USE OF NS-3 CONSENSUS PRIMERS FOR THE POLYMERASE CHAIN-REACTION AMPLIFICATION AND SEQUENCING OF DENGUE VIRUSES AND OTHER FLAVIVIRUSES

Consensus primers for the polymerase chain reaction were designed based on conserved motifs within the serine protease and RNA helicase domains encoded by the NS 3 genes of dengue and other flaviviruses. Target fragments of 470 bp were amplified on cDNA templates synthesized from RNAs of dengue types 1, 2, 3, and 4, Japanese encephalitis, Kunjin, and yellow fever viruses using random or specific downstream primers. PCR of oligo(dT)-primed cDNAs from Japanese encephalitis and Kunjin viral RNAs did not yield target bands. As few as 10(3) copies of dengue viral RNA could be detected. Direct DNA sequencing of PCR products of reference strains of dengue 2 (NGC), Kunjin (MRM 61 C) and yellow fever (17 D) viruses demonstrated complete concurrence with published data. However, 2 nucleotide differences were observed between our data for dengue 3 H 87 strain and the published sequence, resulting in a single amino acid disparity. Differences at 21, 16, and 11 nucleotide positions were noted between dengue 1 Hawaii and S 275/90; dengue 4 H 241 and 814669; Japanese encephalitis Nakayama and JaOArS 982 viral strains, culminating in only 4, 1 and 1 amino acid residue differences, respectively. These amino acid disparities occurred outside putative active sites of the enzymatic domains, emphasizing the important role of the NS 3 protein in flaviviral replication. This RNA-PCR consensus primer strategy coupled with DNA sequencing represents a valuable tool for the molecular diagnosis and epidemiology of dengue and other flaviviral infections.