Additive protection by antioxidant and apoptosis-inhibiting effects on mosquito cells with dengue 2 virus infection

Cytopathic effects (CPEs) in mosquito cells are generally trivial compared to those that occur in mammalian cells, which usually end up undergoing apoptosis during dengue virus (DENV) infection. However, oxidative stress was detected in both types of infected cells. Despite this, the survival of mosquito cells benefits from the upregulation of genes related to antioxidant defense, such as glutathione S transferase (GST). A second defense system, i.e., consisting of antiapoptotic effects, was also shown to play a role in protecting mosquito cells against DENV infection. This system is regulated by an inhibitor of apoptosis (IAP) that is an upstream regulator of caspases-9 and -3. DENV-infected C6/36 cells with double knockdown of GST and the IAP showed a synergistic effect on activation of these two caspases, causing a higher rate of apoptosis (> 20%) than those with knockdown of each single gene (-10%). It seems that the IAP acts as a second line of defense with an additional effect on the survival of mosquito cells with DENV infection. Compared to mammalian cells, residual hydrogen peroxide in DENV-infected C6/36 cells may signal for upregulation of the IAP. This novel finding sheds light on virus/cell interactions and their coevolution that may elucidate how mosquitoes can be a vector of DENV and probably most other arboviruses in nature.     

Comparison of dengue virus type 2-specific small RNAs from RNA interference-competent and -incompetent mosquito cells

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts.     

Fine scale spatiotemporal clustering of dengue virus transmission in children and Aedes aegypti in rural Thai villages

Background

Based on spatiotemporal clustering of human dengue virus (DENV) infections, transmission is thought to occur at fine spatiotemporal scales by horizontal transfer of virus between humans and mosquito vectors. To define the dimensions of local transmission and quantify the factors that support it, we examined relationships between infected humans and Aedes aegypti in Thai villages.

Methodology/Principal Findings

Geographic cluster investigations of 100-meter radius were conducted around DENV-positive and DENV-negative febrile “index” cases (positive and negative clusters, respectively) from a longitudinal cohort study in rural Thailand. Child contacts and Ae. aegypti from cluster houses were assessed for DENV infection. Spatiotemporal, demographic, and entomological parameters were evaluated. In positive clusters, the DENV infection rate among child contacts was 35.3% in index houses, 29.9% in houses within 20 meters, and decreased with distance from the index house to 6.2% in houses 80–100 meters away (p<0.001). Significantly more Ae. aegypti were DENV-infectious (i.e., DENV-positive in head/thorax) in positive clusters (23/1755; 1.3%) than negative clusters (1/1548; 0.1%). In positive clusters, 8.2% of mosquitoes were DENV-infectious in index houses, 4.2% in other houses with DENV-infected children, and 0.4% in houses without infected children (p<0.001). The DENV infection rate in contacts was 47.4% in houses with infectious mosquitoes, 28.7% in other houses in the same cluster, and 10.8% in positive clusters without infectious mosquitoes (p<0.001). Ae. aegypti pupae and adult females were more numerous only in houses containing infectious mosquitoes.

Conclusions/Significance

Human and mosquito infections are positively associated at the level of individual houses and neighboring residences. Certain houses with high transmission risk contribute disproportionately to DENV spread to neighboring houses. Small groups of houses with elevated transmission risk are consistent with over-dispersion of transmission (i.e., at a given point in time, people/mosquitoes from a small portion of houses are responsible for the majority of transmission).     

Aedes aegypti SNAP and a calcium transporter ATPase influence dengue virus dissemination

Dengue virus (DENV) is a flavivirus that causes marked human morbidity and mortality worldwide, and is transmitted to humans by Aedes aegypti mosquitoes. Habitat expansion of Aedes, mainly due to climate change and increasing overlap between urban and wild habitats, places nearly half of the world’s population at risk for DENV infection. After a bloodmeal from a DENV-infected host, the virus enters the mosquito midgut. Next, the virus migrates to, and replicates in, other tissues, like salivary glands. Successful viral transmission occurs when the infected mosquito takes another blood meal on a susceptible host and DENV is released from the salivary gland via saliva into the skin. During viral dissemination in the mosquito and transmission to a new mammalian host, DENV interacts with a variety of vector proteins, which are uniquely important during each phase of the viral cycle. Our study focuses on the interaction between DENV particles and protein components in the A. aegypti vector. We performed a mass spectrometry assay where we identified a set of A. aegypti salivary gland proteins which potentially interact with the DENV virion. Using dsRNA to silence gene expression, we analyzed the role of these proteins in viral infectivity. Two of these candidates, a synaptosomal-associated protein (AeSNAP) and a calcium transporter ATPase (ATPase) appear to play a role in viral replication both in vitro and in vivo, observing a ubiquitous expression of these proteins in the mosquito. These findings suggest that AeSNAP plays a protective role during DENV infection of mosquitoes and that ATPase protein is required for DENV during amplification within the vector.     

Cell membrane enolase of Aedes albopictus C6/36 cells is involved in the entrance mechanism of dengue virus (DENV)

Currently, there are no antiviral drugs that effectively reduce the risks and treat the symptoms associated with dengue virus (DENV). Consequently, efforts remain primarily focused on transmission reduction. One such effort concerns DENV receptors in mosquito vectors Aedes aegypti and Aedes albopictus. Despite a lack of direct evidence demonstrating the binding of DENV to cells in mosquito vectors, one putative DENV binding protein has been α-enolase. To develop a deeper understanding, this study tested whether DENV proteins bind to enolase localized in the cytoplasmic membrane of C6/36 cells using both anti-enolase-specific antibodies, and by colocalization analysis, using confocal microscopy.Additionally, to probe the interaction of enolase with the DENV E protein, we performed a docking analysis using PatchDock and FireDock software packages. Study results demonstrate that the DENV E protein interacts with enolase in the plasma membrane of C6/36 cells of Ae. albopictus. Specific anti-enolase antibodies were found to inhibit DENV infection of these cells. Moreover, enolase was found to be localized to the cytoplasmic membrane, cytoplasm, and nucleus. These combined findings suggest that enolase participates in the entrance mechanism of DENV into vector cells.     

Monocyte Recruitment to the Dermis and Differentiation to Dendritic Cells Increases the Targets for Dengue Virus Replication

Dengue virus (DENV) causes the most prevalent arthropod-borne viral disease in humans. Although Aedes mosquitoes transmit DENV when probing for blood in the skin, no information exists on DENV infection and immune response in the dermis, where the blood vessels are found. DENV suppresses the interferon response, replicates, and causes disease in humans but not wild-type mice. Here, we used mice lacking the interferon-α/β receptor (Ifnar ^–/–), which had normal cell populations in the skin and were susceptible to intradermal DENV infection, to investigate the dynamics of early DENV infection of immune cells in the skin. CD103⁺ classical dendritic cells (cDCs), Ly6C^– CD11b⁺ cDCs, and macrophages in the steady-state dermis were initial targets of DENV infection 12-24 hours post-inoculation but then decreased in frequency. We demonstrated recruitment of adoptively-transferred Ly6C^high monocytes from wild-type and Ifnar ^–/– origin to the DENV-infected dermis and differentiation to Ly6C⁺ CD11b⁺ monocyte-derived DCs (moDCs), which became DENV-infected after 48 hours, and were then the major targets for virus replication. Ly6C^high monocytes that entered the DENV-infected dermis expressed chemokine receptor CCR2, likely mediating recruitment. Further, we show that ∼100-fold more hematopoietic cells in the dermis were DENV-infected compared to Langerhans cells in the epidermis. Overall, these results identify the dermis as the main site of early DENV replication and show that DENV infection in the skin occurs in two waves: initial infection of resident cDCs and macrophages, followed by infection of monocytes and moDCs that are recruited to the dermis. Our study reveals a novel viral strategy of exploiting monocyte recruitment to increase the number of targets for infection at the site of invasion in the skin and highlights the skin as a potential site for therapeutic action or intradermal vaccination.     

Using RNA interference to develop dengue virus resistance in genetically modified Aedes aegypti

Diseases caused by arthropod-borne viruses are significant public health problems, and novel methods are needed to control pathogen transmission. We hypothesize that genetic manipulation of Aedes aegypti mosquitoes can profoundly and permanently reduce vector competence and subsequent transmission of dengue viruses (DENV) to human hosts. We have identified RNA interference (RNAi) as a potential anti-viral, intracellular pathway in the vector that can be triggered by expression of virus-specific, double stranded RNAs (dsRNAs) to reduce vector competence to DENV. We identified DENV-derived RNA segments using recombinant Sindbis viruses to trigger RNAi, that when expressed in mosquitoes ablate homologous DENV replication and transmission. We also demonstrated that heritable expression of DENV-derived dsRNA in cultured mosquito cells can silence virus replication. We now have developed a number of transgenic mosquito lines that transcribe the effector dsRNA from constitutive promoters such as immediate early 1 (baculovirus) and polyubiquitin (Drosophila melanogaster). We have detected DENV-specific small interfering RNAs, the hallmark of RNAi, in at least one of these lines. Surprisingly, none of these lines expressed dsRNA in relevant tissues (e.g., midguts) that will ultimately affect transmission. A major challenge now is to express the effector dsRNA from tissue-specific promoters to allow RNAi to silence virus replication at critical sites in the vector such as midguts and salivary glands. If successful, this strategy has the advantage of harnessing a naturally occurring vector response to block DENV infection in a mosquito vector and profoundly affect virus transmission.     

Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

Background

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.

Methodology and Principal Findings

To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.

Conclusions/Significance

Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.     

Dengue Virus Type 2 Infections of Aedes aegypti Are Modulated by the Mosquito's RNA Interference Pathway

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.     

Tamiflu-resistant but HA-mediated cell-to-cell transmission through apical membranes of cell-associated influenza viruses

The infection of viruses to a neighboring cell is considered to be beneficial in terms of evasion from host anti-virus defense systems. There are two pathways for viral infection to "right next door": one is the virus transmission through cell-cell fusion by forming syncytium without production of progeny virions, and the other is mediated by virions without virus diffusion, generally designated cell-to-cell transmission. Influenza viruses are believed to be transmitted as cell-free virus from infected cells to uninfected cells. Here, we demonstrated that influenza virus can utilize cell-to-cell transmission pathway through apical membranes, by handover of virions on the surface of an infected cell to adjacent host cells. Live cell imaging techniques showed that a recombinant influenza virus, in which the neuraminidase gene was replaced with the green fluorescence protein gene, spreads from an infected cell to adjacent cells forming infected cell clusters. This type of virus spreading requires HA activation by protease treatment. The cell-to-cell transmission was also blocked by amantadine, which inhibits the acidification of endosomes required for uncoating of influenza virus particles in endosomes, indicating that functional hemagglutinin and endosome acidification by M2 ion channel were essential for the cell-to-cell influenza virus transmission. Furthermore, in the cell-to-cell transmission of influenza virus, progeny virions could remain associated with the surface of infected cell even after budding, for the progeny virions to be passed on to adjacent uninfected cells. The evidence that cell-to-cell transmission occurs in influenza virus lead to the caution that local infection proceeds even when treated with neuraminidase inhibitors.     

Dengue and Zika virus infection patterns vary among Aedes aegypti field populations from Belo Horizonte,a Brazilian endemic city

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.     

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.     

Suppressing Dengue-2 Infection by Chemical Inhibition of Aedes aegypti Host Factors

Dengue virus host factors (DENV HFs) that are essential for the completion of the infection cycle in the mosquito vector and vertebrate host represent potent targets for transmission blocking. Here we investigated whether known mammalian DENV HF inhibitors could influence virus infection in the arthropod vector A. aegypti. We evaluated the potency of bafilomycin (BAF; inhibitor of vacuolar H+-ATPase (vATPase)), mycophenolic acid (MPA; inhibitor of inosine-5′-monophosphate dehydrogenase (IMPDH)), castanospermine (CAS; inhibitor of glucosidase), and deoxynojirimycin (DNJ; inhibitor of glucosidase) in blocking DENV infection of the mosquito midgut, using various treatment methods that included direct injection, ingestion by sugar feeding or blood feeding, and silencing of target genes by RNA interference (RNAi). Injection of BAF (5 µM) and MPA (25 µM) prior to feeding on virus-infected blood inhibited DENV titers in the midgut at 7 days post-infection by 56% and 60%, and in the salivary gland at 14 days post-infection by 90% and 83%, respectively, while treatment of mosquitoes with CAS or DNJ did not affect susceptibility to the virus. Ingestion of BAF and MPA through a sugar meal or together with an infectious blood meal also resulted in various degrees of virus inhibition. RNAi-mediated silencing of several vATPase subunit genes and the IMPDH gene resulted in a reduced DENV infection, thereby indicating that BAF- and MPA-mediated virus inhibition in adult mosquitoes most likely occurred through the inhibition of these DENV HFs. The route and timing of BAF and MPA administration was essential, and treatment after exposure to the virus diminished the antiviral effect of these compounds. Here we provide proof-of-principle that chemical inhibition or RNAi-mediated depletion of the DENV HFs vATPase and IMPDH can be used to suppress DENV infection of adult A. aegypti mosquitoes, which may translate to a reduction in DENV transmission.     

A Neuron-Specific Antiviral Mechanism Prevents Lethal Flaviviral Infection of Mosquitoes

Mosquitoes are natural vectors for many etiologic agents of human viral diseases. Mosquito-borne flaviviruses can persistently infect the mosquito central nervous system without causing dramatic pathology or influencing the mosquito behavior and lifespan. The mechanism by which the mosquito nervous system resists flaviviral infection is still largely unknown. Here we report that an Aedes aegypti homologue of the neural factor Hikaru genki (AaHig) efficiently restricts flavivirus infection of the central nervous system. AaHig was predominantly expressed in the mosquito nervous system and localized to the plasma membrane of neural cells. Functional blockade of AaHig enhanced Dengue virus (DENV) and Japanese encephalitis virus (JEV), but not Sindbis virus (SINV), replication in mosquito heads and consequently caused neural apoptosis and a dramatic reduction in the mosquito lifespan. Consistently, delivery of recombinant AaHig to mosquitoes reduced viral infection. Furthermore, the membrane-localized AaHig directly interfaced with a highly conserved motif in the surface envelope proteins of DENV and JEV, and consequently interrupted endocytic viral entry into mosquito cells. Loss of either plasma membrane targeting or virion-binding ability rendered AaHig nonfunctional. Interestingly, Culex pipien pallens Hig also demonstrated a prominent anti-flavivirus activity, suggesting a functionally conserved function for Hig. Our results demonstrate that an evolutionarily conserved antiviral mechanism prevents lethal flaviviral infection of the central nervous system in mosquitoes, and thus may facilitate flaviviral transmission in nature.     

Wolbachia Reduces the Transmission Potential of Dengue-Infected Aedes aegypti

BackgroundDengue viruses (DENV) are the causative agents of dengue, the world’s most prevalent arthropod-borne disease with around 40% of the world’s population at risk of infection annually. Wolbachia pipientis, an obligate intracellular bacterium, is being developed as a biocontrol strategy against dengue because it limits replication of the virus in the mosquito. The Wolbachia strain wMel, which has been introduced into the mosquito vector, Aedes aegypti, has been shown to invade and spread to near fixation in field releases. Standard measures of Wolbachia’s efficacy for blocking virus replication focus on the detection and quantification of virus in mosquito tissues. Examining the saliva provides a more accurate measure of transmission potential and can reveal the extrinsic incubation period (EIP), that is, the time it takes virus to arrive in the saliva following the consumption of DENV viremic blood. EIP is a key determinant of a mosquito’s ability to transmit DENVs, as the earlier the virus appears in the saliva the more opportunities the mosquito will have to infect humans on subsequent bites.Conclusions/SignificanceThe saliva-based traits reported here offer more disease-relevant measures of Wolbachia’s effects on the vector and the virus. The lengthening of EIP highlights another means, in addition to the reduction of infection frequencies and DENV titers in mosquitoes, by which Wolbachia should operate to reduce DENV transmission in the field.     

Rift Valley Fever Virus Incorporates the 78 kDa Glycoprotein into Virions Matured in Mosquito C6/36 Cells

Rift Valley fever virus (RVFV), genus Phlebovirus, family Bunyaviridae is a zoonotic arthropod-borne virus able to transition between distant host species, causing potentially severe disease in humans and ruminants. Viral proteins are encoded by three genomic segments, with the medium M segment coding for four proteins: nonstructural NSm protein, two glycoproteins Gn and Gc and large 78 kDa glycoprotein (LGp) of unknown function. Goat anti-RVFV polyclonal antibody and mouse monoclonal antibody, generated against a polypeptide unique to the LGp within the RVFV proteome, detected this protein in gradient purified RVFV ZH501 virions harvested from mosquito C6/36 cells but not in virions harvested from the mammalian Vero E6 cells. The incorporation of LGp into the mosquito cell line - matured virions was confirmed by immune-electron microscopy. The LGp was incorporated into the virions immediately during the first passage in C6/36 cells of Vero E6 derived virus. Our data indicate that LGp is a structural protein in C6/36 mosquito cell generated virions. The protein may aid the transmission from the mosquitoes to the ruminant host, with a possible role in replication of RVFV in the mosquito host. To our knowledge, this is a first report of different protein composition between virions formed in insect C6/36 versus mammalian Vero E6 cells.     

Proteasome Inhibition Suppresses Dengue Virus Egress in Antibody Dependent Infection

The mosquito-borne dengue virus (DENV) is a cause of significant global health burden, with an estimated 390 million infections occurring annually. However, no licensed vaccine or specific antiviral treatment for dengue is available. DENV interacts with host cell factors to complete its life cycle although this virus-host interplay remains to be fully elucidated. Many studies have identified the ubiquitin proteasome pathway (UPP) to be important for successful DENV production, but how the UPP contributes to DENV life cycle as host factors remains ill defined. We show here that proteasome inhibition decouples infectious virus production from viral RNA replication in antibody-dependent infection of THP-1 cells. Molecular and imaging analyses in β-lactone treated THP-1 cells suggest that proteasome function does not prevent virus assembly but rather DENV egress. Intriguingly, the licensed proteasome inhibitor, bortezomib, is able to inhibit DENV titers at low nanomolar drug concentrations for different strains of all four serotypes of DENV in primary monocytes. Furthermore, bortezomib treatment of DENV-infected mice inhibited the spread of DENV in the spleen as well as the overall pathological changes. Our findings suggest that preventing DENV egress through proteasome inhibition could be a suitable therapeutic strategy against dengue.     

A dengue receptor as possible genetic marker of vector competence in <Emphasis Type="Italic">Aedes aegypti</Emphasis>

Background  

Vector competence refers to the intrinsic permissiveness of an arthropod vector for infection, replication and transmission of a virus. Notwithstanding studies of Quantitative Trait Loci (QTL) that influence the ability of Aedes aegypti midgut (MG) to become infected with dengue virus (DENV), no study to date has been undertaken to identify genetic markers of vector competence. Furthermore, it is known that mosquito populations differ greatly in their susceptibility to flaviviruses. Differences in vector competence may, at least in part, be due to the presence of specific midgut epithelial receptors and their identification would be a significant step forward in understanding the interaction of the virus with the mosquito. The first interaction of DENV with the insect is through proteins in the apical membrane of the midgut epithelium resulting in binding and receptor-mediated endocytosis of the virus, and this determines cell permissiveness to infection. The susceptibility of mosquitoes to infection may therefore depend on their specific virus receptors. To study this interaction in Ae. aegypti strains that differ in their vector competence for DENV, we investigated the DS3 strain (susceptible to DENV), the IBO-11 strain (refractory to infection) and the membrane escape barrier strain, DMEB, which is infected exclusively in the midgut epithelial cells.