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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 including the salivary glands 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. Author summary: When mosquitoes are reared in a laboratory they are typically provided with ample nutrients as larvae so adults can grow to an optimal size; this ensures adults are robust for reproducible experiments. However, in the field not all larvae may have access to equal amounts of food. Studies including ours have shown that by restricting food as larvae, smaller adults can be produced, which can have an altered ability to be infected with and transmit arthropod-borne viruses. Zika virus is ingested into a female mosquito midgut when a blood-meal is acquired from an infected vertebrate host; the virus must infect midgut cells and escape this tissue to secondary tissues via the basal lamina, which surrounds the midgut. Viruses can then infect other organs including the salivary glands, for further transmission. In this study we focus on the impact limited nutrition as a larva has on the adult's transmission potential for Zika virus. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 issue nhibitor of etalloroteinases (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. [ABSTRACT FROM AUTHOR]

In vivo targeted gene disruption is a powerful tool to study gene function. Thus far, two tools for genome editing in Aedes aegypti have been applied, zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN). As a promising alternative to ZFN and TALEN, which are difficult to produce and validate using standard molecular biological techniques, the clustered regularly interspaced short palindromic repeats/CRISPR-associated sequence 9 (CRISPR/Cas9) system has recently been discovered as a "do-it-yourself" genome editing tool. Here, we describe the use of CRISPR/Cas9 in the mosquito vector, Aedes aegypti. In a transgenic mosquito line expressing both Dsred and enhanced cyan fluorescent protein (ECFP) from the eye tissue-specific 3xP3 promoter in separated but tightly linked expression cassettes, we targeted the ECFP nucleotide sequence for disruption. When supplying the Cas9 enzyme and two sgRNAs targeting different regions of the ECFP gene as in vitro transcribed mRNAs for germline transformation, we recovered four different G1 pools (5.5% knockout efficiency) where individuals still expressed DsRed but no longer ECFP. PCR amplification, cloning, and sequencing of PCR amplicons revealed indels in the ECFP target gene ranging from 2-27 nucleotides. These results show for the first time that CRISPR/Cas9 mediated gene editing is achievable in Ae. aegypti, paving the way for further functional genomics related studies in this mosquito species. [ABSTRACT FROM AUTHOR]

In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement. Author Summary: Expression of a DENV2 sequence-derived IR RNA in the mosquito midgut initiates an antiviral intracellular RNAi response that efficiently blocks DENV2 infection and profoundly impairs vector competence for that virus in Aedes aegypti. DENV2-specific IR RNA expression in the Carb109M strain has maintained the RNAi-based, refractory phenotype for 33 generations in laboratory culture. The two transgene integration sites were stable after multiple generations and following introgression into a genetically-diverse (GDLS) Ae. aegypti population. Introgression of the transgene into the GDLS genetic background changed GDLS from a DENV2 susceptible phenotype to a DENV2 refractory phenotype. The DENV2 refractory homozygous line, Carb109M/GDLS.BC5.HZ, exhibits (relative to GDLS) minimal fitness loss associated with the transgene. This line could be a potential candidate for proof-of-principle field studies. [ABSTRACT FROM AUTHOR]

In 2006, we reported a mariner (Mos1)-transformed Aedes aegypti line, Carb77, which was highly resistant to dengue-2 virus (DENV2). Carb77 mosquitoes expressed a DENV2-specific inverted-repeat (IR) RNA in midgut epithelial cells after ingesting an infectious bloodmeal. The IR-RNA formed double-stranded DENV2-derived RNA, initiating an intracellular antiviral RNA interference (RNAi) response. However, Carb77 mosquitoes stopped expressing the IR-RNA after 17 generations in culture and lost their DENV2-refractory phenotype. In the current study, we generated new transgenic lines having the identical transgene as Carb77. One of these lines, Carb109M, has been genetically stable and refractory to DENV2 for >33 generations. Southern blot analysis identified two transgene integration sites in Carb109M. Northern blot analysis detected abundant, transient expression of the IR-RNA 24 h after a bloodmeal. Carb109M mosquitoes were refractory to different DENV2 genotypes but not to other DENV serotypes. To further test fitness and stability, we introgressed the Carb109M transgene into a genetically diverse laboratory strain (GDLS) by backcrossing for five generations and selecting individuals expressing the transgene's EGFP marker in each generation. Comparison of transgene stability in replicate backcross 5 (BC5) lines versus BC1 control lines demonstrated that backcrossing dramatically increased transgene stability. We subjected six BC5 lines to five generations of selection based on EGFP marker expression to increase the frequency of the transgene prior to final family selection. Comparison of the observed transgene frequencies in the six replicate lines relative to expectations from Fisher's selection model demonstrated lingering fitness costs associated with either the transgene or linked deleterious genes. Although minimal fitness loss (relative to GDLS) was manifest in the final family selection stage, we were able to select homozygotes for the transgene in one family, Carb109M/GDLS.BC5.HZ. This family has been genetically stable and DENV2 refractory for multiple generations. Carb109M/GDLS.BC5.HZ represents an important line for testing proof-of-principle vector population replacement. [ABSTRACT FROM AUTHOR]

A number of studies have shown that both innate and adaptive immune defense mechanisms greatly influence the course of human dengue virus (DENV) infections, but little is known about the innate immune response of the mosquito vector Aedes aegypti to arbovirus infection. We present evidence here that a major component of the mosquito innate immune response, RNA interference (RNAi), is an important modulator of mosquito infections. The RNAi response is triggered by double-stranded RNA (dsRNA), which occurs in the cytoplasm as a result of positive-sense RNA virus infection, leading to production of small interfering RNAs (siRNAs). These siRNAs are instrumental in degradation of viral mRNA with sequence homology to the dsRNA trigger and thereby inhibition of virus replication. We show that although dengue virus type 2 (DENV2) infection of Ae. aegypti cultured cells and oral infection of adult mosquitoes generated dsRNA and production of DENV2-specific siRNAs, virus replication and release of infectious virus persisted, suggesting viral circumvention of RNAi. We also show that DENV2 does not completely evade RNAi, since impairing the pathway by silencing expression of dcr2, r2d2, or ago2, genes encoding important sensor and effector proteins in the RNAi pathway, increased virus replication in the vector and decreased the extrinsic incubation period required for virus transmission. Our findings indicate a major role for RNAi as a determinant of DENV transmission by Ae. aegypti. [ABSTRACT FROM AUTHOR]

Current methods for detecting real-time alphavirus (Family Togaviridae) infection in mosquitoes require the use of recombinant viruses engineered to express a visibly detectable reporter protein. These altered viruses expressing fluorescent proteins, usually from a duplicated viral subgenomic reporter, are effective at marking infection but tend to be attenuated due to the modification of the genome. Additionally, field strains of viruses cannot be visualized using this approach unless infectious clones can be developed to insert a reporter protein. To circumvent these issues, we have developed an insect cell-based system for detecting wild-type sindbis virus infection that uses a virus inducible promoter to express a fluorescent reporter gene only upon active virus infection. We have developed an insect expression system that produces sindbis virus minigenomes containing a subgenomic promoter sequence, which produces a translatable RNA species only when infectious virus is present and providing viral replication proteins. This subgenomic reporter RNA system is able to detect wild-type Sindbis infection in cultured mosquito cells. The detection system is relatively species specific and only detects closely related viruses, but can detect low levels of alphavirus specific replication early during infection. A chikungunya virus detection system was also developed that specifically detects chikungunya virus infection. Transgenic Aedes aegypti mosquito families were established that constitutively express the sindbis virus reporter RNA and were found to only express fluorescent proteins during virus infection. This virus inducible reporter system demonstrates a novel approach for detecting non-recombinant virus infection in mosquito cell culture and in live transgenic mosquitoes. [ABSTRACT FROM AUTHOR]