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 61C) and
yellow fever (17 D) viruses demonstrated complete concurrence with published data. However, 2
nucleotide differences were observed between our data for
dengue 3 H87 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 NS3
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.