Immature dengue virus is infectious in human immature dendritic cells via interaction with the receptor molecule DC-SIGN

Abstract

Background: Dengue Virus (DENV) is the most common mosquito-borne viral infection worldwide. Important target cells during DENV infection are macrophages, monocytes, and immature dendritic cells (imDCs). DENV-infected cells are known to secrete a large number of partially immature and fully immature particles alongside mature virions. Fully immature DENV particles are considered non-infectious, but antibodies have been shown to rescue their infectious properties. This suggests that immature DENV particles only contribute to the viral load observed in patients with a heterologous DENV re-infection.

Methodology/principal findings: In this study, we re-evaluated the infectious properties of fully immature particles in absence and presence of anti-DENV human serum. We show that immature DENV is infectious in cells expressing DC-SIGN. Furthermore, we demonstrate that immature dendritic cells, in contrast to macrophage-like cells, do not support antibody-dependent enhancement of immature DENV.

Conclusions/significance: Our data shows that immature DENV can infect imDCs through interaction with DC-SIGN, suggesting that immature and partially immature DENV particles may contribute to dengue pathogenesis during primary infection. Furthermore, since antibodies do not further stimulate DENV infectivity on imDCs we propose that macrophages/monocytes rather than imDCs contribute to the increased viral load observed during severe heterotypic DENV re-infections.

Figure 1. Phenotypic analysis of monocyte-derived immature dendritic cells.

Expression profile of different cell type markers by flow cytometry, details are provided in the text. One representative analysis is shown. White curve area: control antibody. Black curve area: specific staining antibody.

Figure 2. Fully immature DENV-2 particles exhibit basic infectivity on immature dendritic cells.

imDCs were infected with MOG 1000 of standard (std) or immature DENV-2. Supernatant was harvested 43 hpi and analyzed. (A) DENV-2 infectivity on imDCs. (B) Role of DC-SIGN on immature DENV-2 infectivity in imDCs as tested by DC-SIGN blockage. Limit of detection is 18 PFU/ml. Data are expressed as means of at least two independent experiments performed in triplicate; error bars represent standard error of the mean (SEM). N.d. denotes for “not detectable”. Levels of significance (Mann Whitney U test) are presented as **  =  p<0.01.

Figure 3. Immature DENV-2 can infect Raji DC-SIGN cells in a furin-dependent manner.

Raji DC-SIGN cells and Raji wt cells were infected with MOG 1000 of std (grey bars) or immature (black bars) DENV-2. Prior and during infection, selected samples were treated with furin inhibitor. Supernatant was analyzed for viral progeny at 43 hpi. Limit of detection is 20 FFU/ml. Data are expressed as means of at least two independent experiments performed in triplicate; error bars represent SEM. N.d. denotes for “not detectable”. Levels of significance (Mann Whitney U test) are presented as **  =  p<0.01 and ***  =  p<0.001.

Figure 4. DC-SIGN enhances the permissiveness of Raji cells for both std and immature DENV of different serotypes.

Raji DC-SIGN cells and Raji wt cells were infected with MOG 1000 of std (grey bars) or immature (black bars) DENV-1 (A), DENV-2 (B) or DENV-4 (C). Supernatant was analyzed for viral progeny at 43 hpi. Limit of detection is 20 FFU/ml. Data are expressed as means of at least two independent experiments performed in triplicate; error bars represent SEM. N.d. denotes for “not detectable”. Levels of significance (Mann Whitney U test) are presented as **  = p<0.01 and ***  =  p<0.001.

Figure 5. imDCs do not support antibody-dependent enhancement of DENV infection.

(A+B) imDCs were infected with immature DENV-2 (A) at MOG1000 and std DENV-2 (B) at MOG 100 as mentioned in the text. Data of one representative donor is shown. For each donor, experiments were performed at least in duplicate. (C+D) P388D1 cells were infected with immature (C) or std (D) DENV-2 at MOG 1000 under similar conditions as in panel A+B. At least two independent experiments were performed in triplicate. Limit of detection is 18 PFU/ml. Error bars represent SEM. N.d. denotes for “not detectable”. Levels of significance (Mann Whitney U test) are presented as *  =  p<0.05, **  =  p<0.01 and ***  =  p<0.001.

Figure 6. Immature DENV particles of different serotypes can be rendered infectious by heterotypic human serum.

P388D1 cells were infected with MOG 1000 of std (grey bars) and pre-opsonized immature (black bars) DENV-1 (A) or DENV-4 (B) as described in the text. At least two independent experiments were performed in triplicate. Limit of detection is 20 FFU/ml. Error bars represent SEM. N.d. denotes for “not detectable”. Levels of significance (Mann Whitney U test) are presented as **  =  p<0.01.