A method for full genome sequencing of all four serotypes of the dengue virus.
The availability of whole genome sequencing has contributed to many aspects of dengue research, and its use in dengue virus (DENV) surveillance for early epidemic warning has been proposed. Methods to sequence the genomes of individual dengue serotypes have been described previously, but no single method is known to be applicable for all four serotypes. This report describes a method for sequencing the entire genome of all four DENV serotypes. Using tagged oligonucleotide primers designed for the 3' end, viral RNA was reverse transcribed into a cDNA spanning the entire genome of each of the four serotypes (DENV-1 to -4). This was followed by amplification of the entire cDNA in five overlapping amplicons. A sequence tag was added to the sense primer annealing to the 5' UTR sequence and the antisense primer annealing to the 3' UTR sequence to ensure no terminal nucleotides were omitted during PCR. Sixty-one virus isolates were sequenced: 58 DENV-2, one DENV-1, one DENV-4 and one DENV-3 published previously. The method described could be applied readily for viral biology studies and incorporated into proactive dengue virologic surveillance.
Christenbury
Joseph G
JG
Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, 8 College Rd., Singapore 169857, Singapore.
Aw
Pauline P K
PP
Ong
Swee Hoe
SH
Schreiber
Mark J
MJ
Chow
Angelia
A
Gubler
Duane J
DJ
Vasudevan
Subhash G
SG
Ooi
Eng Eong
EE
Hibberd
Martin L
ML
D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D003716:Dengue Virus / Q000145:classification / Q000235:genetics*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012367:RNA, Viral / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*
D017931:DNA Primers; D018076:DNA, Complementary; D012367:RNA, Viral