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 mechanisms contributing to the explosive nature of the current ZIKV outbreak in the Americas are poorly understood. Therefore, we characterized the replication of three strains, one from each phylogenetic clade of ZIKV and evaluated virus strain differences in transmission efficiency by American mosquitoes. Our results suggest that the strain currently circulating in the Americas does not have unusually high infectivity for American Ae. aegypti as compared to the African strains used in this study. Colonized Culex mosquitoes were inefficient vectors. In vitro data suggested slower replication and decreased fitness for the currently circulating American strain compared to African strains isolated decades ago. Therefore, viral adaptation to local mosquitoes does not appear, at present, to be driving the current ZIKV outbreak in the Americas.

We describe the first comprehensive analysis of the midgut metabolome of Aedes aegypti, the primary mosquito vector for arboviruses such as dengue, Zika, chikungunya and yellow fever viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate from several tissues in the mosquito vector. The metabolic environments within these tissues play crucial roles in these processes. Since these viruses are enveloped, viral replication, assembly and release occur on cellular membranes primed through the manipulation of host metabolism. Interference with this virus infection-induced metabolic environment is detrimental to viral replication in human and mosquito cell culture models. Here we present the first insight into the metabolic environment induced during arbovirus replication in Aedes aegypti. Using high-resolution mass spectrometry, we have analyzed the temporal metabolic perturbations that occur following dengue virus infection of the midgut tissue. This is the primary site of infection and replication, preceding systemic viral dissemination and transmission. We identified metabolites that exhibited a dynamic-profile across early-, mid- and late-infection time points. We observed a marked increase in the lipid content. An increase in glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels suggested a diversion of resources during infection from energy storage to synthetic pathways. Elevated levels of acyl-carnitines were observed, signaling disruptions in mitochondrial function and possible diversion of energy production. A central hub in the sphingolipid pathway that influenced dihydroceramide to ceramide ratios was identified as critical for the virus life cycle. This study also resulted in the first reconstruction of the sphingolipid pathway in Aedes aegypti. Given conservation in the replication mechanisms of several flaviviruses transmitted by this vector, our results highlight biochemical choke points that could be targeted to disrupt transmission of multiple pathogens by these mosquitoes. The Aedes aegypti mosquito transmits arboviruses that cause dengue, Zika, chikungunya and yellow fever. These viruses are endemic in tropical and subtropical regions of the world placing 2.5 billion people at risk of infection. Transmission is critically dependent upon the replication of these viruses in both human and mosquito hosts. Successful viral replication is greatly influenced by the biochemical environment of the host cell or tissue and flaviviruses rearrange this environment to benefit their needs. Host-cell derived metabolites such as lipids, sugars and amino acids are utilized to produce progeny virions, help evade the host immune system and enable successful completion of the life cycle. In this study, we applied high-resolution mass spectrometry to understand the alteration of the biochemical landscape of the mosquito during infection by dengue virus. We focused on the mosquito midgut, as this is the initial site of infection. We identified several metabolites that exhibited dynamic profiles during the course of viral infection and replication. By pinpointing biochemical “choke points” required for viral replication, we can devise strategies that will stall virus replication in the mosquito and prevent its transmission to humans.

A rapid, specific, sensitive, and inexpensive method has been developed that detects RNA from a Zika virus strain associated with the current outbreak.

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combine multimodal imaging (cm-nm scale) with single nuclei RNA-sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar^–/–) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection is imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection is limited to grey matter of sensory systems in wildtype mice, but extends into white matter, meninges and choroid plexus in Ifnar^–/– mice. Cells in wildtype display strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+ NK cells, whereas in Ifnar^–/–, the absence of this response contributes to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

The emergence of Zika virus (ZIKV) in the New World has led to more than 200,000 human infections. Perinatal infection can cause severe neurological complications, including fetal and neonatal microcephaly, and in adults there is an association with Guillain-Barré syndrome (GBS). ZIKV is transmitted to humans by Aedes sp. mosquitoes, yet little is known about its enzootic cycle in which transmission is thought to occur between arboreal Aedes sp. mosquitos and non-human primates. In the 1950s and ‘60s, several bat species were shown to be naturally and experimentally susceptible to ZIKV with acute viremia and seroconversion, and some developed neurological disease with viral antigen detected the brain. Because of ZIKV emergence in the Americas, we sought to determine susceptibility of Jamaican fruit bats (Artibeus jamaicensis), one of the most common bats in the New World. Bats were inoculated with ZIKV PRVABC59 but did not show signs of disease. Bats held to 28 days post-inoculation (PI) had detectable antibody by ELISA and viral RNA was detected by qRT-PCR in the brain, saliva and urine in some of the bats. Immunoreactivity using polyclonal anti-ZIKV antibody was detected in testes, brain, lung and salivary glands plus scrotal skin. Tropism for mononuclear cells, including macrophages/microglia and fibroblasts, was seen in the aforementioned organs in addition to testicular Leydig cells. The virus likely localized to the brain via infection of Iba1⁺ macrophage/microglial cells. Jamaican fruit bats, therefore, may be a useful animal model for the study of ZIKV infection. This work also raises the possibility that bats may have a role in Zika virus ecology in endemic regions, and that ZIKV may pose a wildlife disease threat to bat populations. The rapid spread of Zika virus through a naïve population in the Americas resulted in novel and severe disease manifestations, including fetal and neonatal microcephaly, and GBS. These disease complications make understanding the pathology and ecology of ZIKV a priority. Captive Jamaican fruit bats were challenged with ZIKV to determine their susceptibility, to assess whether bats may play a role in virus ecology, and if they might serve as an animal model to better understand ZIKV pathophysiology. The bats became acutely infected and mounted an antibody response. Three terminally euthanized inoculated bats had antibody titers of 3200, 28 days PI. Evidence of virus replication and associated pathologies were found in the brain, testes, lungs and salivary glands of some of the inoculated bats. The virus showed predilection for mononuclear cells, including resident Iba1⁺ macrophage/microglial cells, and Leydig cells. With no discernible disruption to the blood brain barrier nor distribution of viral antigen indicative of circumeventricular neuroinvasion, microglia cells may be a possible route of entry of ZIKV into brains of bats. Further investigations are needed to determine the mechanisms of neuroinvasion of ZIKV in bats, further determine feasibility of bats as an alternative animal-model for congenital Zika syndrome, and what role bats might play in ZIKV viral ecology.

Background: Fatty acids are the building blocks of complex lipids essential for living organisms. In mosquitoes, fatty acids are involved in cell membrane production, energy conservation and expenditure, innate immunity, development and reproduction. Fatty acids are synthesized by a multifunctional enzyme complex called fatty acid synthase (FAS). Several paralogues of FAS were found in the Aedes aegypti mosquito. However, the molecular characteristics and expression of some of these paralogues have not been investigated. Methods: Genome assemblies of Ae. aegypti were analyzed, and orthologues of human FAS was identified. Phylogenetic analysis and in silico molecular characterization were performed to identify the functional domains of the Ae. aegypti FAS (AaFAS). Quantitative analysis and loss-of-function experiments were performed to determine the significance of different AaFAS transcripts in various stages of development, expression following different diets and the impact of AaFAS on dengue virus, serotype 2 (DENV2) infection and transmission. Results: We identified seven putative FAS genes in the Ae. aegypti genome assembly, based on nucleotide similarity to the FAS proteins (tBLASTn) of humans, other mosquitoes and invertebrates. Bioinformatics and molecular analyses suggested that only five of the AaFAS genes produce mRNA and therefore represent complete gene models. Expression levels of AaFAS varied among developmental stages and between male and female Ae. aegypti. Quantitative analyses revealed that expression of AaFAS1, the putative orthologue of the human FAS, was highest in adult females. Transient knockdown (KD) of AaFAS1 did not induce a complete compensation by other AaFAS genes but limited DENV2 infection of Aag2 cells in culture and the midgut of the mosquito. Conclusion: AaFAS1 is the predominant AaFAS in adult mosquitoes. It has the highest amino acid similarity to human FAS and contains all enzymatic domains typical of human FAS. AaFAS1 also facilitated DENV2 replication in both cell culture and in mosquito midguts. Our data suggest that AaFAS1 may play a role in transmission of dengue viruses and could represent a target for intervention strategies. Graphical