A safe insect-based Chikungunya fever vaccine affords rapid and durable protection in cynomolgus macaques.

Chikungunya virus (CHIKV), which induces chikungunya fever and chronic arthralgia, is an emerging public health concern. Safe and efficient vaccination strategies are needed to prevent or mitigate virus-associated acute and chronic morbidities for preparation of future outbreaks. Eilat (EILV)/CHIKV, a chimeric alphavirus which contains the structural proteins of CHIKV and the non-structural proteins of EILV, does not replicate in vertebrate cells. The chimeric virus was previously reported to induce protective adaptive immunity in mice. Here, we assessed the capacity of the virus to induce quick and durable protection in cynomolgus macaques. EILV/CHIKV protected macaques from wild-type (WT) CHIKV infection one year after a single dose vaccination. Transcriptome and in vitro functional analyses reveal that the chimeric virus triggered toll-like receptor signaling and T cell, memory B cell and antibody responses in a dose-dependent manner. Notably, EILV/CHIKV preferentially induced more durable, robust, and broader repertoire of CHIKV-specific T cell responses, compared to a live attenuated CHIKV 181/25 vaccine strain. The insect-based chimeric virus did not cause skin hypersensitivity reactions in guinea pigs sensitized to mosquito bites. Furthermore, EILV/CHIKV induced strong neutralization antibodies and protected cynomolgus macaques from WT CHIKV infection within six days post vaccination. Transcriptome analysis also suggest that the chimeric virus induction of multiple innate immune pathways, including Toll-like receptor signaling, type I IFN and IL-12 signaling, antigen presenting cell activation, and NK receptor signaling. Our findings suggest that EILV/CHIKV is a safe, highly efficacious vaccine, and provides both rapid and long-lasting protection in cynomolgus macaques.

Detection of La Crosse Virus In Situ and in Individual Progeny to Assess the Vertical Transmission Potential in Aedes albopictus and Aedes aegypti.

La Crosse virus (LACV) is circulating in the midwestern and southeastern states of the United States and can cause human encephalitis. The main vector of the virus is the eastern tree-hole mosquito, Aedes triseriatus. Ae. albopictus has been also described as a natural LACV vector, while Ae. aegypti has been infected with the virus under laboratory conditions. Here, we compare the vertical transmission potential of LACV in Ae. albopictus and Ae. aegypti, with emphasis given to the ovarian infection patterns that the virus generates in both species. Both mosquito species received artificial bloodmeals containing LACV. At defined time points post-infection/bloodmeal, midguts, head tissue, and ovaries were analyzed for the presence of virus. Viral infection patterns in the ovaries were visualized via immunofluorescence confocal microscopy and immunohistopathology assays using an LACV-specific monoclonal antibody. In Ae. aegypti, LACV was confronted with midgut infection and escape barriers, which were much less pronounced in Ae. albopictus, resulting in a significantly higher prevalence of infection in the latter. Following the ingestion of a single virus-containing bloodmeal, no progeny larvae were found to be virus-infected. Regardless, females of both species showed the presence of LACV antigen in their ovariole sheaths. Furthermore, in a single Ae. albopictus female, viral antigen was associated with the nurse cells inside the primary follicles. Following the ingestion of a second non-infectious bloodmeal at 7- or 10-days post-ingestion of an LACV-containing bloodmeal, more progeny larvae of Ae. albopictus than of Ae. aegypti were virus-infected. LACV antigen was detected in the egg chambers and ovariole sheaths of both mosquito species. Traces of viral antigen were also detected in a few oocytes from Ae. albopictus. The low level of vertical transmission and the majority of the ovarian infection patterns suggested the transovum rather than transovarial transmission (TOT) of the virus in both vector species. However, based on the detection of LACV antigen in follicular tissue and oocytes, there was the potential for TOT among several Ae. albopictus females. Thus, TOT is not a general feature of LACV infection in mosquitoes. Instead, the TOT of LACV seems to be dependent on its particular interaction with the reproductive tissues of a female.

Transgene-induced cell death following dengue-2 virus infection in Aedes aegypti.

Dengue viruses (DENVs) are mosquito-borne flaviviruses causing millions of human infections each year and pose a challenge for public health systems worldwide. Aedes aegypti is the principal vector species transmitting DENVs to humans. Controlling Ae. aegypti is difficult due to the abundance of breeding sites and increasing insecticide resistance in the vector populations. Developing new vector control strategies is critical for decreasing the disease burden. One potential approach is genetically replacing Ae. aegypti populations with vector populations highly resistant to DENV transmission. Here, we focus on an alternative strategy for generating dengue 2 virus (DENV-2) resistance in genetically-modified Ae. aegypti in which the mosquitoes express an inactive form of Michelob_x (Mx), an antagonist of the Inhibitor of Apoptosis (IAP), to induce apoptosis in those cells in which actively replicating DENV-2 is present. The inactive form of Mx was flanked by the RRRRSAG cleavage motif, which was recognized by the NS2B/NS3 protease of the infecting DENV-2 thereby releasing and activating Mx which then induced apoptosis. Our transgenic strain exhibited a significantly higher mortality rate than the non-transgenic control when infected with DENV-2. We also transfected a DNA construct containing inactive Mx fused to eGFP into C6/36 mosquito cells and indirectly observed Mx activation on days 3 and 6 post-DENV-2 infections. There were clear signs that the viral NS2B/NS3 protease cleaved the transgene, thereby releasing Mx protein into the cytoplasm, as was confirmed by the detection of eGFP expression in infected cells. The present study represents proof of the concept that virus infection can be used to induce apoptosis in infected mosquito cells.

Assessing single-locus CRISPR/Cas9-based gene drive variants in the mosquito Aedes aegypti via single-generation crosses and modeling.

The yellow fever mosquito Aedes aegypti is a major vector of arthropod-borne viruses, including dengue, chikungunya, and Zika viruses. A novel approach to mitigate arboviral infections is to generate mosquitoes refractory to infection by overexpressing antiviral effector molecules. Such an approach requires a mechanism to spread these antiviral effectors through a population, for example, by using CRISPR/Cas9-based gene drive systems. Critical to the design of a single-locus autonomous gene drive is that the selected genomic locus is amenable to both gene drive and appropriate expression of the antiviral effector. In our study, we used reverse engineering to target 2 intergenic genomic loci, which had previously shown to be highly permissive for antiviral effector gene expression, and we further investigated the use of 3 promoters (nanos, ß2-tubulin, or zpg) for Cas9 expression. We then quantified the accrual of insertions or deletions (indels) after single-generation crossings, measured maternal effects, and assessed fitness costs associated with various transgenic lines to model the rate of gene drive fixation. Overall, MGDrivE modeling suggested that when an autonomous gene drive is placed into an intergenic locus, the gene drive system will eventually be blocked by the accrual of gene drive blocking resistance alleles and ultimately be lost in the population. Moreover, while genomic locus and promoter selection were critically important for the initial establishment of the autonomous gene drive, it was the fitness of the gene drive line that most strongly influenced the persistence of the gene drive in the simulated population. As such, we propose that when autonomous CRISPR/Cas9-based gene drive systems are anchored in an intergenic locus, they temporarily result in a strong population replacement effect, but as gene drive-blocking indels accrue, the gene drive becomes exhausted due to the fixation of CRISPR resistance alleles.

Identification of the extracellular metallo-endopeptidases ADAM and ADAMTS in the yellow fever mosquito Aedes aegypti.

The mosquito Aedes aegypti is a major vector for dengue, Zika, yellow fever, and chikungunya (CHIKV) viruses, which cause significant morbidity and mortality among human populations in the tropical regions of the world. Following ingestion of a viremic bloodmeal from a vertebrate host, an arbovirus needs to productively infect the midgut epithelium of the mosquito. De novo synthesized virions then exit the midgut by traversing the surrounding basal lamina (BL) in order to disseminate to secondary tissues and infect those. Once the salivary glands are infected, the virus is transmitted to a vertebrate host along with saliva released during probing of the mosquito. Midgut tissue distention due to bloodmeal ingestion leads to remodeling of the midgut structure and facilitates virus dissemination from the organ. Previously, we described the matrix-metalloproteinases (MMP) of Ae. aegypti as zinc ion dependent endopeptidases (Metzincins) and showed MMP activity during midgut BL rearrangement as a consequence of bloodmeal ingestion and subsequent digestion thereby affecting arbovirus dissemination from the midgut. Here we investigate the ADAM/ADAMTS of Ae. aegypti, which form another major group of multi-domain proteinases within the Metzincin superfamily and are active during extra-cellular matrix (ECM) remodeling. Seven different ADAM and five ADAMTS were identified in Ae. aegypti. The functional protein domain structures of the identified mosquito ADAM resembled those of human ADAM10, ADAM12, and ADAM17, while two of the five mosquito ADAMTS had human orthologs. Expression profiling of Ae. aegypti ADAM/ADAMTS in immature forms, whole body-females, midguts, and ovarian tissues showed transcriptional activity of the proteinases during metamorphosis, bloodmeal ingestion/digestion, and female reproduction. Custom-made antibodies to ADAM10a and ADAM12c showed that both were strongly expressed in midgut and ovarian tissues. Furthermore, transient silencing of ADAM12c significantly reduced the carcass infection rate with CHIKV at 24 h post-infection, while silencing of ADAM12a significantly increased viral titers in secondary tissues at the same time point. Our results indicate a functional specificity for several ADAM/ADAMTS in those selected mosquito tissues.

Cellular diversity and gene expression profiles in the male and female brain of Aedes aegypti.

BACKGROUND: Aedes aegypti is a medically-important mosquito vector that transmits arboviruses including yellow fever, dengue, chikungunya, and Zika viruses to humans. The mosquito exhibits typical sexually dimorphic behaviors such as courtship, mating, host seeking, bloodfeeding, and oviposition. All these behaviors are mainly regulated by the brain; however, little is known about the function and neuron composition of the mosquito brain. In this study, we generated an initial atlas of the adult male and female brain of Ae. aegypti using 10xGenomics based single-nucleus RNA sequencing. RESULTS: We identified 35 brain cell clusters in male and female brains, and 15 of those clusters were assigned to known cell types. Identified cell types include glia (astrocytes), Kenyon cells, (ventral) projection neurons, monoaminergic neurons, medulla neurons, and proximal medulla neurons. In addition, the cell type compositions of male and female brains were compared to each other showing that they were quantitatively distinct, as 17 out of 35 cell clusters varied significantly in their cell type proportions. Overall, the transcriptomes from each cell cluster looked very similar between the male and female brain as only up to 25 genes were differentially expressed in these clusters. The sex determination factor Nix was highly expressed in neurons and glia of the male brain, whereas doublesex (dsx) was expressed in all neuron and glia cell clusters of the male and female brain. CONCLUSIONS: An initial cell atlas of the brain of the mosquito Ae. aegypti has been generated showing that the cellular compositions of the male and female brains of this hematophagous insect differ significantly from each other. Although some of the rare brain cell types have not been detected in our single biological replicate, this study provides an important basis for the further development of a complete brain cell atlas as well as a better understanding of the neurobiology of the brains of male and female mosquitoes and their sexually dimorphic behaviors.

Starvation at the larval stage increases the vector competence of Aedes aegypti females for Zika virus.

Aedes aegypti is the primary vector of Zika virus (ZIKV), a flavivirus which typically presents itself as febrile-like symptoms in humans but can also cause neurological and pregnancy complications. The transmission cycle of mosquito-borne arboviruses such as ZIKV requires that various key tissues in the female mosquito get productively infected with the virus before the mosquito can transmit the virus to another vertebrate host. Following ingestion of a viremic blood-meal from a vertebrate, ZIKV initially infects the midgut epithelium before exiting the midgut after blood-meal digestion to disseminate to secondary tissues including the salivary glands. Here we investigated whether smaller Ae. aegypti females resulting from food deprivation as larvae exhibited an altered vector competence for blood-meal acquired ZIKV relative to larger mosquitoes. Midguts from small 'Starve' and large 'Control' Ae. aegypti were dissected to visualize by transmission electron microscopy (TEM) the midgut basal lamina (BL) as physical evidence for the midgut escape barrier showing Starve mosquitoes with a significantly thinner midgut BL than Control mosquitoes at two timepoints. ZIKV replication was inhibited in Starve mosquitoes following intrathoracic injection of virus, however, Starve mosquitoes exhibited a significantly higher midgut escape and population dissemination rate at 9 days post-infection (dpi) via blood-meal, with more virus present in saliva and head tissue than Control by 10 dpi and 14 dpi, respectively. These results indicate that Ae. aegypti developing under stressful conditions potentially exhibit higher midgut infection and dissemination rates for ZIKV as adults, Thus, variation in food intake as larvae is potentially a source for variable vector competence levels of the emerged adults for the virus.

Intrathoracic Inoculation of Zika Virus in Aedes aegypti.

Aedes aegypti mosquitoes are the main vectors of many medically relevant arthropod-borne (arbo) viruses, including Zika (ZIKV), dengue (DENV), and yellow fever (YFV). Vector competence studies with Ae. aegypti often involve challenging mosquitoes with an artificial bloodmeal containing virus and later quantifying viral titer or infectious plaque-forming units (PFU) in various mosquito tissues at relevant time points post-infection. However, Ae. aegypti mosquitoes are known to exhibit midgut infection and escape barriers (MIB and MEB, respectively), which influence the prevalence and titer of a disseminated infection and can introduce unwanted variability into studies analyzing tissues such as the salivary glands. To surmount this challenge, we describe herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus. Our protocol is advantageous for studies that need a large sample size of infected mosquitoes, need to bypass the midgut, or are analyzing salivary gland infection or escape barriers. Graphic abstract: Cartoon depiction of Aedes aegypti intrathoracic inoculation. Figure made with Biorender.com.

Current Effector and Gene-Drive Developments to Engineer Arbovirus-Resistant Aedes aegypti (Diptera: Culicidae) for a Sustainable Population Replacement Strategy in the Field.

Arthropod-borne viruses (arboviruses) such as dengue, Zika, and chikungunya viruses cause morbidity and mortality among human populations living in the tropical regions of the world. Conventional mosquito control efforts based on insecticide treatments and/or the use of bednets and window curtains are currently insufficient to reduce arbovirus prevalence in affected regions. Novel, genetic strategies that are being developed involve the genetic manipulation of mosquitoes for population reduction and population replacement purposes. Population replacement aims at replacing arbovirus-susceptible wild-type mosquitoes in a target region with those that carry a laboratory-engineered antiviral effector to interrupt arboviral transmission in the field. The strategy has been primarily developed for Aedes aegypti (L.), the most important urban arbovirus vector. Antiviral effectors based on long dsRNAs, miRNAs, or ribozymes destroy viral RNA genomes and need to be linked to a robust gene drive to ensure their fixation in the target population. Synthetic gene-drive concepts are based on toxin/antidote, genetic incompatibility, and selfish genetic element principles. The CRISPR/Cas9 gene editing system can be configurated as a homing endonuclease gene (HEG) and HEG-based drives became the preferred choice for mosquitoes. HEGs are highly allele and nucleotide sequence-specific and therefore sensitive to single-nucleotide polymorphisms/resistant allele formation. Current research efforts test new HEG-based gene-drive designs that promise to be less sensitive to resistant allele formation. Safety aspects in conjunction with gene drives are being addressed by developing procedures that would allow a recall or overwriting of gene-drive transgenes once they have been released.

Heterogeneity of midgut cells and their differential responses to blood meal ingestion by the mosquito, Aedes aegypti.

Mosquitoes are the most notorious hematophagous insects and due to their blood feeding behavior and genetic compatibility, numerous mosquito species are highly efficient vectors for certain human pathogenic parasites and viruses. The mosquito midgut is the principal organ of blood meal digestion and nutrient absorption. It is also the initial site of infection with blood meal acquired parasites and viruses. We conducted an analysis based on single-nucleus RNA sequencing (snRNA-Seq) to assess the cellular diversity of the midgut and how individual cells respond to blood meal ingestion to facilitate its digestion. Our study revealed the presence of 20 distinguishable cell-type clusters in the female midgut of Aedes aegypti. The identified cell types included intestinal stem cells (ISC), enteroblasts (EB), differentiating EB (dEB), enteroendocrine cells (EE), enterocytes (EC), EC-like cells, cardia cells, and visceral muscle (VM) cells. Blood meal ingestion dramatically changed the overall midgut cell type composition, profoundly increasing the proportions of ISC and three EC/EC-like clusters. In addition, transcriptional profiles of all cell types were strongly affected while genes involved in various metabolic processes were significantly upregulated. Our study provides a basis for further physiological and molecular studies on blood digestion, nutrient absorption, and cellular homeostasis in the mosquito midgut.

The Antiviral Small-Interfering RNA Pathway Induces Zika Virus Resistance in Transgenic Aedes aegypti.

The resurgence of arbovirus outbreaks across the globe, including the recent Zika virus (ZIKV) epidemic in 2015-2016, emphasizes the need for innovative vector control methods. In this study, we investigated ZIKV susceptibility to transgenic Aedes aegypti engineered to target the virus by means of the antiviral small-interfering RNA (siRNA) pathway. The robustness of antiviral effector expression in transgenic mosquitoes is strongly influenced by the genomic insertion locus and transgene copy number; we therefore used CRISPR/Cas9 to re-target a previously characterized locus (Chr2:321382225) and engineered mosquitoes expressing an inverted repeat (IR) dsRNA against the NS3/4A region of the ZIKV genome. Small RNA analysis revealed that the IR effector triggered the mosquito's siRNA antiviral pathway in bloodfed females. Nearly complete (90%) inhibition of ZIKV replication was found in vivo in both midguts and carcasses at 7 or 14 days post-infection (dpi). Furthermore, significantly fewer transgenic mosquitoes contained ZIKV in their salivary glands (p = 0.001), which led to a reduction in the number of ZIKV-containing saliva samples as measured by transmission assay. Our work shows that Ae. aegypti innate immunity can be co-opted to engineer mosquitoes resistant to ZIKV.

Quantitative Proteomic Analysis of Chikungunya Virus-Infected Aedes aegypti Reveals Proteome Modulations Indicative of Persistent Infection.

The mosquito-borne chikungunya virus (CHIKV) poses a threat to human health in tropical countries throughout the world. The molecular interactions of CHIKV with its mosquito vector Aedes aegypti are not fully understood. Following oral acquisition of CHIKV via salinemeals, we analyzed changes in the proteome of Ae. aegypti in 12 h intervals by label-free quantification using a timsTOF Pro mass spectrometer. For each of the seven time points, between 2647 and 3167 proteins were identified among CHIKV-infected and noninfected mosquito samples, and fewer than 6% of those identified proteins were affected by the virus. Functional enrichment analysis revealed that the three pathways, Endocytosis, Oxidative phosphorylation, and Ribosome biogenesis, were enriched during CHIKV infection. On the other hand, three pathways of the cellular RNA machinery and five metabolism related pathways were significantly attenuated in the CHIKV-infected samples. Furthermore, proteins associated with cytoskeleton and vesicular transport, as well as various serine-type endopeptidases and metallo-proteinases, were modulated in the presence of CHIKV. Our study reveals biological pathways and novel proteins interacting with CHIKV in the mosquito. Overall, CHIKV infection caused minor changes to the mosquito proteome demonstrating a high level of adaption between the vector and the virus, essentially coexisting in a nonpathogenic relationship. The mass spectrometry data have been deposited to the MassIVE repository (https://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=abfd14f7015243c69854731998d55df1) with the data set identifier MSV000085115.

Antiviral Effectors and Gene Drive Strategies for Mosquito Population Suppression or Replacement to Mitigate Arbovirus Transmission by Aedes aegypti.

The mosquito vector Aedes aegypti transmits arthropod-borne viruses (arboviruses) of medical importance, including Zika, dengue, and yellow fever viruses. Controlling mosquito populations remains the method of choice to prevent disease transmission. Novel mosquito control strategies based on genetically manipulating mosquitoes are being developed as additional tools to combat arbovirus transmission. Genetic control of mosquitoes includes two basic strategies: population suppression and population replacement. The former aims to eliminate mosquito populations while the latter aims to replace wild populations with engineered, pathogen-resistant mosquitoes. In this review, we outline suppression strategies being applied in the field, as well as current antiviral effector genes that have been characterized and expressed in transgenic Ae. aegypti for population replacement. We discuss cutting-edge gene drive technologies that can be used to enhance the inheritance of effector genes, while highlighting the challenges and opportunities associated with gene drives. Finally, we present currently available models that can estimate mosquito release numbers and time to transgene fixation for several gene drive systems. Based on the recent advances in genetic engineering, we anticipate that antiviral transgenic Ae. aegypti exhibiting gene drive will soon emerge; however, close monitoring in simulated field conditions will be required to demonstrate the efficacy and utility of such transgenic mosquitoes.

Zika Virus Dissemination from the Midgut of Aedes aegypti is Facilitated by Bloodmeal-Mediated Structural Modification of the Midgut Basal Lamina.

The arboviral disease cycle requires that key tissues in the arthropod vector become persistently infected with the virus. The midgut is the first organ in the mosquito that needs to be productively infected with an orally acquired virus. Following midgut infection, the virus then disseminates to secondary tissues including the salivary glands. Once these are productively infected, the mosquito is able to transmit the virus to a vertebrate host. Recently, we described the midgut dissemination pattern for chikungunya virus in Aedes aegypti. Here we assess the dissemination pattern in the same mosquito species for Zika virus (ZIKV), a human pathogenic virus belonging to the Flaviviridae. ZIKV infection of secondary tissues, indicative of dissemination from the midgut, was not observed before 72 h post infectious bloodmeal (pibm). Virion accumulation at the midgut basal lamina (BL) was only sporadic, although at 96-120 h pibm, virions were frequently observed between strands of the BL indicative of their dissemination. Our data suggest that ZIKV dissemination from the mosquito midgut occurs after digestion of the bloodmeal. Using gold-nanoparticles of 5 nm and 50 nm size, we show that meal ingestion leads to severe midgut tissue distention, causing the mesh width of the BL to remain enlarged after complete digestion of the meal. This could explain how ZIKV can exit the midgut via the BL after bloodmeal digestion. Ingestion of a subsequent, non-infectious bloodmeal five days after acquisition of an initial, dengue 4 virus containing bloodmeal resulted in an increased number of virions present in the midgut epithelium adjacent to the BL. Thus, subsequent bloodmeal ingestion by an infected mosquito may primarily stimulate de novo synthesis of virions leading to increased viral titers in the vector.

Infection Pattern of Mayaro Virus in Aedes aegypti (Diptera: Culicidae) and Transmission Potential of the Virus in Mixed Infections With Chikungunya Virus.

Mayaro virus (MAYV; Togaviridae; Alphavirus) has drawn increasing attention as an arthropod-borne virus with potential to cause outbreaks among the human populations of the Western Hemisphere. In the tropical regions of Central and South America, the virus exists in sylvatic cycles between mosquitoes and primate reservoirs such as marmosets. Although forest-dwelling mosquitoes are regarded as important vectors for MAYV, it has been shown previously that the virus can infect and potentially be transmitted by the mosquitoes, Aedes aegypti and Aedes albopictus (Diptera: Culicidae). Here, we compare the infection and transmission efficiencies of two MAYV strains, IQT 4235 from Iquitos, Peru ('IQT') and the type strain of MAYV from Trinidad, TRVL 4675 ('TRVL') in two laboratory-adapted Ae. aegypti strains, Higgs White Eye and Orlando. The TRVL strain was less efficiently transmitted by both mosquito strains than MAYV IQT. Based on the full-length nucleotide sequences of the two viral genomes, we show that the TRVL prototype strain of MAYV is phylogenetically ancestral and more distantly related to the IQT strain. The TRVL strain efficiently infected wild-type Ae. albopictus from Missouri and readily disseminated in those. Considering scenarios in which natural MAYV transmission cycles may overlap with those of chikungunya virus (CHIKV; Togaviridae; Alphavirus), we assessed the effects of mixed infections of the two viruses in mosquitoes based on coinfection or superinfection. Although coinfection had no measurable effect on the transmission potential of either virus, we observed superinfection exclusion for CHIKV in MAYV-infected mosquitoes but not for MAYV in CHIKV-infected mosquitoes.

Ultrastructural Analysis of Chikungunya Virus Dissemination from the Midgut of the Yellow Fever Mosquito, Aedes aegypti.

The transmission cycle of chikungunya virus (CHIKV) requires that mosquito vectors get persistently infected with the virus, following its oral acqsuisition from a vertebrate host. The mosquito midgut is the initial organ that gets infected with orally acquired CHIKV. Following its replication in the midgut epithelium, the virus exits the midgut and infects secondary tissues including the salivary glands before being transmitted to another host. Here, we investigate the pattern of CHIKV dissemination from the midgut of Aedes aegypti at the ultrastructural level. Bloodmeal ingestion caused overstretching of the midgut basal lamina (BL), which was disrupted in areas adjacent to muscles surrounding the midgut as shown by scanning electron microscopy (SEM). Using both transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM) to analyze midgut preparations, mature chikungunya (CHIK) virions were found accumulating at the BL and within strands of the BL at 24⁻32 h post-infectious bloodmeal (pibm). From 48 h pibm onwards, virions no longer congregated at the BL and became dispersed throughout the basal labyrinth of the epithelial cells. Ingestion of a subsequent, non-infectious bloodmeal caused mature virions to congregate again at the midgut BL. Our study suggests that CHIKV needs a single replication cycle in the midgut epithelium before mature virions directly traverse the midgut BL during a relatively narrow time window, within 48 h pibm.

African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast.

Zika virus (ZIKV) drew worldwide attention when a recent epidemic was linked to fetal microcephaly. Here we used human embryonic stem cell derived trophoblasts as a model for primitive placental trophoblast to test the hypothesis that there are differences in how the two genetically distinct ZIKV lineages, African (AF) and Asian (AS), target the human placenta. Upon infection with three AF (ib-H30656, SEN/1984/41525-DAK, and MR-766) and three AS (FSS13025, MexI-44, and PANcdc259249) ZIKV strains, we observed that severe placental cell lysis was only induced after infection with AF strains, while viral replication rates remained similar between both lineages. Differences in cytopathic effects (CPE) were not observed in Vero cells, indicating that the AF strains were not inherently superior at cell lysis. Taken together, we propose that infection with AF strains of ZIKV early in pregnancy would likely result in pregnancy loss, rather than allow further fetal development with accompanying brain damage. Our results also suggest that the long term laboratory-adapted MR-766 strain does not behave aberrantly in cell culture relative to other AF lineage strains.

Chikungunya virus dissemination from the midgut of Aedes aegypti is associated with temporal basal lamina degradation during bloodmeal digestion.

In the mosquito, the midgut epithelium is the initial tissue to become infected with an arthropod-borne virus (arbovirus) that has been acquired from a vertebrate host along with a viremic bloodmeal. Following its replication in midgut epithelial cells, the virus needs to exit the midgut and infect secondary tissues including the salivary glands before it can be transmitted to another vertebrate host. The viral exit mechanism from the midgut, the midgut escape barrier (MEB), is poorly understood although it is an important determinant of mosquito vector competence for arboviruses. Using chikungunya virus (CHIKV) as a model in Aedes aegypti, we demonstrate that the basal lamina (BL) of the extracellular matrix (ECM) surrounding the midgut constitutes a potential barrier for the virus. The BL, predominantly consisting of collagen IV and laminin, becomes permissive during bloodmeal digestion in the midgut lumen. Bloodmeal digestion, BL permissiveness, and CHIKV dissemination are coincident with increased collagenase activity, diminished collagen IV abundance, and BL shredding in the midgut between 24-32 h post-bloodmeal. This indicates that there may be a window-of-opportunity during which the MEB in Ae. aegypti becomes permissive for CHIKV. Matrix metalloproteinases (MMPs) are the principal extracellular endopeptidases responsible for the degradation/remodeling of the ECM including the BL. We focused on Ae. aegypti (Ae)MMP1, which is expressed in midgut epithelial cells, is inducible upon bloodfeeding, and shows collagenase (gelatinase) activity. However, attempts to inhibit AeMMP activity in general or specifically that of AeMMP1 did not seem to affect its function nor produce an altered midgut escape phenotype. As an alternative, we silenced and overexpressed the Ae. aegypti tissue inhibitor of metalloproteinases (AeTIMP) in the mosquito midgut. AeTIMP was highly upregulated in the midgut during bloodmeal digestion and was able to inhibit MMP activity in vitro. Bloodmeal-inducible, midgut-specific overexpression of AeTIMP or its expression via a recombinant CHIKV significantly increased midgut dissemination rates of the virus. Possibly, AeTIMP overexpression affected BL degradation and/or restoration thereby increasing the midgut dissemination efficiency of the virus.

The midgut transcriptome of Aedes aegypti fed with saline or protein meals containing chikungunya virus reveals genes potentially involved in viral midgut escape.

BACKGROUND: The mosquito Aedes aegypti is the primary vector for medically important arthropod-borne viruses, including chikungunya virus (CHIKV). Following oral acquisition, an arbovirus has to persistently infect several organs in the mosquito before becoming transmissible to another vertebrate host. A major obstacle an arbovirus has to overcome during its infection cycle inside the mosquito is the midgut escape barrier, representing the exit mechanism arboviruses utilize when disseminating from the midgut. To understand the transcriptomic basis of midgut escape and to reveal genes involved in the process, we conducted a comparative transcriptomic analysis of midgut samples from mosquitoes which had received a saline meal (SM) or a protein meal (PM) (not) containing CHIKV. RESULTS: CHIKV which was orally acquired by a mosquito along with a SM or PM productively infected the midgut epithelium and disseminated to secondary tissues. A total of 27 RNA-Seq libraries from midguts of mosquitoes that had received PM or SM (not) containing CHIKV at 1 and 2 days post-feeding were generated and sequenced. Fewer than 80 genes responded differentially to the presence of CHIKV in midguts of mosquitoes that had acquired the virus along with SM or PM. SM feeding induced differential expression (DE) of 479 genes at day 1 and 314 genes at day 2 when compared to midguts of sugarfed mosquitoes. By comparison, PM feeding induced 6029 DE genes at day 1 and 7368 genes at day 2. Twenty-three DE genes encoding trypsins, metalloproteinases, and serine-type endopeptidases were significantly upregulated in midguts of mosquitoes at day 1 following SM or PM ingestion. Two of these genes were Ae. aegypti late trypsin (AeLT) and serine collagenase 1 precursor (AeSP1). In vitro, recombinant AeLT showed strong matrix metalloproteinase activity whereas recombinant AeSP1 did not. CONCLUSIONS: By substituting a bloodmeal for SM, we identified midgut-expressed genes not involved in blood or protein digestion. These included genes coding for trypsins, metalloproteinases, and serine-type endopeptidases, which could be involved in facilitating midgut escape for arboviruses in Ae. aegypti. The presence of CHIKV in any of the ingested meals had relatively minor effects on the overall gene expression profiles in midguts.

Vulnerability of primitive human placental trophoblast to Zika virus.

Infection of pregnant women by Asian lineage strains of Zika virus (ZIKV) has been linked to brain abnormalities in their infants, yet it is uncertain when during pregnancy the human conceptus is most vulnerable to the virus. We have examined two models to study susceptibility of human placental trophoblast to ZIKV: cytotrophoblast and syncytiotrophoblast derived from placental villi at term and colonies of trophoblast differentiated from embryonic stem cells (ESC). The latter appear to be analogous to the primitive placenta formed during implantation. The cells from term placentas, which resist infection, do not express genes encoding most attachment factors implicated in ZIKV entry but do express many genes associated with antiviral defense. By contrast, the ESC-derived trophoblasts possess a wide range of attachment factors for ZIKV entry and lack components of a robust antiviral response system. These cells, particularly areas of syncytiotrophoblast within the colonies, quickly become infected, produce infectious virus and undergo lysis within 48 h after exposure to low titers (multiplicity of infection > 0.07) of an African lineage strain (MR766 Uganda: ZIKVU) considered to be benign with regards to effects on fetal development. Unexpectedly, lytic effects required significantly higher titers of the presumed more virulent FSS13025 Cambodia (ZIKVC). Our data suggest that the developing fetus might be most vulnerable to ZIKV early in the first trimester before a protective zone of mature villous trophoblast has been established. Additionally, MR766 is highly trophic toward primitive trophoblast, which may put the early conceptus of an infected mother at high risk for destruction.