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

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience

IntroductionOptical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited.MethodsT1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images.ResultsVolumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR.DiscussionThe possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging

Characterization of changes in metabolic pathways during dengue virus serotype 2 infection of the Aedes aegypti mosquito vector to identify control points for interrupting virus transmission

2018 Summer.Includes bibliographical references.Dengue viruses (DENV) are mosquito-borne viruses that cause a wide range of acute symptoms from mild fever to lethal dengue shock syndrome in humans. DENV are transmitted primarily by Aedes aegypti (Ae. aegypti). These mosquitoes are widely distributed throughout tropical and subtropical areas around the world. Increasing globalization, urbanization and global warming are factors that enhance the spread of these vectors placing over 2.5 billion people at risk of contracting these viruses. Transmission of these viruses depends on their ability to infect, replicate and disseminate into several tissues in the mosquito vector. During DENV infection of its human and mosquito hosts, a visible rearrangement of lipid membrane architecture and alterations of the metabolic repertoire is induced. These events occur to facilitate efficient viral replication and virus assembly within the cell and to circumvent antiviral responses from the host. Interference with these virus-induced processes can be detrimental to virus replication and can prevent viral transmission. In this dissertation, we present the first insight into the metabolic environment induced during DENV serotype 2 (DENV2) replication in Ae. aegypti. Using untargeted high-resolution liquid chromatography-mass spectrometry, we explored the temporal metabolic perturbations that occur following dengue virus infection of the midgut, the primary site of the virus infection in the mosquito vector. Temporal changes of metabolites across early-, mid- and late-infection time points were identified. A marked increase in the /content of glycerophospholipids, sphingolipids and fatty acyls was coincident with the kinetics of viral replication. Elevation of glycerolipid levels and the accumulation of medium-chain acyl-carnitines suggested a diversion of resources during infection from energy storage to synthetic pathways and energy production. From the observations above, two active pathways, sphingolipid and de novo fatty acid synthesis pathways, were further validated to identify metabolic control hubs. Using inhibitor screening of the sphingolipid pathway, we determined that sphingolipid Δ-4 desaturase (DEGS), the enzyme that converts dihydroceramide to ceramide was important for DENV2 infection in cultured Ae. aegypti cells (Aag2). Long, double-stranded RNA-mediated knockdown of DEGS expression led to the imbalance of ceramide to dihydroceramide ratios and affected DENV2 infection in cell culture. However, the inhibitory effect to DENV2 replication was not observed during DEGS-knockdown in mosquito vectors. De novo fatty acid biosynthesis is the pathway that synthesizes the first lipid molecules, fatty acids, required in synthesizing complex lipid molecules, such as glycerophospholipids, glycerolipids and sphingolipids. As a result, this pathway serves as a bottle neck for the control of lipid metabolism. In this study, we annotated and characterized the expression of seven Ae. aegypti fatty acid synthase (AaFAS) genes in the different stages of mosquito development and upon exposure to different diets. We found that AaFAS1 shares the highest amino acid similarity to human fatty acid synthase (FAS) and is the dominant AaFAS that expressed in female mosquitoes. Knockdown expression of AaFAS1 expression showed a reduction in DENV2 replication in the Aag2 cells and in the midgut of Ae. aegypti mosquitoes during early infection. However, the correlation between viral infection and levels of AaFAS1 expression was difficult to elucidate. The work in this dissertation has highlighted metabolic pathways that are induced by DENV2 infection and the metabolic control points within these pathways that are critical for DENV2 infection in Ae. aegypti. Successful perturbation of metabolic homeostasis can potentially limit virus replication in the vector, presenting a novel avenue to block the transmission of DENV2 from the mosquitoes to humans

Molecular determinants of dengue virus type-2 critical for early events in antibody-dependent enhancement of infection

2013 Summer.Includes bibliographical references.Antibody-dependent enhancement (ADE) of infection might be one of the major factors in the development of more severe forms of dengue disease in patients undergoing a secondary infection of dengue virus (DENV). The ADE is caused by cross-reactivity of subneutralizing or non-neutralizing antibodies (Abs), which form virus-Ab complexes and enhance virus infection by binding to the Fcγ receptors (FCγR) on FcγR-bearing cells. The early events in non-ADE infection have been previously studied, but the virus entry pathway and the DENV molecular determinants involved in ADE are still largely unclear. There are two hypotheses for the early entry pathway of ADE infection: (1) Ab opsonized DENV binds to FcγR and directly enters cells through phagocytic pathway; (2) FcγR plays an auxiliary role in concentrating the opsonized virus to the cell surface, but other cellular receptors are still required for virus entry into the endocytosis pathway. Herein, we investigated contributions of the DENV2 E proteins to ADE infection, as well as the role of FcγRIIA in the enhancement of infection and possible entry route of the Ab-opsonized virion. ADE of wild type (WT) DENV2 infection can be promoted in FcγRIIA-bearing K562 cells by using subneutralizing cross-reactive flaviviral monoclonal antibody (MAb), subcomplex DENV MAb or serotype-specific DENV2 MAb against the E protein, as well as non-neutralizing anti-DENV-prM MAb. The enhancement of infection was analyzed by comparing DENV2 infection under ADE conditions with DENV-Ab complex and non-ADE conditions with DENV alone. Numbers of DENV infected cells were determined by flow cytometry of infected cells stained with labeled MAb 2H2-AlexaFluor-488, while increases in viral output were quantified by qRT-PCR of viral genomes. We investigated multiple DENV mutants generated by reverse genetic technology to identify molecular determinants in the envelope protein (E) of DENV-2 that are critical for DENV attachment and viral-endosomal membrane fusion in both non-ADE and ADE infection of the virus in K562 cells. We determined that binding of virus-Ab complex with FcγRIIA alone is not sufficient for virus entry during ADE infection. Furthermore, the molecular determinants of E protein critical for virus entry and virus-mediated endosomal membrane fusion involved in non-ADE infection were also required for ADE infection. ADE was also tested in FcγRIIA-transfected CV-1 cells (CV-1- FcγRIIA). Surprisingly, no enhancement occurred with any of the tested MAb in this cell type. Numbers of cells expressing FcγRIIA and density of FcγRIIA molecules expressed on CV-1- FcγRIIA cell surface were similar to those of K562 cells. These results supported our findings that FcγRIIA alone was not sufficient to enhance viral infection. The results also suggested that cellular components that are present in K562 cells but absent in CV-1- FcγRIIA cells play a major role in ADE of infection

Differential intra-host infection kinetics in Aedes aegypti underlie superior transmissibility of African relative to Asian Zika virus

International audienceDespite numerous studies highlighting the higher transmissibility of the African Zika virus (ZIKV) lineage compared to the Asian lineage in mosquito vectors, little is known about how the viruses interact with different tissues during the early steps of mosquito infection. To address this gap, we aimed to characterize intra-host infection barriers by combining tissue-level monitoring of infection using plaque assays and a novel quantitative analysis of single-cell-level infection kinetics by in situ immunofluorescent staining. Our results revealed that, in Aedes aegypti , an African ZIKV strain exhibited a higher replication rate across various tissues than an Asian ZIKV strain. This difference was potentially due to a higher virus production in individual cells, faster spread within tissues, or a combination of both factors. Furthermore, we observed that higher blood meal titers resulted in a faster viral spread to neighboring cells suggesting that intra-host infection dynamics depend on inoculum size. We also identified a significant bottleneck during midgut infection establishment for both ZIKV lineages, with only a small percentage of the virus population successfully initiating infection. Finally, the in situ immunofluorescent staining technique enabled the examination of virus infection characteristics in different cell types and revealed heterogeneity in viral replication. Together, these findings demonstrate that differences in intra-host infection kinetics underlie differential transmissibility between African and Asian ZIKV lineages. This information could serve as a starting point to further investigate the underlying mechanisms and ultimately inform the development of alternative control strategies. IMPORTANCE The recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell-level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency

Experimental Zika virus infection of Jamaican fruit bats (Artibeus jamaicensis) and possible entry of virus into brain via activated microglial cells.

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 in 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

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy in neuroscience

Introduction: Optical Projection Tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high resolution optical imaging techniques, ideally suited for ex vivo 3D whole mouse brain imaging. Although they exhibit high specificity for their targets, the anatomical detail provided by tissue autofluorescence remains limited. Methods: T1-weighted images were acquired from 19 BABB or DBE cleared brains to create an MR template using serial longitudinal registration. Afterwards, fluorescent OPT and LSFM images were coregistered/normalized to the MR template to create fusion images. Results: Volumetric calculations revealed a significant difference between BABB and DBE cleared brains, leading to develop two optimized templates, with associated tissue priors and brain atlas, for BABB (OCUM) and DBE (iOCUM). By creating fusion images, we identified virus infected brain regions, mapped dopamine transporter and translocator protein expression, and traced innervation from the eye along the optic tract to the thalamus and superior colliculus using cholera toxin B. Fusion images allowed for precise anatomical identification of fluorescent signal in the detailed anatomical context provided by MR. Discussion: The possibility to anatomically map fluorescent signals on magnetic resonance (MR) images, widely used in clinical and preclinical neuroscience, would greatly benefit applications of optical imaging of mouse brain. These specific MR templates for cleared brains enable a broad range of neuroscientific applications integrating 3D optical brain imaging

Type I interferon shapes brain distribution and tropism of tick-borne flavivirus

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

Select strains of East and West African ZIKV have higher fitness <i>in vitro</i> than the American strain PRVABC59.

<p>Equal amounts of virus (MOI 0.01) was used to infect the indicated cell line. Supernatant was harvested and proportion of each virus was determined by sequencing and analyzing chromatograms. Data are presented as proportion of the first virus listed present at the indicated time point in (A) Vero, (B) Huh7, C6/36 (C), and (D) Aag2 cells.</p