Use of NS 3 consensus primers for the polymerase chain reaction amplification and sequencing of dengue viruses and other flaviviruses

Abstract

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.

Comparison of Flavivirus Universal Primer Pairs and Development of a Rapid, Highly Sensitive Heminested Reverse Transcription-PCR Assay for Detection of Flaviviruses Targeted to a Conserved Region of the NS5 Gene Sequences

“…Several RT-PCRs have been developed for detection of flavivirus RNA by using different pairs of primers for differentiating between species of viruses (12), including flavi-universal primers for mosquito-borne flaviviruses (29) and six published primer pairs permitting complete detection of the Flavivirus genus (7,8,15,19,24,26). Our purpose was first to compare these flavivirus genus diagnosis references.…”

Zika Virus

“…Most protocols target the terminal portion of the NS5-encoding gene or the 3= NCR of the Flavivirus genome because of highly conserved regions in this part of the genome (340). ZIKV was successfully detected with Flavivirus RT-PCR assays targeting the E-encoding gene (341), the NS1-encoding gene (342), the NS3-encoding gene (343), and the NS5-encoding gene (47,(344)(345)(346)(347). After detection of Flavivirus RNA, identification to the species level requires additional testing.…”

Comparative Analysis of NS3 Sequences of Temporally Separated Dengue 3 Virus Strains Isolated from Southeast Asia

“…A previously reported RT-PCR protocol [4] was modified and performed in a single tube containing 50 pi of reaction mix compris ing 15 pi of resuspended RNA. 1 x buffer with 1.5 m.V/ MgCh, 0.2 m.V/ of each dNTP, 0.15 pA/cach of DV1 primer (5'-GGRACKT-CAGGWTCTCC-3') and DV3 primer (5'-AARTGIGCYTCRTC-CAT-3'), 25 U of MMLV reverse transcriptase and 0.15 U of Taq polymerase (HT Biotechnology, Cambridge, UK).…”