Characterization of hepatitis C virus quasispecies using heteroduplex analysis

Hepatitis C virus (HCV) exists as a population of distinct but related viruses (quasispecies). Increased numbers of quasispecies have been found in persons not responding to therapy and with rapid progression of disease. However, the clinical and epidemiologic application of HCV quasispecies assessments has been limited by the expense and effort of traditional evaluation by sequencing HCV clones. Using TA cloning and cDNA sequencing as a gold standard, the heteroduplex assay (HDA) was evaluated as a method of assessing HCV quasispecies in subjects with acute HCV infection. HCV was extracted using TRIzol and amplified with nested RT-PCR. Clones were established from PCR product by TA cloning (Invitrogen), from three genomic regions: core-El (nucleotide 502-974), hypervariable region 1 (HVR1) in HCV envelope (1087-1262), and E2 (1415-1866). HDA was assessed using isotope (³²P labeling probe) and nonisotope methods. Assay sensitivity. 41 HCV clones were screened with HDA and cDNA sequencing. HDA identified as distinct (gel shift recognized) all clones with less than and up to 99 percent nucleotide identity. Genomic region. 18 clones were screened using primers for each of three genomic regions in 2 patients with 2-4 serial samples. Equivalent HDA results were obtained on clones screened with HVR1 and E2 primers, while the gel shift for core-El region was not optimal for delineation of HCV quasispecies. Reproducibility and Detection system. Identical results were obtained when HDA was repeated on 108 clones using the E2 primers. Identical quasispecies assessments were obtained among 34 clones assessed by isotope and nonisotope detection. Conclusion. Compared to cDNA sequencing, nonisotope HDA is a sensitive and reproducible method of assessing HCV quasispecies. Future studies of clinical applications are indicated.