Short report: detection of Japanese encephalitis virus in mouse peripheral blood mononuclear cells using an in situ reverse transcriptase-polymerase chain reaction.

Japanese encephalitis (JE) is an important mosquito-borne viral disease in Southeast Asia. Isolation of JE virus from peripheral blood is usually difficult because of transient and low titer of viremia. An in situ reverse transcriptase-polymerase chain reaction (RT-PCR) method was designed to amplify gene (envelope) fragments of JE virus residing in peripheral blood mononuclear cells (PBMCs) without extraction of RNA. Baby hamster kidney-21 cells infected with the T1P1 strain of JE virus (an isolate from Armigeres subalbatus collected in Taiwan) were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The RT-PCR was then performed in microtubes using digoxigenin-labeled primers. Virus-positive PBMCs were detected in mice at day 1 and day 3, but not day 5, after intravenous inoculation with JE virus, suggesting that detectable virus circulating in the blood of mice is present for only 2-3 days. On examination of mouse brain tissues, viral RNAs were absent until day 3 post-inoculation. This implied that virus migration from the peripheral blood into the central nervous system occurs at or after day 3 post-inoculation. This method is unique in that the reactions can be conducted in tubes; this makes it convenient, accurate, and efficient compared with the conventional in situ RT-PCR on slides.