Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay - PubMed (original) (raw)

Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay

Wei-Kung Wang et al. J Clin Microbiol. 2002 Dec.

Abstract

While dengue virus is thought to replicate in mononuclear phagocytic cells in vivo, attempts to detect it in peripheral blood mononuclear cells (PBMC) by virus isolation or antigen detection have had variable and generally low rates. In this study, we developed a reverse transcription (RT)-real-time PCR assay to quantify positive- and negative-sense RNA of dengue virus type 2 within the cells. The assay includes an RT step using either sense or antisense primer followed by a real-time PCR step using the designed primers and probe, which target a capsid region highly conserved in dengue virus type 2 strains. It can be used to monitor the dynamic change of intracellular dengue virus RNA species during the course of infection. When this assay is employed in quantification of dengue virus RNA species in PBMC from 10 patients infected with dengue virus type 2, both positive- and negative-sense dengue RNA can be detected, indicating that dengue virus is actively replicating in PBMC in vivo. Moreover, the amounts of negative-sense dengue virus RNA in PBMC correlate very well with the viral load of dengue virus in plasma, suggesting that quantification of negative-sense dengue virus RNA in PBMC may provide another indicator of dengue virus replication in vivo. Use of this convenient, sensitive, and accurate method of quantification in clinical samples from patients with different disease severity would further our understanding of the pathogenesis of dengue.

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Figures

FIG. 1.

FIG. 1.

Alignment of the designed primers and probe with DEN-2 sequences available in GenBank and representative DEN-1, DEN-3, and DEN-4 sequences. The genome positions according to the DEN-2 Jamaica strain (5) are shown at the top. Dashes indicate identity.

FIG.2.

FIG.2.

(A) Schematic diagram of the construct, CPrM/pCRII-TOPO, and the protocol used in generating the positive-sense RNA (+RNA) and negative-sense RNA (−RNA) as standards for the real-time RT-PCR and the RT-real-time PCR assays. The relative positions of the primers and probe are shown. (B) Relationship of known input RNA copies to the threshold cycle (CT) in the real-time RT-PCR assay. (C) Relationship between the RNA copy number determined by the real-time RT-PCR assay (copies per milliliter) and the virus titer (PFU per milliliter). RNA templates derived from serial 10-fold dilutions of the DEN-2 New Guinea virus were subjected to the real-time RT-PCR assay. (D) Relationship between the initial positive-sense RNA copies used in generating cDNA and the threshold cycle (CT) in the real-time PCR assay. r is the correlation coefficient.

FIG. 3.

FIG. 3.

Replication kinetics of DEN-2 virus in BHK cells. Quantification of dengue virus in the culture supernatants (A) and dengue virus RNA species within the cells (B) is shown. BHK cells were infected with DEN-2 virus (16681 strain) at MOI of 0.1, and culture supernatants collected at different time points were subjected to a plaque assay and real-time RT-PCR assay. Solid circles: DEN-2 virus infections; open circles: mock infections. Cells were counted and subjected to the RT-real-time PCR assay. Amounts of positive- and negative-sense RNA per million cells are shown.

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