Identification of conserved nucleotide sequences within the GB virus C 5'-untranslated region: design of PCR primers for detection of viral RNA.
Recently, the discovery of a new human RNA virus, GB virus C (GBV-C), was reported. GBV-C was isolated from the serum of a West African individual using degenerate oligonucleotide PCR primers designed from a consensus sequence of the NS3 helicase genes of hepatitis C virus (HCV), GBV-A, and GBV-B. Seven other individuals were shown to be infected with GBV-C via RT-PCR using these primers. Subsequently, degenerate PCR primers based upon a consensus sequence of the eight original isolates were designed. These primers were shown to be superior to the original set. However, since they were derived from a region of the viral genome exhibiting up to 17% nucleotide sequence divergence, mismatch between the primers and template may result in an underestimation of the true GBV-C prevalence. To overcome this potential problem, we obtained the sequences at the 5'-untranslated region (UTR) of the GBV-C genome from 35 infected individuals and identified regions of high sequence conservation among the isolates. We describe the design and testing of PCR primers derived from conserved sequences within the 5'-UTR of the GBV-C genome. These primers were shown to be as effective as the helicase-derived primers in detecting GBV-C RNA in human sera.
Muerhoff
A S
AS
Virus Discovery Group, Experimental Biology Research, Abbott Laboratories, North Chicago, IL USA. muerhoffs@randb.abbott.com
Simons
J N
JN
Erker
J C
JC
Desai
S M
SM
Mushahwar
I K
IK
D001483:Base Sequence; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics*; D018067:Flaviviridae / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D016415:Sequence Alignment; D017421:Sequence Analysis
D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D012367:RNA, Viral