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

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.Dengue virus belongs to the genus Flavivirus of the family Flaviviridae. It contains a positive-sense single-stranded RNA genome of approximately 11 kb (9,22). Flanked by the two nontranslated regions at both ends, the single open reading frame consists of three structural genes at the 5Ј one-fourth and seven nonstructural genes at the 3Ј three-fourths. Among the 80 or so arthropod-borne flaviviruses, epidemics of infection by the four serotypes of dengue virus (DEN-1, DEN-2, DEN-3, and DEN-4) continue to be a major public health problem in tropical and subtropical areas (4,9,14,25). It has been estimated that approximately 100 million dengue infections occur annually worldwide (9,11,25).The clinical presentations of dengue virus infection range from asymptomatic, or a mild self-limited illness, dengue fever (DF), to a severe and potentially life-threatening disease, dengue hemorrhage fever/dengue shock syndrome (DHF/DSS) (9, 14, 37). Following an incubation period of 3 to 14 days, fever and a variety of symptoms occur, coinciding with the appearance of dengue virus in blood (9,14). Based on the pathological findings in experimentally infected rhesus monkeys and in humans with fatal infections, dengue virus is thought to replicate in mononuclear phagocytic cells in vivo (1,2,9,10,11,14,23,26). Several studies have attempted to detect dengue virus in peripheral blood mononuclear cells (PBMC). Scott et al. (15). Overall, the detection rates of dengue virus in PBMC through either virus isolation or antigen detection method were variable and generally low (3,15,30,36).Rec...

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