Analysis of plasma viral RNA levels during acute dengue virus infection using quantitative competitor reverse transcription-polymerase chain reaction

There is increasing recognition of the potential importance of viral burden in the pathogenesis of dengue hemorrhagic fever (DHF). There is little dat a available, however, describing the kinetics of viral replication in human s with natural dengue virus (DV) infection. Standard procedures for measuri ng titers of infectious virus in clinical specimens are either laborious or insensitive. We developed a method for measurement of DV RNA in plasma sam ples based on reverse transcription-polymerase chain reaction (RTPCR) using a mutant RNA target as a competitor. This technique was reproducible and a ccurate for samples containing any of the four DV serotypes, and could be a pplied to samples containing as few as 250 copies of RNA per reaction. We e xamined plasma viral RNA levels in 80 children with acute DV infection; seq uential plasma samples were tested in 34 of these children. Plasma viral RN A levels ranged as high as 10(9) RNA copies/ml, and correlated with titers of infectious virus measured in mosquitoes (r= 0.69). Plasma viral RNA leve ls fell rapidly during the last several days of the febrile period. We did not find a significant difference in maximal plasma viral RNA levels betwee n children with DHF and children with dengue fever, but peak viral RNA leve ls were identified in only 16 subjects. We conclude that this quantitative RT-PCR method will be valuable for further studies of natural DV infections . (C) 2001 Wiley-Liss, Inc.