A mouse-passaged dengue virus strain with reduced affinity for heparan sulfate causes severe disease in mice by establishing increased systemic viral loads - PubMed (original) (raw)

A mouse-passaged dengue virus strain with reduced affinity for heparan sulfate causes severe disease in mice by establishing increased systemic viral loads

Tyler R Prestwood et al. J Virol. 2008 Sep.

Abstract

The four serotypes of dengue virus (DENV1 to DENV4) cause extensive morbidity and mortality. A major obstacle to studying disease pathogenesis and developing therapies has been the lack of a small-animal model. We previously reported isolation of a DENV2 strain, obtained by passaging a clinical isolate between mosquito cells and mice, that caused severe DENV disease in mice and contained multiple mutations, including many in domain II of the envelope (E) protein. Here, we describe a recombinant virus, differing from the non-mouse-passaged virus by two mutations in the E protein, that induces vascular leakage and tumor necrosis factor alpha (TNF-alpha)-mediated lethality, while the non-mouse-passaged virus causes paralysis. This recombinant virus has a weaker affinity for heparan sulfate, resulting in an increased serum half-life, higher systemic viral loads, and high levels of TNF-alpha in the serum of infected mice. These results exemplify the role of the E protein in modulating virion clearance and connect the effect of clearance on the systemic viral loads responsible for severe disease manifestations.

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Figures

FIG. 1.

Comparison of uncloned DENV2 strains PL046 and D2S10 with recombinant viruses derived from pPL046-IC (PL046-IC) and pE124/128-IC (E124/128-IC). (A) Plaque assays using BHK-21 cells were performed. Plaques were visualized day 5 p.i. for PL046 and PL046-IC and day 7 p.i. for D2S10 and E124/128-IC. (B) Virus was quantified by real-time RT-PCR in five different uncloned and four different recombinant preparations. The same preparations were used for plaque assay on BHK-21 cells to calculate viral RNA copies per PFU. The data represent means ± the standard errors of the mean (SEM). The asterisk indicates a difference that is statistically significant (P < 0.05). (C) Western blot of uncloned PL046 and D2S10 and recombinant viruses derived from pPL046-IC and pE124/128-IC. A monoclonal antibody (clone E18) was used to detect the E protein in 105 PFU of PL046 (lane 1), D2S10 (lane 2), PL046-IC (lane 3), and E124/128-IC (lane 4) and 104 PFU of PL046 (lane 5), D2S10 (lane 6), PL046-IC (lane 7), and E124/128-IC (lane 8). In this denaturing gel, the E monomer is detected as the major band of approximately 55 kDa.

FIG. 2.

Infection of IFN-α/β and IFN-γ receptor-deficient 129/Sv (AG129) mice with recombinant DENV2 PL046-IC or E124/128-IC. (A) Survival curves of AG129 mice infected with 1.5 × 1011 GE of PL046-IC or E124/128-IC. All PL046-IC-challenged mice developed paralysis, whereas all E124/128-IC mice died without manifesting paralysis. The P value between the two viral strains is indicated. The data from three independent experiments, each with different viral preparations, were pooled (n = the total number of mice per group). (B) Vascular permeability was assessed by measuring albumin concentrations in the large intestine of mice day 3 p.i. by ELISA. The results represent the mean values ± the SEM of five mice per group, except for the naive group with three mice. The dotted line shows the limit of detection of the assay. **, P < 0.01. (C) TNF-α levels in the serum were analyzed by ELISA. Mean values ± the SEM of six to nine mice per group for each time point are shown. The dotted line represents the limit of detection of the assay. **, P < 0.01. (D) Anti-TNF-α or isotype control antibody was administered to E124/128-IC-infected mice on days 1, 2, and 3 p.i. Isotype control-treated mice succumbed to infection without exhibiting signs of paralysis, whereas anti-TNF-α-treated mice developed paralysis. The data were pooled from two experiments (n = the total number of mice per group; the P value is indicated).

FIG. 3.

Infection of BHK and K562 cells by the recombinant viral strains. (A) BHK-21 cells were infected with 5,000 GE of PL046-IC or E124/128-IC, and the presence of DENV prM was detected by flow cytometry. The percentages of positive cells are shown. The data from one representative out of five independent experiments using different viral preparations are shown. (B) K562/DC-SIGN cells were infected with 5,000 or 500 GE of PL046-IC or E124/128-IC, incubated for 24 h, and analyzed by flow cytometry. The percentages of DENV antigen positive cells are shown from three independent experiments with different viral preparations and asterisks denote statistical significance (*, P < 0.05; **, P < 0.01).

FIG. 4.

Localization of K128 and N124E mutations in three-dimensional structure of PL046 E. Positions of N124 and K128 are viewed from the top of the E monomer structure with domain I (red), domain II (yellow), domain III (blue), and fusion peptide (green) based on the published coordinates (39).

FIG. 5.

Interaction between the recombinant DENV strains and HS proteoglycans. (A) Inhibition of viral attachment to BHK-21 cells by heparinase. BHK-21 cells were pretreated or not with heparinase I, exposed to 5,000 GE per cell of virus for 1 h at 4°C, washed at 4°C, and homogenized, and RNA was isolated. The quantity of DENV present was determined by real-time RT-PCR. The results are expressed as percentages of DENV particles bound to cells relative to the number bound to untreated PL046-IC-infected cells. Error bars represent the SEM of two independent experiments using a total of five or six different viral preparations of E124/128-IC or PL046-IC, respectively. (B) Inhibition of BHK-21 infectivity by heparinase. BHK-21 cells were pretreated or not with heparinase I or chondroitinase ABC, exposed to 5,000 GE per cell of virus for 1 h at 4°C, washed at 4°C, plated, and incubated at 37°C. At 24 h p.i., the presence of DENV prM was detected via flow cytometry. The results are shown as percentages of DENV-positive cells relative to the untreated control. Error bars indicate the SEM corresponding to three independent experiments using four or six different viral preparations of E124/128-IC or PL046-IC, respectively. (C) Inhibition of BHK-21 infectivity with soluble GAG. A total of 5,000 GE per cell of PL046-IC or E124/128-IC was mixed at different concentrations of the indicated soluble GAG prior to incubation with BHK-21 cells for 24 h at 37°C, followed by detection of DENV infection via flow cytometry. Data from one representative out of three independent experiments using eight different viral preparations are shown. (D) Viral binding to heparin. Approximately 1010 GE of sucrose-gradient-purified virus was added to heparin-Sepharose columns equilibrated with a phosphate buffer. After a washing step, bound virus was eluted with an increasing NaCl concentration, ranging from 0 to 500 mM. Viral RNA in each fraction was then quantified by real-time RT-PCR. Asterisks denote statistical significance of results (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG. 6.

Clearance of the recombinant DENV virions from circulation. AG129 mice were intravenously infected with 1.5 × 1011 GE of PL046-IC or E124/128-IC. Serum was collected at various time points p.i., and viral RNA levels were quantified by real-time RT-PCR. The data are expressed as GE of DENV RNA per ml of serum. Each symbol represents an individual mouse. Asterisks denote viral RNA levels that are statistically different between PL046-IC- and E124/128-IC-injected mice at 3 h p.i. (**, P < 0.01).

FIG. 7.

Levels of viral RNA in tissues of PL046-IC or E124/128-IC-infected mice. (A) Serum; (B) liver, kidneys, lungs, and spleen; (C) bone marrow, mesenteric lymph nodes (MLN), and peripheral lymph nodes (PLN); (D) stomach, large intestine, and small intestine. AG129 mice were intravenously infected with 1.5 × 1011 GE of PL046-IC or E124/128-IC. The indicated tissues were harvested at 0.5, 3, 5, 7, 12, 24, and 72 h p.i., and viral RNA was quantified by real-time RT-PCR. The data are expressed as the geometric mean of DENV RNA copies relative to 18S of 3 to 10 mice per group for each time point. Asterisks denote viral RNA levels that are statistically different between PL046-IC- and E124/128-IC-infected mice (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

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A mouse-passaged dengue virus strain with reduced affinity for heparan sulfate causes severe disease in mice by establishing increased systemic viral loads - PubMed (original) (raw)