Longitudinal Analysis of Natural Killer Cells in Dengue Virus-Infected Patients in Comparison to Chikungunya and Chikungunya/Dengue Virus-Infected Patients

BackgroundDengue virus (DENV) is the most prominent arbovirus worldwide, causing major epidemics in South-East Asia, South America and Africa. In 2010, a major DENV-2 outbreak occurred in Gabon with cases of patients co-infected with chikungunya virus (CHIKV). Although the innate immune response is thought to be of primordial importance in the development and outcome of arbovirus-associated pathologies, our knowledge of the role of natural killer (NK) cells during DENV-2 infection is in its infancy.MethodologyWe performed the first extensive comparative longitudinal characterization of NK cells in patients infected by DENV-2, CHIKV or both viruses. Hierarchical clustering and principal component analyses were performed to discriminate between CHIKV and DENV-2 infected patients.Conclusions/SignificanceAlthough specific differences were observed between CHIKV and DENV-2 infections, the significant remodeling of NK cell populations observed here suggests their potential roles in the control of both infections.     

登革2型病毒衣壳蛋白在哺乳动物细胞中的表达与鉴定

从构建的重组质粒pLEX—C中高保真PCR获得编码登革2型病毒43株C基／E／（D2C）DNA片段,通过基因重组的方法将其克隆入真核表达载体pcDNA6／V5-His获得了重组真核表达载体pc／D2C。经电穿孔的方法转染BHK21细胞后,分别通过RT—PCR、免疫荧光和western印迹鉴定表达的蛋白。结果重组蛋白在BHK21细胞中获得表达,表达的蛋自主要存在于胞浆中,并具有较好的抗原性,能够被抗登革病毒衣壳蛋白单克隆抗体特异识别。此研究为深入了解登革病毒衣壳蛋白在病毒复制及组装过程中的生物学功能奠定了基础。     

登革病毒衣壳蛋白靶向核酸酶表达系统的建立及应用

根据登革 2型病毒衣壳蛋白C基因和葡萄球菌核酸酶SN基因序列设计引物 ,从构建的原核表达载体pLEX D2C SN中扩增获得编码登革病毒衣壳蛋白和葡萄球菌核酸酶的融合基因D2C SN ,将其插入到真核表达载体pcDNA6 V5 His中 ,筛选获得重组质粒pcDNA D2C SN .电穿孔转染BHK细胞后 ,5mg Lblasticidin压力筛选 ,通过RT PCR、间接免疫荧光和免疫印迹鉴定表达的蛋白 ,体外DNA消化试验检测核酸酶活性 .结果表明 ,融合蛋白D2C SN在BHK细胞中获得了稳定表达 ,表达的融合蛋白能够被抗登革病毒衣壳蛋白的单克隆抗体特异识别 ,并具有良好的核酸酶活性 ,能够对DNA进行切割 .同时 ,BHK细胞中稳定表达的融合蛋白D2C SN能够有效抑制登革病毒的增殖 ,使其感染性降低 10 3 ～ 10 4倍 .这些结果为进一步将衣壳蛋白靶向病毒灭活策略应用于人类抗登革病毒感染奠定了基础     

Targeted Delivery of Small Interfering RNA to Human Dendritic Cells To Suppress Dengue Virus Infection and Associated Proinflammatory Cytokine Production

Dengue is a common arthropod-borne flaviviral infection in the tropics, for which there is no vaccine or specific antiviral drug. The infection is often associated with serious complications such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS), in which both viral and host factors have been implicated. RNA interference (RNAi) is a potent antiviral strategy and a potential therapeutic option for dengue if a feasible strategy can be developed for delivery of small interfering RNA (siRNA) to dendritic cells (DCs) and macrophages, the major in vivo targets of the virus and also the source of proinflammatory cytokines. Here we show that a dendritic cell-targeting 12-mer peptide (DC3) fused to nona-d-arginine (9dR) residues (DC3-9dR) delivers siRNA and knocks down endogenous gene expression in heterogenous DC subsets, (monocyte-derived DCs [MDDCs], CD34⁺ hematopoietic stem cell [HSC])-derived Langerhans DCs, and peripheral blood DCs). Moreover, DC3-9dR-mediated delivery of siRNA targeting a highly conserved sequence in the dengue virus envelope gene (siFvE^D) effectively suppressed dengue virus replication in MDDCs and macrophages. In addition, DC-specific delivery of siRNA targeting the acute-phase cytokine tumor necrosis factor alpha (TNF-α), which plays a major role in dengue pathogenesis, either alone or in combination with an antiviral siRNA, significantly reduced virus-induced production of the cytokine in MDDCs. Finally to validate the strategy in vivo, we tested the ability of the peptide to target human DCs in the NOD/SCID/IL-2Rγ^−/− mouse model engrafted with human CD34⁺ hematopoietic stem cells (HuHSC mice). Treatment of mice by intravenous (i.v.) injection of DC3-9dR-complexed siRNA targeting TNF-α effectively suppressed poly(I:C)-induced TNF-α production by DCs. Thus, DC3-9dR can deliver siRNA to DCs both in vitro and in vivo, and this delivery approach holds promise as a therapeutic strategy to simultaneously suppress virus replication and curb virus-induced detrimental host immune responses in dengue infection.Dengue is a mosquito-borne flavivirus infection that has emerged as a serious public health problem worldwide. Four serotypes of dengue virus (DEN-1 to DEN-4) are capable of causing human disease varying in severity from acute self-limiting febrile illness to life-threatening dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). The plasma leakage, hemorrhagic manifestations, and shock that characterize DHF/DSS are considered to have an immunological basis, as they are more common during secondary infection with a heterologous dengue virus strain (15, 28, 33). However, severe clinical manifestations can also occur during primary dengue infection, pointing to a contributory role of viral virulence factors. The WHO estimates that more than 20,000 people worldwide, mainly children, die each year from serious complications of dengue. No specific antiviral therapies are currently available for treating the infection, and efforts to develop a safe prophylactic vaccine have been hindered by the complex role of the immune system in disease pathogenesis (39, 52, 57). Thus, novel treatment strategies that block viral replication and/or to attenuate the exaggerated cytokine response associated with DHF/DSS complications are urgently needed.Potent and specific gene silencing mediated by RNA interference (RNAi) has generated a great deal of interest in development of RNAi as a therapeutic strategy against viral infections (50, 54). Many studies have demonstrated the effectiveness of the RNAi approach to suppress flavivirus infection, including dengue virus replication in experimental cell lines (3, 23, 26, 42, 60). In addition, the versatility of RNAi could also be exploited to block important host mediators that contribute to dengue pathogenesis. However, the existence of four distinct dengue virus serotypes and the ability of viruses to develop resistance to RNAi by mutating their sequences will have to be taken into account before clinical use can be contemplated. A more serious hurdle for RNAi therapeutics is the specific delivery of small interfering RNA (siRNA) to relevant cell types.Even though dengue virus antigens have been detected in many tissues, including liver, spleen, lymph node, and skin of patients with DHF/DSS, macrophages and dendritic cells (DCs) are considered the predominant infected cell types (9, 36, 59). Following the bite of an infected Aedes mosquito, the initial local viral replication is believed to take place in the skin DCs, including myeloid DCs and Langerhans cells (31, 53, 59). Dengue-infected DCs play a key role in the immunopathogenesis of DHF/DSS, as, along with macrophages, they release proinflammatory cytokines and soluble factors that mediate plasma leakage, thrombocytopenia, and hypovolemic shock associated with severe dengue infection (14, 15, 29, 38). Therefore, development of a method to introduce siRNA into DCs would be an important step toward using RNAi therapeutically to suppress viral replication and/or to attenuate the vigorous host cytokine responses in dengue infection (7, 19).To target DCs, we used a previously characterized 12-amino-acid peptide identified from a phage display peptide library that specifically binds to a ligand expressed on DCs (10). In an earlier study, we demonstrated that fusing nucleic acid-binding nine d-arginine residues to a neuronal cell-targeting peptide enabled siRNA delivery to neuronal cells (27). Here, in a similar approach, we synthesized a chimeric peptide consisting of the DC-targeting peptide fused to nona-D-arginines (9dR) to target siRNA selectively to DCs. We investigated whether the DC3-9dR peptide could deliver siRNA targeting a dengue virus envelope sequence to reduce the viral load in DCs. As tumor necrosis factor alpha (TNF-α) is one of the acute-phase cytokines with a major role in inducing plasma leakage in dengue infection (8, 12, 17, 20), we also explored the possibility of reducing TNF-α expression in DC in vitro and in vivo. Our findings demonstrate the potential of a targeted RNAi-based approach for simultaneously decreasing viral load and reducing aberrant cytokine responses in DCs.     

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.     

Selective Susceptibility of Human Skin Antigen Presenting Cells to Productive Dengue Virus Infection

Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14⁺ and CD1c⁺ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response.     

Easy and Rapid Detection of Mumps Virus by Live Fluorescent Visualization of Virus-Infected Cells

Mumps viruses show diverse cytopathic effects (CPEs) of infected cells and viral plaque formation (no CPE or no plaque formation in some cases) depending on the viral strain, highlighting the difficulty in mumps laboratory studies. In our previous study, a new sialidase substrate, 2-(benzothiazol-2-yl)-4-bromophenyl 5-acetamido-3,5-dideoxy-α-D-glycero-D-galacto-2-nonulopyranosidonic acid (BTP3-Neu5Ac), was developed for visualization of sialidase activity. BTP3-Neu5Ac can easily and rapidly perform histochemical fluorescent visualization of influenza viruses and virus-infected cells without an antiviral antibody and cell fixation. In the present study, the potential utility of BTP3-Neu5Ac for rapid detection of mumps virus was demonstrated. BTP3-Neu5Ac could visualize dot-blotted mumps virus, virus-infected cells, and plaques (plaques should be called focuses due to staining of infected cells in this study), even if a CPE was not observed. Furthermore, virus cultivation was possible by direct pick-up from a fluorescent focus. In conventional methods, visible appearance of the CPE and focuses often requires more than 6 days after infection, but the new method with BTP3-Neu5Ac clearly visualized infected cells after 2 days and focuses after 4 days. The BTP3-Neu5Ac assay is a precise, easy, and rapid assay for confirmation and titration of mumps virus.     

Seoul Virus-Infected Rat Lung Endothelial Cells and Alveolar Macrophages Differ in Their Ability To Support Virus Replication and Induce Regulatory T Cell Phenotypes

Hantaviruses cause a persistent infection in reservoir hosts that is attributed to the upregulation of regulatory responses and downregulation of proinflammatory responses. To determine whether rat alveolar macrophages (AMs) and lung microvascular endothelial cells (LMVECs) support Seoul virus (SEOV) replication and contribute to the induction of an environment that polarizes CD4⁺ T cell differentiation toward a regulatory T (Treg) cell phenotype, cultured primary rat AMs and LMVECs were mock infected or infected with SEOV and analyzed for viral replication, cytokine and chemokine responses, and expression of cell surface markers that are related to T cell activation. Allogeneic CD4⁺ T cells were cocultured with SEOV-infected or mock-infected AMs or LMVECs and analyzed for helper T cell (i.e., Treg, Th17, Th1, and Th2) marker expression and Treg cell frequency. SEOV RNA and infectious particles in culture media were detected in both cell types, but at higher levels in LMVECs than in AMs postinfection. Expression of Ifnβ, Ccl5, and Cxcl10 and surface major histocompatibility complex class II (MHC-II) and MHC-I was not altered by SEOV infection in either cell type. SEOV infection significantly increased Tgfβ mRNA in AMs and the amount of programmed cell death 1 ligand 1 (PD-L1) in LMVECs. SEOV-infected LMVECs, but not AMs, induced a significant increase in Foxp3 expression and Treg cell frequency in allogeneic CD4⁺ T cells, which was virus replication and cell contact dependent. These data suggest that in addition to supporting viral replication, AMs and LMVECs play distinct roles in hantavirus persistence by creating a regulatory environment through increased Tgfβ, PD-L1, and Treg cell activity.     

A Role for the Insulin Receptor in the Suppression of Dengue Virus and Zika Virus in Wolbachia-Infected Mosquito Cells

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IFI6 Inhibits Apoptosis via Mitochondrial-Dependent Pathway in Dengue Virus 2 Infected Vascular Endothelial Cells

Dengue hemorrhagic fever (DHF)/Dengue shock syndrome (DSS) is a fatal infectious disease that demands an effective treatment. Interferon (IFN)-stimulated genes (ISGs) induced by dengue virus (DENV) exert antiviral effects. Among ISGs, IFN-α inducible gene 6 (IFI6) was increased in DENV infected human umbilical vascular endothelial cells (HUVECs) by microarray analysis in our previous study. However, its function is incompletely understood. In this study, we confirmed that IFI6 was markedly induced in DENV infection of both primary HUVECs and EA.hy926 cell lines. Recombinant EA.hy926 cell lines in which IFI6 was either over-expressed (IFI6+/+) or knocked-down (IFI6-/-) were generated. The activation of caspase-3 and intrinsic apoptosis-related protein caspase-9 were down-regulated in IFI6+/+ but up-regulated in IFI6-/- cells at 24–48 hrs post-infection. After incubation with DENV for 48 hrs, the mitochondrial membrane potential (Δψ(m)) was more stable in IFI6+/+ cells but reduced in IFI6-/- cells, as assayed by fluorescence staining with JC-1. We observed that Bcl-2 expression was increased in IFI6+/+ and decreased in IFI6-/- cells. By contrast, Bax expression was decreased in IFI6+/+ and increased in IFI6-/- cells. It is presumed that the anti-apoptotic function of IFI6 is expressed by regulating the rheostatic balance between bcl-2/bax expression and inhibition of Δψ(m) depolarization during DENV infection of vascular endothelial cells(VECs). In addition, the pro-apoptotic protein X-linked Inhibitor of Apoptosis (XIAP)-Associated Factor 1(XAF1) expression had been reported to be up-regulated and led to the induction of apoptosis in DENV2-infected VECs,but the relationship between XAF1 and IFI6 dengue virus-induced apoptosis in VECs warrants further study.     

Green Fluorescent Protein as a Reporter To Monitor Gene Expression and Food Colonization by Aspergillus flavus

Transformants of Aspergillus flavus containing the Aequorea victoria gfp gene fused to a viral promoter or the promoter region and 483 bp of the coding region of A. flavus aflR expressed green fluorescence detectable without a microscope or filters. Expression of green fluorescent protein fluorescence was correlated with resistance to aflatoxin accumulation in five corn genotypes inoculated with these transformants.