Chloroquine interferes with dengue-2 virus replication in U937 cells

Kleber Juvenal Silva Farias, Paula Renata Lima Machado, Renato Ferreira de Almeida Junior, Ana Alice de Aquino, Benedito Antônio Lopes da Fonseca, Kleber Juvenal Silva Farias, Paula Renata Lima Machado, Renato Ferreira de Almeida Junior, Ana Alice de Aquino, Benedito Antônio Lopes da Fonseca

Abstract

The objective of this study was to investigate the use of chloroquine (CLQ) as an antiviral agent against dengue. Chloroquine, an amine acidotropic drug known to affect intracellular exocytic pathways by increasing endosomal pH, was used in the in vitro treatment of U937 cells infected with dengue virus type 2 (DENV-2). Viral replication was assessed by quantification of virus produced through detection of copy numbers of DENV-2 RNA, plaque assay and indirect immunofluorescence. qRT-PCR and plaque assays were used to quantify the DENV-2 load in infected U937 cells after CLQ treatment. It was found that a dose of 50 μg/mL of CLQ was not toxic to the cells and resulted in significantly less virus production in infected U937 cells than occurred in untreated cells. In the present work, CLQ was effective against DENV-2 replication in U937 cells, and also caused a statistically significant reduction in expression of proinflammatory cytokines. The present study indicates that CLQ may be used to reduce viral yield in U937 cells.

Keywords: DENV-2; chloroquine; plaque assay; quantitative reverse transcription polymerase chain reaction.

© 2014 The Societies and Wiley Publishing Asia Pty Ltd.

Figures

Figure 1
Figure 1
The effect of CLQ on the cytotoxicity in U937 cells. Concentrations equal or higher than 500 μg/mL (CLQ) were highly cytotoxic to U937 cells, while concentrations equal or lower than 50 μg/mL (CLQ) did not induce significant cytotoxicity.
Figure 2
Figure 2
Action of CLQ on DENV‐2 replication in U937 cells. (a) The viral RNA present in the culture supernatants of U937cells infected with DENV‐2, treated and untreated CLQ post‐infection, was extracted and analyzed by qRT‐PCR. (b) Culture supernatants of U937cells infected with DENV‐2, treated and untreated CLQ post‐infection, were analyzed by plaque assay. The results represent the average values of the viral RNA copy number (P  < 0.001).
Figure 3
Figure 3
Action of CLQ added at 24‐hr intervals on DENV‐2 replication in U937 cells. (a) The viral RNA present in the culture supernatants of U937 cells infected with DENV‐2, treated and untreated CLQ (every 24 hr post‐infection), was extracted and analyzed by qRT‐PCR. (b) Culture supernatants of U937 cells infected with DENV‐2, treated and untreated CLQ (every 24 hr post‐infection), was analyzed by plaque assay. The results represent the average values of the viral RNA copy number (P  < 0.05).
Figure 4
Figure 4
Action of CLQ added at 12‐hr intervals on DENV‐2 replication in U937 cells. The viral RNA present in the culture supernatants of U937 cells infected with DENV‐2, treated and untreated CLQ (every 12 hr post‐infection), was extracted and analyzed by qRT‐PCR. The results represent the average values of the viral RNA copy number (P  < 0.05).
Figure 5
Figure 5
Indirect immunofluorescence assay. Detection of DENV‐2 virus in U937 cells infected or uninfected, treated and untreated CLQ (every 24 hr post‐infection). (Original magnification 200–1,000×).
Figure 6
Figure 6
Relative quantification of cytokines by real‐time PCR in U937 cells infected or not with DENV‐2 in the presence or absence of CLQ added at 24‐hr intervals. IFN‐α (a), IFN‐β (b), IFN‐γ (c), TNF‐α (d), IL‐6 (e), IL‐12 (f), and IL‐10 (g) mRNA expression in DENV‐2 infected or uninfected U937 cells, treated and untreated CLQ were analyzed by quantitative RT‐PCR, and normalized to mouse beta actin gene. Values were submitted to t test in which *P < 0.05 vs. U937 cells infected with DENV‐2s.

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