Sequence diversity in the 5'-UTR region of GB virus C/hepatitis G virus assessed using sequencing, heteroduplex mobility analysis and single-strand conformation polymorphism.
GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense RNA virus belonging to the Flaviviridae family identified recently. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect GBV-C/HGV RNA using nested primers designed to amplify 245 bp of the 5'-untranslated region (UTR). GBV-C/HGV RNA was detected in 20.7% of 101 HCV-RNA positive and 6.8% of 44 HCV-RNA negative specimens. Sequencing of the PCR products demonstrated they had between 84.3 and 100% nucleotide identity. Most of the diversity corresponded to two variable regions identified within the 5'-UTR. Phylogenetic analysis indicated that GBV-C/HGV subtypes present in Australia belonged to group 2 and were closest in evolutionary terms to isolates from the USA and Europe. All isolates were analysed using single-strand conformation polymorphism (SSCP) and heteroduplex mobility analysis (HMA) on 8% non-denaturing polyacrylamide gels. SSCP of the isolates identified a number of distinct conformation polymorphisms that corresponded with sequence-determined genetic diversity. HMA was developed to assess the amount of genetic diversity between isolates without the need for sequencing. The average difference between the predicted divergence of two isolates calculated from the mobility of the heteroduplex and the actual value (based on nucleotide sequence) was 2.3% in this sample of isolates, where the mean sequence divergence was 8.52%.
White
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PA
Department of Microbiology, SEALS, Prince of Wales Hospital, Randwick, Sydney, NSW, Australia.
Li
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Z
Rawlinson
W D
WD
D020121:5' Untranslated Regions; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D005069:Evaluation Studies as Topic; D018067:Flaviviridae / Q000235:genetics* / Q000302:isolation & purification; D014644:Genetic Variation; D016174:Hepacivirus / Q000302:isolation & purification; D006801:Humans; D008969:Molecular Sequence Data; D009692:Nucleic Acid Heteroduplexes / Q000302:isolation & purification; D010802:Phylogeny; D018807:Polymorphism, Single-Stranded Conformational; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D014773:Virology / Q000379:methods*
D020121:5' Untranslated Regions; D017931:DNA Primers; D009692:Nucleic Acid Heteroduplexes; D012367:RNA, Viral