Infection of human cells by dengue virus is modulated by different cell types and viral strains

Abstract

Although prior studies have investigated cellular infection by dengue virus (DV), many have used highly passaged strains. We have reassessed cellular infection by DV type 2 (DV2) using prototype and low-passage isolates representing genotypes from different geographic areas. We observed marked variation in the susceptibility to infection among cell types by different DV2 strains. HepG2 hepatoma cells were susceptible to infection by all DV2 strains assayed. Although the prototype strain generated higher titers of secreted virus than the low-passage isolates, this difference did not correspond to positive- or negative-strand viral RNA levels and thus may reflect variation in efficiency among DV2 isolates to translate viral proteins or package and/or secrete virus. In contrast, human foreskin fibroblasts were susceptible to the prototype and low-passage Thai isolates but not to five Nicaraguan strains tested, as reflected by the absence of accumulation of negative-strand viral RNA, viral antigen, and infectious virus. A similar pattern was observed with the antibody-dependent pathway of infection. U937 and THP-1 myeloid cells and peripheral blood monocytes were infected in the presence of enhancing antibodies by the prototype strain but not by low-passage Nicaraguan isolates. Again, the barrier appeared to be prior to negative-strand accumulation. Thus, depending on the cell type and viral isolate, blocks that limit the production of infectious virus in vitro may occur at distinct steps in the pathway of cellular infection.

FIG. 1

Time course of infection of SW13 cells by DV2. SW13 cells were exposed to DV2 (strain 16681; MOI of 1) and incubated for 1, 2, 4, and 6 days. Cells (2 × 10⁵) were processed for flow cytometry after fixation and detergent permeabilization and incubated with a MAb that recognizes the E protein of DV3 (top) or DV2 (bottom). The flow-cytometric data are expressed as the log of fluorescence intensity. One representative experiment of two is shown.

FIG. 2

Infection of HepG2 cells by different strains of DV2. HepG2 cells were exposed to the prototype 16681 strain or recent Thai (K0049 and C0477) and Nicaraguan (N9622) isolates over a range of MOIs and incubated for 72 h. Cells (10⁶) were processed for flow cytometry (A), and supernatants were harvested for plaque assays (B). The flow-cytometric data are expressed as the percentages of positive cells. The plaque assay data are expressed as the numbers of PFU per milliliter based on cytopathic effect in BHK21 cells. The data are the averages of three experiments, and the error bars represent the standard errors of the means.

FIG. 3

Infection of HFF cells by different strains of DV2. HFF cells were exposed to the prototype 16681 strain or recent Thai (K0049 and C0477) and Nicaraguan (N9622) isolates over a range of MOIs and incubated for 72 h. Cells (2 × 10⁵) were processed for flow cytometry (A), and supernatants were harvested for plaque assays (B). The data are expressed as described for Fig. 2.

FIG. 4

Infection of HepG2 and HFF cells by Nicaraguan DV2 isolates. HFF cells and HepG2 cells were exposed to the 16681 prototype Thai strain or recent Nicaraguan isolates (N1042, N1043, N1047, N1064) at an MOI of 3 and incubated for 72 h. Cells (HepG2, 6 × 10⁵; HFF, 2 × 10⁵) were processed for flow cytometry. The data are expressed as described for Fig. 2.

FIG. 5

Asymmetric, competitive RT-PCR quantification of positive- and negative-strand viral RNA. HepG2 and HFF cells were exposed to Thai (16681, C0477) or Nicaraguan (N1064) strains and incubated for 72 h. Cells (HepG2, 6 × 10⁵; HFF, 6 × 10⁵) were harvested, total RNA was isolated, and quantitative asymmetric RT-PCR was performed with fixed amounts of cellular RNA in the presence of 10-fold-decreasing concentrations of positive- or negative-strand competitor. The RT-PCR product was subjected to agarose gel electrophoresis. The number above each lane represents the log of the number of copies of competitor used. The amount of viral RNA was determined from the competitor concentration that produces competitor and DV bands of equal intensities (asterisk). For the positive strand, the equivalence points and numbers of RNA copies per cell were as follows. HepG2 cells: 16681, 10⁷, 1,666 RNA copies/cell; C0477, 10⁷, 1,666 RNA copies/cell; N1064, 10⁷, 1,666 RNA copies/cell; HFF cells: 16681, 10⁶, 167 RNA copies/cell; C0477, 5 × 10⁵, 83 RNA copies/cell; N1064, 10⁶, 167 RNA copies/cell. For the negative strand, the equivalence points and numbers of RNA copies per cell were as follows. HepG2 cells: 16681, 10⁶, 83 RNA copies/cell; C0477, 10⁶, 83 RNA copies/cell; N1064, 5 × 10⁵, 42 RNA copies/cell; HFF cells: 16681, 10⁵, 16 RNA copies/cell; C0477, 10⁴, 0.8 RNA copies/cell; N1064, <10¹, <0.01 RNA copies/cell. The limit of sensitivity of the assay is 0.01 under these conditions.