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Am. J. Trop. Med. Hyg., 69(6), 2003, pp. 648-651
Copyright © 2003 by The American Society of Tropical Medicine and Hygiene

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SHORT REPORT: DETECTION OF JAPANESE ENCEPHALITIS VIRUS IN MOUSE PERIPHERAL BLOOD MONONUCLEAR CELLS USING AN IN SITU REVERSE TRANSCRIPTASE–POLYMERASE CHAIN REACTION

CHING-KAI CHUANG, SHYAN-SONG CHIOU, LI-CHING LIANG, AND WEI-JUNE CHEN
Department of Public Health and Parasitology, College of Medicine, Chang Gung University, Kwei-San, Tao-Yuan, Taiwan; Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei, Taiwan

 

ABSTRACT

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.

Received February 17, 2003. Accepted for publication July 25, 2003.

Acknowledgments: We thank Mei-Hwei Fan-Chiang and Jing-Gi Hwang for their technical assistance.

Financial support: This work was supported by a grant from Chang Gung Memorial Hospital (CMRP 1212) and in part by the Department of Health, Republic of China (DOH88-TD-1018).

Authors’ addresses: Ching-Kai Chuang, Li-Ching Liang, and Wei-June Chen, Department of Public Health and Parasitology, College of Medicine, Chang Gung University, 259 Wen-Hwa First Road, Kwei-San, Tao-Yuan 33332, Taiwan. Shyan-Song Chiou, Institute of Epidemiology, College of Public Health, National Taiwan University, Taipei 10018, Taiwan.

Reprint requests: Wei-June Chen, Department of Public Health and Parasitology, College of Medicine, Chang Gung University, 259 Wen-Hwa First Road, Kwei-San, Tao-Yuan 33332, Taiwan, Fax: 886-3-211-8700, E-mail: wjchen{at}mail.cgu.edu.tw.






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Copyright © 2003 by the American Society of Tropical Medicine and Hygiene.