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.
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.
Wang
Wei-Kung
WK
Institute of Microbiology, College of Medicine, National Taiwan University, Taipei, Taiwan. wwang60@yahoo.com
Sung
Tzu-Ling
TL
Tsai
Yu-Chen
YC
Kao
Chuan-Liang
CL
Chang
Shu-Mei
SM
King
Chwan-Chuen
CC
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D003715:Dengue / Q000821:virology*; D003716:Dengue Virus / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology*; D008969:Molecular Sequence Data; D012367:RNA, Viral / Q000097:blood*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication
D017931:DNA Primers; D012367:RNA, Viral