Clinical implications of viral quasispecies heterogeneity in chronic hepatitis C

✍ Scribed by González-Peralta, Regino P.; Qian, Keping; She, Jan Y.; Davis, Gary L.; Ohno, Tomoyoshi; Mizokami, Masashi; Lau, Johnson Y.N.

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 648 KB
• Volume: 49
• Category: Article
• ISSN: 0146-6615
• DOI: 10.1002/(sici)1096-9071(199607)49:3<242::aid-jmv14>3.0.co;2-e

✦ Synopsis

To determine the clinical significance of viral quasispecies heterogeneity, 59 patients with chronic hepatitis C were studied using singlestranded conformational polymorphism (SSCP) analysis of the HCV E2 hypervariable region 1 (HVRI); of these, 48 were subsequently treated with interferon-a. The SSCP method was validated using clones of known nucleotide sequence. HVRI was amplified i n 54 of 59 (92%) patients. The median number of SSCP bands per sample was 6 (range: 2-12). Increased quasispecies heterogeneity correlated with the estimated duration of HCV infection ( P < 0.05), parenteralacquired HCV infection (vs. sporadic, P < 0.05), serum HCV RNA levels ( P < 0.05), and HCV geno-

Methods

Three technical requirements for the development of a SSCP assay were anticipated. First, to assure sensitivity and avoid quasispecies selection bias, PCR primers designed from relatively well-conserved regions of the HCV E2 gene were used. However, to allow for assessment of heterogeneity, the primers flanked a hypervariable region (HVR1) [Kato et al., 19921. Second, the 'nested' amplicons were around 200 bp long, the optimum size for SSCP since it allows easy discrimination of bands after gel separation [Hayashi, 19921. Third, a predetermined quantity of cDNA was gel-loaded for SSCP analysis. This is of particular importance since patients with high level HCV viremia would have more amplicon per 0 1996 WILEY-LISS, INC.