IDENTIFICATION OF MOSQUITO-BORNE FLAVIVIRUS SEQUENCES USING UNIVERSALPRIMERS AND REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION

A reverse transcription/polymerase chain reaction (RT/PCR) protocol fo r the rapid detection and identification of flaviviruses was developed using a set of universal oligonucleotide primers. These primers corre spond to sequences in the 3' non-coding region and in the NS5 gene whi ch are highly conserved among the mosquito-borne flaviviruses. The seq uences of the resulting amplified products were analysed for dengue 1, dengue 2, dengue 3, dengue 4, Japanese encephalitis, West Nile, yello w fever and Zika viruses, and compared with the published sequences of other flaviviruses. The 291-297 nucleotides corresponding to the C-te rminus of NS5 gene showed 56 to 76 % similarity, whereas the 3' non-co ding region (190 to 421 nucleotides) showed only 20 to 36 % similarity . Genetic classification of the Zika virus supported its traditional s erelogical grouping. Recombinant plasmids containing the flavivirus se quences were used in a nucleic acid hybridization test to identify the RT/PCR products derived from viral RNA extracted from experimentally infected mosquitoes. The plasmids were dotted on a strip of nitrocellu lose membrane and incubated with the RT/PCR product labelled with digo xigenin during the PCR step. This is a valuable method for the rapid a nd specific identification of mosquito-borne flaviviruses in biologica l specimens and for subsequent sequence analysis.