A simple method for Alexa Fluor dye labelling of dengue virus

Zhang, Summer L.; Tan, Hwee-Cheng; Hanson, Brendon J.; Ooi, Eng Eong

doi:10.1016/j.jviromet.2010.04.001

Cited by 41 publications

(34 citation statements)

References 11 publications

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“…Fluorescent DENV was prepared by labeling with Alexa Fluor 594 succinimidyl ester (AF594SE, Molecular Probes, Invitrogen, Carlsbad, CA, USA) according to the method described in a previous study 36 . The labeled viruses were purified using Amicon Ultra-15 PLTK Ultracel-PL Membrane (30 kDa) centrifugal filter units (Millipore) to remove excess dye.…”

Section: Methodsmentioning

confidence: 99%

Dengue virus infection increases microglial cell migration

Jhan

Tsai

Chen

et al. 2017

Sci Rep

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Activated microglial cells are present in dengue virus (DENV)-infected brains; however, the possible effects of DENV on microglia remain unclear. Here, we demonstrated DENV caused infection, including viral entry, RNA replication, viral protein expression, and virus release, in the murine microglial cell line BV2. DENV infection caused an increase in the formation of the multipolar phenotype in vitro and in vivo without affecting cell growth and cytotoxicity. DENV infection considerably increased cell motility and disrupting either actin filaments or clathrin retarded such effect. Increase in cell migration was only occurred by DENV infection following a clathrin-regulated endocytosis of DENV entry. Ultraviolet-inactivated DENV did not affect cell migration, and pharmacologically blocking toll-like receptor (TLR) 3 and TLR3-related signaling pathways reduced the DENV-induced increase in cell migration. These results demonstrate an advanced effect of DENV infection on microglial migration via a mechanism involving viral entry, RNA release, and TLR3 signal activation.

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Section: Methodsmentioning

confidence: 99%

Dengue virus infection increases microglial cell migration

Jhan

Tsai

Chen

et al. 2017

Sci Rep

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“…DENV was incubated with media, antibodies, or serum for 1h at 37°C before adding to cells at indicated MOI. Uptake was assessed using DiD and Alexa 488-labeled DENV as described (9,30). For drug assays, cells were pretreated with piceatannol (Sigma-Aldrich) or sodium stibogluconate (Santa Cruz Biotechnology) 6 h before infection.…”

Section: Methodsmentioning

confidence: 99%

Leukocyte immunoglobulin-like receptor B1 is critical for antibody-dependent dengue

Chan

Ong

Tan

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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104

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Viruses must evade the host innate defenses for replication and dengue is no exception. During secondary infection with a heterologous dengue virus (DENV) serotype, DENV is opsonized with sub-or nonneutralizing antibodies that enhance infection of monocytes, macrophages, and dendritic cells via the Fc-gamma receptor (FcγR), a process termed antibody-dependent enhancement of DENV infection. However, this enhancement of DENV infection is curious as cross-linking of activating FcγRs signals an early antiviral response by inducing the type-I IFN-stimulated genes (ISGs). Entry through activating FcγR would thus place DENV in an intracellular environment unfavorable for enhanced replication. Here we demonstrate that, to escape this antiviral response, antibody-opsonized DENV coligates leukocyte Ig-like receptor-B1 (LILRB1) to inhibit FcγR signaling for ISG expression. This immunoreceptor tyrosine-based inhibition motif-bearing receptor recruits Src homology phosphatase-1 to dephosphorylate spleen tyrosine kinase (Syk). As Syk is a key intermediate of FcγR signaling, LILRB1 coligation resulted in reduced ISG expression for enhanced DENV replication. Our findings suggest a unique mechanism for DENV to evade an early antiviral response for enhanced infection.early innate immune response | innate immune signaling | immune evasion D espite long-lived serotype-specific immunity upon initial infection, predicted global prevalence of dengue now surpasses World Health Organization estimates by more than threefold with 390 million cases annually (1). Furthermore, the risk of severe disease is augmented by cross-reactive or subneutralizing levels of antibody (2, 3), which opsonize dengue virus (DENV) to ligate Fc-gamma receptor (FcγR) for entry into monocytes, macrophages, and dendritic cells, a phenomenon known as antibody-dependent enhancement (ADE) of DENV infection (4, 5). The resultant greater viral burden leads to increased systemic inflammation that precipitates plasma leakage, a hallmark of dengue hemorrhagic fever (6). However, ligation of the activating FcγRs by immune complexes has been shown to induce type-I IFN stimulated genes (ISGs), independent of autocrine or paracrine IFN activity, unless the inhibitory FcγRIIB is coligated (7). We and others reported recently that coligation of FcγRIIB by DENV immune complexes requires high antibody concentration, and such coligation inhibited the entry of DENV immune complexes into monocytes (8, 9). At low antibody concentrations where ADE occurs, the inhibitory FcγRIIB is not coligated (9). Ligation of the activating FcγRs by DENV opsonized with subneutralizing levels of antibody would thus induce the expression of ISGs and hinder DENV replication (10). Here, we demonstrate that DENV employs a unique evasive mechanism by coligating LILRB1 to down-regulate the early antiviral responses triggered by activating FcγRs for ADE.Results ADE Differs in THP-1 Subclones. Our work was enabled by the isolation of subclones of THP-1 cells with different phenotypes to ADE. The low rate of...

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“…The method for DiD labeling and Alexa594 labeling of DENV was as previously described (22) and as detailed in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

“…However, because the DiD signal is quenched before fusion with cellular membranes (16), it is not possible to visualize DENV outside of the cell. To overcome this limitation, we labeled DENV with Alexa Fluor (22) and demonstrated the presence of antibodybound viruses clustered outside THP-1 cells (Fig. 2E).…”

Section: Antibody Concentration Effects On Fcγr-mediated Uptake Of Immentioning

confidence: 99%

Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection

Chan

Zhang²,

Tan

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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126

136

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The interaction of antibodies, dengue virus (DENV), and monocytes can result in either immunity or enhanced virus infection. These opposing outcomes of dengue antibodies have hampered dengue vaccine development. Recent studies have shown that antibodies neutralize DENV by either preventing virus attachment to cellular receptors or inhibiting viral fusion intracellularly. However, whether the antibody blocks attachment or fusion, the resulting immune complexes are expected to be phagocytosed by Fc gamma receptor (FcγR)-bearing cells and cleared from circulation. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by monocytes whereas other antibodies would have resulted in enhancement of DENV replication. Using convalescent sera from dengue patients, we observed that neutralization of the homologous serotypes occurred despite FcγR-mediated uptake. However, FcγR-mediated uptake appeared to be inhibited when neutralized heterologous DENV serotypes were used instead. We demonstrate that this inhibition occurred through the formation of viral aggregates by antibodies in a concentration-dependent manner. Aggregation of viruses enabled antibodies to cross-link the inhibitory FcγRIIB, which is expressed at low levels but which inhibits FcγR-mediated phagocytosis and hence prevents antibody-dependent enhancement of DENV infection in monocytes. D engue is the most common mosquito-borne viral disease globally. The lack of an effective preventive measure, especially a licensed vaccine, has resulted in the global spread of this virus (1, 2). Although neutralizing antibodies can confer lifelong immunity against reinfection by one of the four dengue virus (DENV) serotypes, subneutralizing antibody levels or crossreactive antibodies appear to enhance the risk of severe dengue in subsequent infections (3-6). DENV bound with subneutralizing concentrations of antibody has been shown to result in increased virus uptake and replication in Fc gamma receptor (FcγR)-bearing cells such as monocytes/macrophages (4, 7). Thus, defining the determinants for virus neutralization will be important for the design of an effective dengue vaccine that protects against all four DENV serotypes while minimizing the risk of antibodydependent enhancement of DENV infection.Neutralization of flavivirus infection is a multiple-hit phenomenon. Recent stoichiometric studies have shown that both antibody affinity and epitope accessibility are important determinants for virus neutralization (8-10). Antibodies neutralize DENV by either preventing virus attachment to cellular receptors (11) or inhibiting viral fusion intracellularly (12). However, whether the antibody blocks attachment or fusion, the resulting immune complex is expected to be cleared from the circulation by professional phagocytes, especially the FcγR-bearing cells. This suggests that only antibodies that are able to block fusion intracellularly would be able to neutralize DENV upon FcγR-mediated uptake by ...

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A simple method for Alexa Fluor dye labelling of dengue virus

Cited by 41 publications

References 11 publications

Dengue virus infection increases microglial cell migration

Dengue virus infection increases microglial cell migration

Leukocyte immunoglobulin-like receptor B1 is critical for antibody-dependent dengue

Ligation of Fc gamma receptor IIB inhibits antibody-dependent enhancement of dengue virus infection

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