6 research outputs found

    ApoA-I promotes DV attachment/entry.

    No full text
    <p>Huh-7 cells infected with DV/SFM, DV/HSM or DV/ApoAI-FLAG were harvested at 30 minutes postinfection. Dengue antigen on the surface of infected cells was detected by indirect immunofluorescence assay and the percentage of DV-bound cells was analyzed by FACS.</p

    ApoA-I is able to enhance DV infectivity.

    No full text
    <p>DV/SFM was pre-incubated with an increasing volume of supernatants collected at 2 dpt from AD293 cells transfected with pApoAI-FLAG and grown in serum-free DMEM (ApoAI-FLAG/SFM) (A) or pre-incubated with human delipidated ApoA-I (De-ApoAI) from <i>Calbiochem</i> for 1 h at 4°C, followed by infection of U937 cells at an MOI of 0.1. Total RNA was extracted at 1 dpi, and viral replication was measured by real-time PCR. The results represent the average standard deviation of three independent experiments. NS, no significance; **p<0.01; ***p<0.001.</p

    Co-precipitation of DV with ApoA-I.

    No full text
    <p>(A) Vero cells were infected with DV at a MOI of 1 and culture medium were changed to DMEM with 10% human serum HS (HSM) at 2 dpi. The mock-infected cells by DMEM was used as a control and also subjected to the same medium change. Culture supernatants were harvested at 7 dpi and purified by sucrose cushion ultracentrifugation (UC). The virus pellets were resuspended in serum-free DMEM. Presence of ApoA-I was analyzed by Western blotting using anti-ApoA-I antibody. (B) Human serum was added into DV/SFM to a final concentration of 10% and the mixture was incubated at 4° for 1 hour, followed by sucrose cushion ultracentrifugation. The pellets were analyzed by Western blotting using anti-ApoA-I and anti-E antibodies respectively. (C) Co-immunoprecipitation of ApoA-I with DV. AD-293 cells were transfected with a plasmid expressing FLAG-tagged ApoA-I (pApoAI-FLAG) and cultured in serum-free DMEM. At 3 dpt, secreted ApoA-I in the culture supernatant was purified with anti-FLAG M2 Affinity Gel. The resulting ApoAI-FLAG/M2 beads were washed twice with 1×TBS and incubated with DV/SFM at 4°C for over night. The co-immunoprecipitates were eluted and detected by Western blotting with anti-E and anti-FLAG antibodies. As a control, co-immunoprecipitation was also performed using the supernatant from cells transfected with empty vector p3×FLAG-CMV-14 (pFLAG). M, pre-stained protein marker.</p

    Interaction of ApoA-I and DV prior to infection is important for enhancement of virus infectivity.

    No full text
    <p>(A) U937 cells were infected with DV/SFM that was pre-incubated with the serum-free supernatant from ApoAI-FLAG expressing cells (ApoAI-FLAG/SFM) at 4°C for 1 hour before infection. (B) U937 cells were infected with DV/SFM in which ApoAI-FLAG/SFM was added at the time of infection. (C) U937 cells were pre-incubated with ApoAI-FLAG/SFM or De-ApoAI at 37°C for 1 h, followed by washing with 1×PBS and infection with DV/SFM. All infections were performed at an MOI of 0.1. Total RNA was extracted at 1 dpi and viral replication was measured by real-time PCR. The results represent the average standard deviation of three independent experiments. NS, no significance; ***p<0.001.</p

    Down-regulation of SR-BI reduces DV infection in Huh-7 cells.

    No full text
    <p>(A) U937 cells and (B) Huh-7 cells were transfected with siRNA CTL or siRNA SR-BI. At 2 dpt, U937 cells and Huh-7 cells were infected with DV/SFM or DV/HSM at an MOI of 0.1. Total RNA was extracted at 1 dpi. Viral replication was measured by real-time PCR. The results represent the average standard deviation of three independent experiments. NS, no significance; **p<0.01. (C) Knockdown of SR-BI by siRNA. At 2 dpt, Huh-7 cell lysates were prepared and analyzed by Western blotting using anti-SR-BI and anti-Actin antibodies. The relative band density of Western blotting was analyzed with Image J.</p

    Human serum enhances infectivity of DV.

    No full text
    <p>(A) U937, (B) PBMCs, (E) Huh7 and (F) HepG2 cells were infected with DV collected from infected Vero cells cultured in serum-free medium (DV/SFM) or DV/SFM containing 10% human serum (DV/HSM) at an MOI of 0.1. Total RNA was extracted at 1 dpi, 2 dpi, 3 dpi for infected U937 cells respectively, and at 1 dpi for other infected cells. Viral replication was measured by real-time PCR. The results represent the average standard deviation of three independent experiments. NS, no significance; *p<0.05; **p<0.01; ***p<0.001. (C) DV/SFM and DV/HSM were ultracentrifuged (UC) over a 30% sucrose cushion, and resulting virus pellets (UC DV/SFM and UC DV/HSM) were resuspended in serum-free medium followed by infection of PBMCs. At 3 dpi, virus infection of cells was observed by IFA and virus RNA copy number was measure by real-time PCR. (D) Detection of dengue IgG antibodies in human serum. Dengue IgG ELISA kit (<i>Abnova</i>) was used to measure dengue IgG antibodies in the human pooled serum, and results were presented as antibody index (Ab index) values, which were calculated by the value of OD450 of the tested sample divided by the cut-off value that was generated from the calibrator in the kit. Ab index <0.9, no detectable IgG antibody to DV; 0.9–1.1, borderline positive; >1.1, detectable IgG antibody to DV. Positive Control (CTL) and negative control (CTL) are supplied in the kit.</p
    corecore