RAPID GENOTYPING OF HEPATITIS-C VIRUS ISOLATES BY DIDEOXY FINGERPRINTING

A number of distinct hepatitis C virus (HCV) types and subtypes have b een identified by DNA sequencing of multiple genome regions. It has be en postulated that these might also reflect phenotypic differences in the nature of HCV infection. Recent evidence suggests a relationship b etween HCV genotype and alpha-interferon response in patients with chr onic hepatitis C. A simplified method of genotyping in comparison to d irect DNA sequencing was investigated with the intention of providing a rapid, less labour-intensive method for routine genotyping. HCV RNA was extracted from serum by a modified guanidinium/acid-phenol extract ion and peripheral blood lymphocytes using RNAzol B (Cinna-Biotecx). T he RNA was reverse transcribed and a 287 bp segment of the 5' non-codi ng region (5' NCR) amplified using a nested-PCR reaction. PCR products were purified using Qiaquick spin columns. Products were directly seq uenced by cycle sequencing. Dideoxy termination analysis was carried o ut by cyclic extension of a P-33-labelled primer by Tth polymerase wit h termination by dideoxy thiamine (ddT) or cytosine (ddC). Reaction pr oducts were analysed by electrophoresis on denaturing 7 M urea/6% acry lamide gels followed by autoradiography. Computer aided sequence analy sis indicated that conserved 5' NCR sequence variation alone was suffi cient to identify HCV types 1a, 1b, 2a, 2b, 3 and 4. Dideoxy fingerpri nting improved greatly the efficiency of genotyping with an approximat e four-fold increase in throughput. In addition, the results were very easily analysed although it was essential to run appropriate controls for each genotype. Reactions incorporating ddT distinguished types 1, 2a, 2b, 3 (provisionally 1a and 1b); a ddC reaction confirmed 1a and 1b typing. Standard denaturing gels gave superior results than a varie ty of non-denaturing ('SSCP') gels. Our results show that dideoxy fing erprinting is a reliable and efficient alternative to direct sequencin g for HCV genotyping which is adaptable to semi-routine screening.