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.     

Broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells

Background

Dendritic cells (DC), present in the skin, are the first target cells of dengue virus (DENV). Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) is present on DC and recognizes N-glycosylation sites on the E-glycoprotein of DENV. Thus, the DC-SIGN/E-glycoprotein interaction can be considered as an important target for inhibitors of viral replication. We evaluated various carbohydrate-binding agents (CBAs) against all four described serotypes of DENV replication in Raji/DC-SIGN⁺ cells and in monocyte-derived DC (MDDC).

Methodology/Principal Findings

A dose-dependent anti-DENV activity of the CBAs Hippeastrum hybrid (HHA), Galanthus nivalis (GNA) and Urtica dioica (UDA), but not actinohivin (AH) was observed against all four DENV serotypes as analyzed by flow cytometry making use of anti-DENV antibodies. Remarkably, the potency of the CBAs against DENV in MDDC cultures was significantly higher (up to 100-fold) than in Raji/DC-SIGN⁺ cells. Pradimicin-S (PRM-S), a small-size non-peptidic CBA, exerted antiviral activity in MDDC but not in Raji/DC-SIGN⁺ cells. The CBAs act at an early step of DENV infection as they bind to the viral envelope of DENV and subsequently prevent virus attachment. Only weak antiviral activity of the CBAs was detected when administered after the virus attachment step. The CBAs were also able to completely prevent the cellular activation and differentiation process of MDDC induced upon DENV infection.

Conclusions/Significance

The CBAs exerted broad spectrum antiviral activity against the four DENV serotypes, laboratory-adapted viruses and low passage clinical isolates, evaluated in Raji/DC-SIGN⁺ cells and in primary MDDC.     

Asymmetric competitive suppression between strains of dengue virus

Background  

Within-host competition between strains of a vector-borne pathogen can affect strain frequencies in both the host and vector, thereby affecting viral population dynamics. However little is known about inter-strain competition in one of the most genetically diverse and epidemiologically important mosquito-borne RNA virus: dengue virus (DENV). To assess the strength and symmetry of intra-host competition among different strains of DENV, the effect of mixed infection of two DENV serotypes, DENV2 and DENV4, on the replication of each in cultured mosquito cells was tested. The number of infectious particles produced by each DENV strain in mixed infections was compared to that in single infections to determine whether replication of each strain was decreased in the presence of the other strain (i.e., competition). The two DENV strains were added to cells either simultaneously (coinfection) or with a 1 or 6-hour time lag between first and second serotype (superinfection).     

Production of Infectious Dengue Virus in Aedes aegypti Is Dependent on the Ubiquitin Proteasome Pathway

Dengue virus (DENV) relies on host factors to complete its life cycle in its mosquito host for subsequent transmission to humans. DENV first establishes infection in the midgut of Aedes aegypti and spreads to various mosquito organs for lifelong infection. Curiously, studies have shown that infectious DENV titers peak and decrease thereafter in the midgut despite relatively stable viral genome levels. However, the mechanisms that regulate this decoupling of infectious virion production from viral RNA replication have never been determined. We show here that the ubiquitin proteasome pathway (UPP) plays an important role in regulating infectious DENV production. Using RNA interference studies, we show in vivo that knockdown of selected UPP components reduced infectious virus production without altering viral RNA replication in the midgut. Furthermore, this decoupling effect could also be observed after RNAi knockdown in the head/thorax of the mosquito, which otherwise showed direct correlation between infectious DENV titer and viral RNA levels. The dependence on the UPP for successful DENV production is further reinforced by the observed up-regulation of key UPP molecules upon DENV infection that overcome the relatively low expression of these genes after a blood meal. Collectively, our findings indicate an important role for the UPP in regulating DENV production in the mosquito vector.     

BST2/Tetherin Inhibits Dengue Virus Release from Human Hepatoma Cells

Type I interferons (IFN) have been shown to play an important role for inhibiting Dengue virus (DENV) infection. Identifying IFN-induced cellular proteins are essential for understanding its mechanisms against DENV. Here we established stable Huh7-derived cell lines expressing the IFN-induced cell membrane protein BST2 (Huh7-BST2) or its variant bearing a V5 tag at the C-terminal (Huh7-BST5CV5). These cell lines were infected with DENV to determine proteins modulating their anti-DENV response. We found that expression of BST2 did not affect the efficiency of DENV infection and intracellular replication. Rather, it significantly reduced the virion yield of the infected cells, particularly at low MOI infection. In addition, BST2 also decreased the foci formation and the size of infectious foci in cultured Huh7 monolayers with media containing methocellulose. The addition of the V5 tag at C-terminal inhibited the GPI modification of BST2 and blocked its shift from endoplasm to cytoplastic membrane. BST2CV5 did not affect DENV infection and foci formation in Huh7 cells but reduced virion yield by 1 log at low MOI infection. Interestingly, intracellular BST2CV5 expression was reduced by high level of DENV production.

Conclusion

Our results imply that BST2 is a functional mediator of the IFN response against DENV infection. BST2 inhibits the release of DENV virions from Huh7 cells and limits viral cell-to-cell transmission. BST2CV5 variant is unable to inhibit DENV release but impairs viral infection in cells.     

Antiviral activity of an N-allyl acridone against dengue virus

Background

Dengue virus (DENV), a member of the family Flaviviridae, is at present the most widespread causative agent of a human viral disease transmitted by mosquitoes. Despite the increasing incidence of this pathogen, there are no antiviral drugs or vaccines currently available for treatment or prevention. In a previous screening assay, we identified a group of N-allyl acridones as effective virus inhibitors. Here, the antiviral activity and mode of action targeted to viral RNA replication of one of the most active DENV-2 inhibitors was further characterized.

Results

The compound 10-allyl-7-chloro-9(10H)-acridone, designated 3b, was active to inhibit the in vitro infection of Vero cells with the four DENV serotypes, with effective concentration 50% (EC₅₀) values in the range 12.5-27.1 μM, as determined by virus yield inhibition assays. The compound was also effective in human HeLa cells. No cytotoxicity was detected at 3b concentrations up to 1000 μM. Mechanistic studies demonstrated that virus entry into the host cell was not affected, whereas viral RNA synthesis was strongly inhibited, as quantified by real time RT-PCR. The addition of exogenous guanosine together with 3b rescued only partially the infectivity of DENV-2.

Conclusions

The acridone derivative 3b selectively inhibits the infection of Vero cells with the four DENV serotypes without a direct interaction with the host cell or the virion but interfering specifically with the intracellular virus multiplication. The mode of antiviral action for this acridone apparently involves the cellular enzyme inosine-monophospahe dehydrogenase together with another still unidentified target related to DENV RNA synthesis.     

Activation of the innate immune response against DENV in normal non-transformed human fibroblasts

Background

When mosquitoes infected with DENV are feeding, the proboscis must traverse the epidermis several times (“probing”) before reaching a blood vessel in the dermis. During this process, the salivary glands release the virus, which is likely to interact first with cells of the various epidermal and dermal layers, cells which could be physiologically relevant to DENV infection and replication in humans. However, important questions are whether more abundant non-hematopoietic cells such as fibroblasts become infected, and whether they play any role in antiviral innate immunity in the very early stages of infection, or even if they might be used by DENV as primary replication cells.

Methodology/Principal Findings

Fibroblasts freshly released from healthy skin and infected 12 hours after their isolation show a positive signal for DENV. In addition, when primary skin fibroblast cultures were established and subsequently infected, we showed DENV-2 antigen-positive intracellular signal at 24 hours and 48 hours post-infection. Moreover, the fibroblasts showed productive infection in a conventional plaque assay. The skin fibroblasts infected with DENV-2 underwent potent signaling through both TLR3 and RIG- 1, but not Mda5, triggering up-regulation of IFNβ, TNFα, defensin 5 (HB5) and β defensin 2 (HβD2). In addition, DENV infected fibroblasts showed increased nuclear translocation of interferon (IFN) regulatory factor 3 (IRF3), but not interferon regulatory factor 7 (IRF7), when compared with mock-infected fibroblasts.

Conclusions/Significance

In this work, we demonstrated the high susceptibility to DENV infection by primary fibroblasts from normal human skin, both in situ and in vitro. Our results suggest that these cells may contribute to the pro-inflammatory and anti-viral microenvironment in the early stages of interaction with DENV-2. Furthermore, the data suggest that fibroblast may also be used as a primary site of DENV replication and provide viral particles that may contribute to subsequent viral dissemination.     

Limited Dengue Virus Replication in Field-Collected Aedes aegypti Mosquitoes Infected with Wolbachia

Introduction

Dengue is one of the most widespread mosquito-borne diseases in the world. The causative agent, dengue virus (DENV), is primarily transmitted by the mosquito Aedes aegypti, a species that has proved difficult to control using conventional methods. The discovery that A. aegypti transinfected with the wMel strain of Wolbachia showed limited DENV replication led to trial field releases of these mosquitoes in Cairns, Australia as a biocontrol strategy for the virus.

Methodology/Principal Findings

Field collected wMel mosquitoes that were challenged with three DENV serotypes displayed limited rates of body infection, viral replication and dissemination to the head compared to uninfected controls. Rates of dengue infection, replication and dissemination in field wMel mosquitoes were similar to those observed in the original transinfected wMel line that had been maintained in the laboratory. We found that wMel was distributed in similar body tissues in field mosquitoes as in laboratory ones, but, at seven days following blood-feeding, wMel densities increased to a greater extent in field mosquitoes.

Conclusions/Significance

Our results indicate that virus-blocking is likely to persist in Wolbachia-infected mosquitoes after their release and establishment in wild populations, suggesting that Wolbachia biocontrol may be a successful strategy for reducing dengue transmission in the field.     

Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.     

Nonstructural protein Pns4 of rice dwarf virus is essential for viral infection in its insect vector

Background

Rice dwarf virus (RDV), a plant reovirus, is mainly transmitted by the green rice leafhopper, Nephotettix cincticeps, in a persistent-propagative manner. Plant reoviruses are thought to replicate and assemble within cytoplasmic structures called viroplasms. Nonstructural protein Pns4 of RDV, a phosphoprotein, is localized around the viroplasm matrix and forms minitubules in insect vector cells. However, the functional role of Pns4 minitubules during viral infection in insect vector is still unknown yet.

Methods

RNA interference (RNAi) system targeting Pns4 gene of RDV was conducted. Double-stranded RNA (dsRNA) specific for Pns4 gene was synthesized in vitro, and introduced into cultured leafhopper cells by transfection or into insect body by microinjection. The effects of the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene on viral replication and spread in cultured cells and insect vector were analyzed using immunofluorescence, western blotting or RT-PCR assays.

Results

In cultured leafhopper cells, the knockdown of Pns4 expression due to RNAi induced by synthesized dsRNA from Pns4 gene strongly inhibited the formation of minitubules, preventing the accumulation of viroplasms and efficient viral infection in insect vector cells. RNAi induced by microinjection of dsRNA from Pns4 gene significantly reduced the viruliferous rate of N. cincticeps. Furthermore, it also strongly inhibited the formation of minitubules and viroplasms, preventing efficient viral spread from the initially infected site in the filter chamber of intact insect vector.

Conclusions

Pns4 of RDV is essential for viral infection and replication in insect vector. It may directly participate in the functional role of viroplasm for viral replication and assembly of progeny virions during viral infection in leafhopper vector.

     

Rise and fall of vector infectivity during sequential strain displacements by mosquito‐borne dengue virus

Each of the four serotypes of mosquito‐borne dengue virus (DENV‐1‐4) comprises multiple, genetically distinct strains. Competitive displacement between strains within a serotype is a common feature of DENV epidemiology and can trigger outbreaks of dengue disease. We investigated the mechanisms underlying two sequential displacements by DENV‐3 strains in Sri Lanka that each coincided with abrupt increases in dengue haemorrhagic fever (DHF) incidence. First, the post‐DHF strain displaced the pre‐DHF strain in the 1980s. We have previously shown that post‐DHF is more infectious than pre‐DHF for the major DENV vector, Aedes aegypti. Then, the ultra‐DHF strain evolved in situ from post‐DHF and displaced its ancestor in the 2000s. We predicted that ultra‐DHF would be more infectious for Ae. aegypti than post‐DHF but found that ultra‐DHF infected a significantly lower percentage of mosquitoes than post‐DHF. We therefore hypothesized that ultra‐DHF had effected displacement by disseminating in Ae. aegypti more rapidly than post‐DHF, but this was not borne out by a time course of mosquito infection. To elucidate the mechanisms that shape these virus–vector interactions, we tested the impact of RNA interference (RNAi), the principal mosquito defence against DENV, on replication of each of the three DENV strains. Replication of all strains was similar in mosquito cells with dysfunctional RNAi, but in cells with functional RNAi, replication of pre‐DHF was significantly suppressed relative to the other two strains. Thus, differences in susceptibility to RNAi may account for the differences in mosquito infectivity between pre‐DHF and post‐DHF, but other mechanisms underlie the difference between post‐DHF and ultra‐DHF.     

Autophagy Facilitates Antibody-Enhanced Dengue Virus Infection in Human Pre-Basophil/Mast Cells

Background

Dengue virus (DENV) infection can cause severe hemorrhagic disease in humans. Although the pathogenic mechanisms underlying severe DENV disease remain unclear, one of the possible contributing factors is antibody-dependent enhancement (ADE) which occurs when sub-neutralizing antibodies derived from a previous DENV infection enhance viral infection through interaction between virus-antibody complexes and FcR-bearing cells, such as macrophages and basophil/mast cells. Although recent reports showed that DENV induces autophagy, the relationship between antibody-enhanced DENV infection and autophagy is not clear.

Methodology/Principal Findings

We showed that sub-neutralizing antibodies derived from dengue patient sera enhanced DENV infection and autophagy in the KU812 pre-basophil-like cell line as well as the HMC-1 immature mast cell line. Antibody-enhanced DENV infection of KU812 cells increased the number of autophagosome vesicles, LC3 punctation, LC3-II accumulation, and p62 degradation over that seen in cells infected with DENV alone. The percentages of DENV envelope (E) protein-positive cells and LC3 puncta following antibody-enhanced DENV infection of KU812 cells were reduced by the autophagy inhibitor 3-MA. Antibody-enhanced DENV infection of HMC-1 cells showed co-localization of DENV E protein and dsRNA with autophagosomes, which was inhibited by 3-MA treatment. Furthermore, DENV infection and replication were reduced when KU812 cells were transfected with the autophagy-inhibiting Atg4B^C74A mutant.

Conclusions/Significance

Our results demonstrate a significant induction of autophagy in antibody-enhanced DENV infection of pre-basophil-like KU812 and immature mast cell-like HMC-1 cells. Also, autophagy plays an important role in DENV infection and replication in these cells. Given the importance of ADE and FcR-bearing cells such as monocytes, macrophages and basophil/mast cells in dengue disease, the results provide insights into dengue pathogenesis and therapeutic means of control.     

The Native Wolbachia Symbionts Limit Transmission of Dengue Virus in Aedes albopictus

Background

The chikungunya (CHIK) outbreak that struck La Reunion Island in 2005 was preceded by few human cases of Dengue (DEN), but which surprisingly did not lead to an epidemic as might have been expected in a non-immune population. Both arboviral diseases are transmitted to humans by two main mosquito species, Aedes aegypti and Aedes albopictus. In the absence of the former, Ae. albopictus was the only species responsible for viral transmission on La Reunion Island. This mosquito is naturally super-infected with two Wolbachia strains, wAlbA and wAlbB. While Wolbachia does not affect replication of CHIK virus (CHIKV) in Ae. albopictus, a similar effect was not observed with DEN virus (DENV).

Methods/Principal Findings

To understand the weak vectorial status of Ae. albopictus towards DENV, we used experimental oral infections of mosquitoes from La Reunion Island to characterize the impact of Wolbachia on DENV infection. Viral loads and Wolbachia densities were measured by quantitative PCR in different organs of Ae. albopictus where DENV replication takes place after ingestion. We found that: (i) Wolbachia does not affect viral replication, (ii) Wolbachia restricts viral density in salivary glands, and (iii) Wolbachia limits transmission of DENV, as infectious viral particles were only detected in the saliva of Wolbachia-uninfected Ae. albopictus, 14 days after the infectious blood-meal.

Conclusions

We show that Wolbachia does not affect the replication of DENV in Ae. albopictus. However, Wolbachia is able to reduce viral infection of salivary glands and limit transmission, suggesting a role of Wolbachia in naturally restricting the transmission of DENV in Ae. albopictus from La Reunion Island. The extension of this conclusion to other Ae. albopictus populations should be investigated.     

Multiple recombinants in two dengue virus, serotype-2 isolates from patients from Oaxaca, Mexico

Background  

Dengue (DEN) is a serious cause of mortality and morbidity in the world including Mexico, where the infection is endemic. One of the states with the highest rate of dengue cases is Oaxaca. The cause of DEN is a positive-sense RNA virus, the dengue virus (DENV) that evolves rapidly increasing its variability due to the absence of a repair mechanism that leads to approximately one mutational event per genome replication; which results in enhancement of viral adaptation, including the escape from host immune responses. Additionally, recombination may play a role in driving the evolution of DENV, which may potentially affect virulence and cause host tropism changes. Recombination in DENV has not been described in Mexican strains, neither has been described the relevance in virus evolution in an endemic state such as Oaxaca where the four serotypes of DENV are circulating.     

Antibody-Dependent Enhancement Infection Facilitates Dengue Virus-Regulated Signaling of IL-10 Production in Monocytes

Background

Interleukin (IL)-10 levels are increased in dengue virus (DENV)-infected patients with severe disorders. A hypothetical intrinsic pathway has been proposed for the IL-10 response during antibody-dependent enhancement (ADE) of DENV infection; however, the mechanisms of IL-10 regulation remain unclear.

Principle Finding

We found that DENV infection and/or attachment was sufficient to induce increased expression of IL-10 and its downstream regulator suppressor of cytokine signaling 3 in human monocytic THP-1 cells and human peripheral blood monocytes. IL-10 production was controlled by activation of cyclic adenosine monophosphate response element-binding (CREB), primarily through protein kinase A (PKA)- and phosphoinositide 3-kinase (PI3K)/PKB-regulated pathways, with PKA activation acting upstream of PI3K/PKB. DENV infection also caused glycogen synthase kinase (GSK)-3β inactivation in a PKA/PI3K/PKB-regulated manner, and inhibition of GSK-3β significantly increased DENV-induced IL-10 production following CREB activation. Pharmacological inhibition of spleen tyrosine kinase (Syk) activity significantly decreased DENV-induced IL-10 production, whereas silencing Syk-associated C-type lectin domain family 5 member A caused a partial inhibition. ADE of DENV infection greatly increased IL-10 expression by enhancing Syk-regulated PI3K/PKB/GSK-3β/CREB signaling. We also found that viral load, but not serotype, affected the IL-10 response. Finally, modulation of IL-10 expression could affect DENV replication.

Significance

These results demonstrate that, in monocytes, IL-10 production is regulated by ADE through both an extrinsic and an intrinsic pathway, all involving a Syk-regulated PI3K/PKB/GSK-3β/CREB pathway, and both of which impact viral replication.     

Decreased dengue replication and an increased anti-viral humoral response with the use of combined Toll-like receptor 3 and 7/8 agonists in macaques

Background

Pathogenic versus protective outcomes to Dengue virus (DENV) infection are associated with innate immune function. This study aimed to determine the role of increased TLR3- and TLR7/8-mediated innate signaling after Dengue infection of rhesus macaques in vivo to evaluate its impact on disease and anti-DENV immune responses.

Methodology/Principal Findings

TLR3 and TLR7/8 agonists (emulsified in Montanide) were administered subcutaneously to rhesus macaques at 48 hours and 7 days after DENV infection. The Frequency and activation of myeloid dendritic cells, plasmacytoid dendritic cells, and B cells were measured by flow cytometry while the serum levels of 14 different cytokines and chemokines were quantified. Adaptive immune responses were measured by DENV-specific antibody subtype measurements. Results showed that the combined TLR agonists reduced viral replication and induced the development of a proinflammatory reaction, otherwise absent in Dengue infection alone, without any clear signs of exacerbated disease. Specifically, the TLR-induced response was characterized by activation changes in mDC subsets concurrent with higher serum levels of CXCL-10 and IL-1Ra. TLR stimulation also induced higher titers of anti-DENV antibodies and acted to increase the IgG2/IgG1 ratio of anti-DENV to favor the subtype associated with DENV control. We also observed an effect of DENV-mediated suppression of mDC activation consistent with prior in vitro studies.

Conclusions/Significance

These data show that concurrent TLR3/7/8 activation of the innate immune response after DENV infection in vivo acts to increase antiviral mechanisms via increased inflammatory and humoral responses in rhesus macaques, resulting in decreased viremia and melioration of the infection. These findings underscore an in vivo protective rather than a pathogenic role for combined TLR3/7/8-mediated activation in Dengue infection of rhesus macaques. Our study provides definitive proof-of-concept into the mechanism by which DENV evades immune recognition and activation in vivo.     

Dengue Virus Infection and Virus-Specific HLA-A2 Restricted Immune Responses in Humanized NOD-scid IL2rγnull Mice

Background

The lack of a suitable animal model to study viral and immunological mechanisms of human dengue disease has been a deterrent to dengue research.

Methodology/Principal Findings

We sought to establish an animal model for dengue virus (DENV) infection and immunity using non-obese diabetic/severe combined immunodeficiency interleukin-2 receptor γ-chain knockout (NOD-scid IL2rγ^null) mice engrafted with human hematopoietic stem cells. Human CD45⁺ cells in the bone marrow of engrafted mice were susceptible to in vitro infection using low passage clinical and established strains of DENV. Engrafted mice were infected with DENV type 2 by different routes and at multiple time points post infection, we detected DENV antigen and RNA in the sera, bone marrow, spleen and liver of infected engrafted mice. Anti-dengue IgM antibodies directed against the envelope protein of DENV peaked in the sera of mice at 1 week post infection. Human T cells that developed following engraftment of HLA-A2 transgenic NOD-scid IL2rγ^null mice with HLA-A2⁺ human cord blood hematopoietic stem cells, were able to secrete IFN-γ, IL-2 and TNF-α in response to stimulation with three previously identified A2 restricted dengue peptides NS4b 2353_(111–119), NS4b 2423_(181–189), and NS4a 2148_(56–64).

Conclusions/Significance

This is the first study to demonstrate infection of human cells and functional DENV-specific T cell responses in DENV-infected humanized mice. Overall, these mice should be a valuable tool to study the role of prior immunity on subsequent DENV infections.     

Thrombin induces Egr-1 expression in fibroblasts involving elevation of the intracellular Ca2+ concentration, phosphorylation of ERK and activation of ternary complex factor

Background

The Arthropods are a diverse group of organisms including Chelicerata (ticks, mites, spiders), Crustacea (crabs, shrimps), and Insecta (flies, mosquitoes, beetles, silkworm). The cattle tick, Rhipicephalus (Boophilus) microplus, is an economically significant ectoparasite of cattle affecting cattle industries world wide. With the availability of sequence reads from the first Chelicerate genome project (the Ixodes scapularis tick) and extensive R. microplus ESTs, we investigated evidence for putative RNAi proteins and studied RNA interference in tick cell cultures and adult female ticks targeting Drosophila homologues with known cell viability phenotype.

Results

We screened 13,643 R. microplus ESTs and I. scapularis genome reads to identify RNAi related proteins in ticks. Our analysis identified 31 RNAi proteins including a putative tick Dicer, RISC associated (Ago-2 and FMRp), RNA dependent RNA polymerase (EGO-1) and 23 homologues implicated in dsRNA uptake and processing. We selected 10 R. microplus ESTs with >80% similarity to D. melanogaster proteins associated with cell viability for RNAi functional screens in both BME26 R. microplus embryonic cells and female ticks in vivo. Only genes associated with proteasomes had an effect on cell viability in vitro. In vivo RNAi showed that 9 genes had significant effects either causing lethality or impairing egg laying.

Conclusion

We have identified key RNAi-related proteins in ticks and along with our loss-of-function studies support a functional RNAi pathway in R. microplus. Our preliminary studies indicate that tick RNAi pathways may differ from that of other Arthropods such as insects.