Vol 33 (Suppl 3) 2002 139 MOLECULAR SEROTYPING OF DENGUE VIRUSES IN FIELD- CAUGHT AEDES MOSQUITOS BY IN-HOUSE RNA EXTRACTION/ RT-PCR REAGENT KITS P Pankhong 1 , W Siriprasertkul 1 , S Patpoparn 2 , M Srisuphanunt 2 , J Rojanapremsuk 2 , R Sithiprasasna 3 , RE Coleman 3 , A Nisalak 4 , TP Endy 4 , MK Attatippaholkun 5 and WH Attatippaholkun 1 1 Department of Clinical Chemistry, Faculty of Medical Technology, Mahidol University, Bangkok; 2 Department of Parasitology, Faculty of Public Health, Mahidol University, Bangkok; 3 Department of Entomology, 4 Department of Virology, The US Army Medical Component (Armed Forces Research Institute of Medical Sciences), Bangkok; 5 Thailand Institute of Scientific and Technological Research, Bangkok, Thailand Abstract. We developed in-house RNA extraction and RT-PCR reagent kits for the molecular serotyping of dengue viruses in field-caught Aedes mosquitos. Mosquitos that showed positive results by ELISA or IFA were selected for the identification of dengue viruses in order to predict the distribution of the four dengue serotypes. Total RNA was extracted from one whole mosquito as well as from one dissected mosquito by our in-house RNA extraction reagents using the modified method of guanidinium thiocyanate denaturation and isopropanol precipitation. The extracted RNA was amplified by our in-house RT-PCR reagents specific for each dengue serotype under optimized conditions. Dengue viral RNA extracted from a single mosquito as well as from the head and thorax of one dissected mosquito could be detected successfully; it could not be found in the abdomen, however. These results indicated that most of the dengue viruses were located in the head and thorax rather than in the abdomen. The results of dengue serotyping showed a pure specific PCR product for each dengue serotype at 490, 230, 320 and 398bp for DEN-1, DEN-2, DEN-3, and DEN-4 respectively. In addition, the detection sensitivity was very high: an amount of RNA template equivalent to approximately 1/80 of a single mosquito could be detected by agarose gel electrophoresis and ethidium bromide staining. The coupling of RT-PCR- based surveillance of dengue viral infection with disease mapping data (Geograpical Information System, GIS) could serve as an alternative epidemiological means of providing an early warning of dengue fever/ dengue hemorrhagic fever epidemics. Disposito, 1988). The flavivirus genome encodes an uninterrupted open reading frame (ORF), flanked by 5´ and 3´ non-coding regions. The order of proteins encoded in dengue viruses ORF is: 5´-non coding region (5´-NC)-capsid(C)-premembrane/membrane (prM/M)-envelope (E)-nonstructural proteins -NS1- NS2A-NS2B-NS3-NS4A-NS4B-NS5- 3´-non coding region (3´- NC) (Westaway et al, 1985). Dengue viruses are transmitted to human by domestic Aedes mosquitos, which inhabit the tropics and subtropics, raising the spectre of endemic dengue in these areas. The previous data suggested that dengue viruses were isolated from Aedes mosquitos collected in nature (Khin and Than, 1983; Hull et al, 1984). Therefore, the identification and typing of dengue viruses isolated from the field-caught Aedes mosquitos and from clinical specimens are important for epidemiological and clinical investigations. The technique that is simple and rapid for the detection, identification, and typing of dengue viruses is reverse transcription - polymerase chain reaction (RT-PCR). We present our development of an in-house RNA extraction and RT-PCR reagent kits for the molecular serotyping of dengue viruses in field-caught Aedes mosquitos. INTRODUCTION The genus Flavivirus of the family Flaviviridae includes viruses that cause diseases of major health importance, such as yellow fever (YF), Japanese encephalitis (JE), tick-borne encephalitis (TBE), and dengue (DEN). There are four antigenically related, but distinct, dengue virus serotypes (DEN-1, DEN-2, DEN-3, DEN-4), all of which can cause a range of diseases: dengue fever (DF), dengue hemorrhagic fever (DHF), and dengue shock syndrome (DSS). DEN diseases cause death and hospitalization in tropical and subtropical countries around the world. Dengue viruses are enveloped RNA viruses that contain a single- stranded, positive-sense RNA of approximately 11kb in length. The genomic RNA has type-I 5´ cap of m 7 Gppp A and lacks a 3´ -end poly(A) track (Wengler and Wengler, 1981; Brinton et al, 1986; Brinton and Correspondence: WH Attatippaholkun, Department of Clinical Chermistry, Faculty of Medical Technology, Mahidol University, Bangkok 10700, Thailand. Tel: ++66 (0) 2419 7168; Fax: ++66 (0) 2412 4110 E-mail: mtwap@mahidol.ac.th