登革病毒对人血管内皮细胞感染性的研究

用登革病毒Ⅱ型（DV2）感染体外培养和传代的人脐静脉内皮细胞（HUVEC）,研究发现,HUVEC是登革病毒的允许性细胞。病毒感染后12h即可在培养上清中用微量蚀斑法测出病毒,病毒滴度48h达高峰,以后迅速下降。并发现在一定范围内病毒产量随病毒感染复数（MOI）的增加而增高。间接免疫荧光法证明感染的HUVEC胞浆及胞膜上携带DV2抗原。电镜和光镜下,感染细胞未见明显的形态和结构改变。     

Differential RNA Sequence Requirement for Dengue Virus Replication in Mosquito and Mammalian Cells

Dengue virus cycles between mosquitoes and humans. Each host provides a different environment for viral replication, imposing different selective pressures. We identified a sequence in the dengue virus genome that is essential for viral replication in mosquito cells but not in mammalian cells. This sequence is located at the viral 3′ untranslated region and folds into a small hairpin structure. A systematic mutational analysis using dengue virus infectious clones and reporter viruses allowed the determination of two putative functions in this cis-acting RNA motif, one linked to the structure and the other linked to the nucleotide sequence. We found that single substitutions that did not alter the hairpin structure did not affect dengue virus replication in mammalian cells but abolished replication in mosquito cells. This is the first sequence identified in a flavivirus genome that is exclusively required for viral replication in insect cells.     

Morphological Variants of Sindbis Virus Obtained from Infected Mosquito Tissue Culture Cells

Tissue-cultured Aedes albopictus cells infected with morphologically homogeneous Sindbis virus were found to produce progeny virions which could be divided into three classes based on size. The thickness of the envelope was constant on all three sizes of progeny virions suggesting that the variability in size rested with the viral nucleocapsid. It is suggested that the three classes of virions have icosahedral nucleocapsids composed of common subunits organized in decreasing triangulation numbers.     

Multiploid CD61+ Cells Are the Pre-Dominant Cell Lineage Infected during Acute Dengue Virus Infection in Bone Marrow

Depression of the peripheral blood platelet count during acute infection is a hallmark of dengue. This thrombocytopenia has been attributed, in part, to an insufficient level of platelet production by megakaryocytes that reside in the bone marrow (BM). Interestingly, it was observed that dengue patients experience BM suppression at the onset of fever. However, few studies focus on the interaction between dengue virus (DENV) and megakaryocytes and how this interaction can lead to a reduction in platelets. In the studies reported herein, BM cells from normal healthy rhesus monkeys (RM) and humans were utilized to identify the cell lineage(s) that were capable of supporting virus infection and replication. A number of techniques were employed in efforts to address this issue. These included the use of viral RNA quantification, nonstructural protein and infectivity assays, phenotypic studies utilizing immunohistochemical staining, anti-differentiation DEAB treatment, and electron microscopy. Cumulative results from these studies revealed that cells in the BM were indeed highly permissive for DENV infection, with human BM having higher levels of viral production compared to RM. DENV-like particles were predominantly observed in multi-nucleated cells that expressed CD61+. These data suggest that megakaryocytes are likely the predominant cell type infected by DENV in BM, which provides one explanation for the thrombocytopenia and the dysfunctional platelets characteristic of dengue virus infection.     

Analysis of Dengue Virus Genetic Diversity during Human and Mosquito Infection Reveals Genetic Constraints

Dengue viruses (DENV) cause debilitating and potentially life-threatening acute disease throughout the tropical world. While drug development efforts are underway, there are concerns that resistant strains will emerge rapidly. Indeed, antiviral drugs that target even conserved regions in other RNA viruses lose efficacy over time as the virus mutates. Here, we sought to determine if there are regions in the DENV genome that are not only evolutionarily conserved but genetically constrained in their ability to mutate and could hence serve as better antiviral targets. High-throughput sequencing of DENV-1 genome directly from twelve, paired dengue patients’ sera and then passaging these sera into the two primary mosquito vectors showed consistent and distinct sequence changes during infection. In particular, two residues in the NS5 protein coding sequence appear to be specifically acquired during infection in Ae. aegypti but not Ae. albopictus. Importantly, we identified a region within the NS3 protein coding sequence that is refractory to mutation during human and mosquito infection. Collectively, these findings provide fresh insights into antiviral targets and could serve as an approach to defining evolutionarily constrained regions for therapeutic targeting in other RNA viruses.     

Use of Centrifugal Filter Devices to Concentrate Dengue Virus in Mosquito per os Infection Experiments

Dengue virus (DENV) is an arbovirus transmitted to humans by the bite of infected Aedes mosquitoes. Experimental per os infection of mosquitoes with DENV is usually a preliminary step in virus/vector studies but it requires being able to prepare artificial blood-meals with high virus titers. We report here the convenient use of centrifugal filter devices to quickly concentrate DENV particles in cell-culture supernatants. The median viral titer in concentrated-supernatants was 8.50 log₁₀ TCID₅₀/mL. By using these DENV concentrated-supernatants to prepare infectious blood-meals in Aedes aegypti per os infection experiments, we obtained a mean mosquito-infection rate of 94%. We also evaluated the use of centrifugal filter devices to recover DENV particles from non-infectious blood-meals presented to infected mosquitoes through a feeding membrane to collect their saliva.     

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.     

Ⅲ型登革病毒在THP-1细胞上的ADE模型建立

登革热(dengue fever,DF)是由Ⅰ、Ⅱ、Ⅲ和Ⅳ型登革病毒(dengue virus,DENV)引起的急性传染病,抗体依赖增强感染(antibody-dependent enhancement,ADE)是自限性的登革热以及危及生命的登革出血热(dengue hemorrhagic fever,DHF)或登革休克综合症(dengue shock syndrome,DSS)等重症的主要原因。采用不同稀释度的Ⅱ型登革病毒prM前膜抗体与分离自云南西双版纳重症病人的Ⅲ型登革病毒复合感染THP-1细胞,通过实时荧光定量PCR发现亚中和浓度prM前膜抗体诱发THP-1细胞液中更高浓度的病毒载量。在THP-1细胞系上的研究可为后续研究登革病毒ADE打下坚实的基础。     

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.     

Activation of Peripheral Blood Mononuclear Cells by Dengue Virus Infection Depotentiates Balapiravir

In a recent clinical trial, balapiravir, a prodrug of a cytidine analog (R1479), failed to achieve efficacy (reducing viremia after treatment) in dengue patients, although the plasma trough concentration of R1479 remained above the 50% effective concentration (EC₅₀). Here, we report experimental evidence to explain the discrepancy between the in vitro and in vivo results and its implication for drug development. R1479 lost its potency by 125-fold when balapiravir was used to treat primary human peripheral blood mononuclear cells (PBMCs; one of the major cells targeted for viral replication) that were preinfected with dengue virus. The elevated EC₅₀ was greater than the plasma trough concentration of R1479 observed in dengue patients treated with balapiravir and could possibly explain the efficacy failure. Mechanistically, dengue virus infection triggered PBMCs to generate cytokines, which decreased their efficiency of conversion of R1479 to its triphosphate form (the active antiviral ingredient), resulting in decreased antiviral potency. In contrast to the cytidine-based compound R1479, the potency of an adenosine-based inhibitor of dengue virus (NITD008) was much less affected. Taken together, our results demonstrate that viral infection in patients before treatment could significantly affect the conversion of the prodrug to its active form; such an effect should be calculated when estimating the dose efficacious for humans.     

Study of Dengue Virus Infection in SCID Mice Engrafted with Human K562 Cells

Here we report that severe combined immunodeficient (SCID) mice engrafted with human K562 cells (K562-SCID mice) can be used as an animal model to study dengue virus (DEN) infection. After intratumor injection into K562 cell masses of PL046, a Taiwanese DEN-2 human isolate, the K562-SCID mice showed neurological signs of paralysis and died at approximately 2 weeks postinfection. In addition to being detected in the tumor masses, high virus titers were detected in the peripheral blood and the brain tissues, indicating that DEN had replicated in the infected K562-SCID mice. In contrast, the SCID mice were resistant to DEN infection and the mock-infected K562-SCID mice survived for over 3 months. These data illustrate that DEN infection contributed directly to the deaths of the infected K562-SCID mice. Other serotypes of DEN were also used to infect the K562-SCID mice, and the mortality rates of the infected mice varied with different challenge strains, suggesting the existence of diverse degrees of virulence among DENs. To determine whether a neutralizing antibody against DEN in vitro was also protective in vivo, the K562-SCID mice were challenged with DEN-2 and received antibody administration at the same time or 1 day earlier. Our results revealed that the antibody-treated mice exhibited a reduction in mortality and a delay of paralysis onset after DEN infection. In contrast to K562-SCID, the persistently DEN-infected K562 cells generated in vitro invariably failed to be implanted in the mice. It seems that in the early stage of implantation, a gamma interferon activated, nitric oxide-mediated anti-DEN effect might play a role in the innate immunity against DEN-infected cells. The system described herein offers an opportunity to explore DEN replication in vivo and to test various antiviral protocols in infected hosts.     

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.     

Sensing of Immature Particles Produced by Dengue Virus Infected Cells Induces an Antiviral Response by Plasmacytoid Dendritic Cells

Dengue virus (DENV) is the leading cause of mosquito-borne viral illness and death in humans. Like many viruses, DENV has evolved potent mechanisms that abolish the antiviral response within infected cells. Nevertheless, several in vivo studies have demonstrated a key role of the innate immune response in controlling DENV infection and disease progression. Here, we report that sensing of DENV infected cells by plasmacytoid dendritic cells (pDCs) triggers a robust TLR7-dependent production of IFNα, concomitant with additional antiviral responses, including inflammatory cytokine secretion and pDC maturation. We demonstrate that unlike the efficient cell-free transmission of viral infectivity, pDC activation depends on cell-to-cell contact, a feature observed for various cell types and primary cells infected by DENV, as well as West Nile virus, another member of the Flavivirus genus. We show that the sensing of DENV infected cells by pDCs requires viral envelope protein-dependent secretion and transmission of viral RNA. Consistently with the cell-to-cell sensing-dependent pDC activation, we found that DENV structural components are clustered at the interface between pDCs and infected cells. The actin cytoskeleton is pivotal for both this clustering at the contacts and pDC activation, suggesting that this structural network likely contributes to the transmission of viral components to the pDCs. Due to an evolutionarily conserved suboptimal cleavage of the precursor membrane protein (prM), DENV infected cells release uncleaved prM containing-immature particles, which are deficient for membrane fusion function. We demonstrate that cells releasing immature particles trigger pDC IFN response more potently than cells producing fusion-competent mature virus. Altogether, our results imply that immature particles, as a carrier to endolysosome-localized TLR7 sensor, may contribute to regulate the progression of dengue disease by eliciting a strong innate response.     

Small RNA Profiling in Dengue Virus 2-Infected Aedes Mosquito Cells Reveals Viral piRNAs and Novel Host miRNAs

In Aedes mosquitoes, infections with arthropod-borne viruses (arboviruses) trigger or modulate the expression of various classes of viral and host-derived small RNAs, including small interfering RNAs (siRNAs), PIWI interacting RNAs (piRNAs), and microRNAs (miRNAs). Viral siRNAs are at the core of the antiviral RNA interference machinery, one of the key pathways that limit virus replication in invertebrates. Besides siRNAs, Aedes mosquitoes and cells derived from these insects produce arbovirus-derived piRNAs, the best studied examples being viruses from the Togaviridae or Bunyaviridae families. Host miRNAs modulate the expression of a large number of genes and their levels may change in response to viral infections. In addition, some viruses, mostly with a DNA genome, express their own miRNAs to regulate host and viral gene expression. Here, we perform a comprehensive analysis of both viral and host-derived small RNAs in Aedes aegypti Aag2 cells infected with dengue virus 2 (DENV), a member of the Flaviviridae family. Aag2 cells are competent in producing all three types of small RNAs and provide a powerful tool to explore the crosstalk between arboviral infection and the distinct RNA silencing pathways. Interestingly, besides the well-characterized DENV-derived siRNAs, a specific population of viral piRNAs was identified in infected Aag2 cells. Knockdown of Piwi5, Ago3 and, to a lesser extent, Piwi6 results in reduction of vpiRNA levels, providing the first genetic evidence that Aedes PIWI proteins produce DENV-derived small RNAs. In contrast, we do not find convincing evidence for the production of virus-derived miRNAs. Neither do we find that host miRNA expression is strongly changed upon DENV2 infection. Finally, our deep-sequencing analyses detect 30 novel Aedes miRNAs, complementing the repertoire of regulatory small RNAs in this important vector species.     

Immunodiagnosis of Dengue Virus Infection Using Saliva

Keeping in view the complications and the case fatality associated with dengue virus, several serologic tests have been developed. However, the major drawback of these serologic tests is the need for a venous blood sample obtained by invasive venipuncture. As a noninvasive alternative, saliva provides a body fluid that contains antibodies of diagnostic importance. Hence, the detection of DEN-specific IgM and IgG antibodies in serum and saliva from 80 patients was compared. Salivary IgM antibodies were detected in 100% of the serum IgM-positive samples and in 30% of the serum samples that were negative for IgM antibodies. Salivary IgG antibodies were detected in 93.3% of the serum samples that were positive for anti-dengue IgG antibodies and in none of the serum IgG-negative cases. None of the specimens from the healthy controls showed the presence of IgM or IgG antibodies. The detection of both IgG and IgM antibodies in saliva correlated well with the serum IgG and IgM detection by the ELISA test (r = 0.6322 and r = 0.4227). Detection of salivary IgM antibodies by ELISA showed 100% sensitivity, 70% specificity, 90.9% positive predictive value, and 100% negative predictive value. The detection of IgG in saliva proved to be a promising tool as the sensitivity, specificity, positive predictive value, and negative predictive value were found out to be 93.3%, 100%, 100%, and 83.3%, respectively. Thus, from this study we conclude that the detection of DEN-specific salivary IgG and IgM antibodies are useful markers for dengue infection.     

Lipid Abnormalities in Patients Infected With HumanImmunodeficiency Virus

ObjectiveTo review the types, mechanisms, clinical implications, and management of lipid abnormalities associated with human immunodeficiency virus (HIV) infection and its treatment.MethodsReview of the relevant literature using MEDLINE data sources from 1985 to February 2008, endocrinology textbooks, and hand-searching of cross-references from original articles and reviews. Clinical trials, animal studies, in vitro studies, case reports, reviews, and guidelines of major medical associations were included.ResultsAdvanced stages of HIV infection are characterized by low plasma levels of total cholesterol, highdensity lipoprotein cholesterol, low-density lipoprotein cholesterol, and elevated triglycerides. Antiretroviral agents can exert negative effects on lipids that vary substantially between different drug classes and between individual drugs within each class. Prospective studies suggest that the use of protease inhibitors may be associated with increased risk of myocardial infarction that is mediated in part by dyslipidemia. Target levels of plasma lipids and management of HIV–related dyslipidemia generally follow the same guidelines as in the general population. However, dyslipidemia in this setting is often difficult to control with a single lipid-lowering agent, and potentially serious drug interactions may exist between some statins, such as simvastatin, and protease inhibitors.ConclusionsPlasma lipids should be measured in all patients infected with HIV before and 3 to 4 months after starting antiretroviral drugs. Statins are the initial drugs of choice in most patients. The concomitant use of statins and antiretroviral drugs should take into account various interactions between these agents. (Endocr Pract. 2008;14:492-500)