surrounding IVM MDAs were calculated, and these data were inserted into a previously developed, mosquito age-structured model of malaria transmission.ResultsAnopheles gambiae s.s. (P < 0.0001) and Anopheles arabiensis (P = 0.0191) from the treated villages had significantly reduced survival compared to those from control villages. Furthermore, An gambiae s.s. caught 1-6 days after MDA in treated villages had significantly reduced survival compared to control village collections (P = 0.0003), as well as those caught pre-MDA (P < 0.0001) and >7 days post-MDA (P < 0.0001). The daily probability of mosquito survival dropped >10% for the six days following MDA. The mosquito age-structured model of malaria transmission demonstrated that a single IVM MDA would reduce malaria transmission (Ro) below baseline for at least eleven days, and that repeated IVM MDAs would result in a sustained reduction in malaria Ro.ConclusionsIvermectin MDA significantly reduced the survivorship of An. gambiae s.s. for six days past the date of the MDA, which is sufficient to temporarily reduce malaria transmission. Repeated IVM MDAs could be a novel and integrative malaria control tool in areas with seasonal transmission, and which would have simultaneous impacts on neglected tropical diseases in the same villages.

Ivermectin Mass Drug Administration to Humans Disrupts Malaria Parasite Transmission in Senegalese Villages

Ivermectin mass drug administration (MDA) to humans is used to control onchocerciasis and lymphatic filariasis. Recent field studies have shown an added killing effect of ivermectin MDA against malaria vectors. We report that ivermectin MDA reduced the proportion of Plasmodium falciparum infectious Anopheles gambiae sensu stricto (s.s.) in treated villages in southeastern Senegal. Ivermectin MDA is a different delivery method and has a different mode of action from current malaria control agents. It could be a powerful and synergistic new tool to reduce malaria transmission in regions with epidemic or seasonal malaria transmission, and the prevalence and intensity of neglected tropical diseases.

Endectocides for malaria control

Systemic endectocidal drugs, used to control nematodes in humans and other vertebrates, can be toxic to Anopheles spp. mosquitoes when they take a blood meal from a host that has recently received one of these drugs. Recent laboratory and field studies have highlighted the potential of ivermectin to control malaria parasite transmission if this drug is distributed strategically and more often. There are important theoretical benefits to this strategy, as well as caveats. A better understanding of drug effects against vectors and malaria ecologies are needed. In the near future, ivermectin and other endectocides could serve as potent and novel malaria transmission control tools that are directly linked to the control of neglected tropical diseases in the same communities.