Twenty five consecutive patients (16 men and 9 women) with chronic hepatitis C, with a mean age of 47 years (range: 23–58 years), were included in the study. All had persistently elevated serum alanine aminotransferase (ALT) levels of at least twice the upper normal limit of the normal range for at least 6 months, histological evidence of chronic hepatitis (chronic active hepatitis in 23 patients, chronic persistent hepatitis in 2 patients) and seropositivity for both anti-HCV [second generation enzyme immuno assay (EIA), Abbott Laboratories, North Chicago, IL, USA] and HCV RNA [reverse transcription-polymerase chain reaction (RT-PCR)]. Criteria for exclusion included evidence of infection with hepatitis A or B virus, decompensated liver disease, pregnancy or lactation, the presence of any serious medical or psychiatric illness and evidence of other forms of liver disease. Informed consent for participation was obtained from all patients before treatment.

Clinical and laboratory evaluation were performed monthly before and during treatment and then monthly between 6 and 36 months after treatment was completed.

Patients were randomly assigned to receive recombinant IFN alfa-2a (Intermax alpha; Lucky Biotec. Taejeon, Korea)according to three different protocols: group A (n=7), 3 million units (MU), subcutaneously (s. c.), every day for 3 months; group B (n=8), 3MU, s. c., every other day for 3 months; group C (n=10), 3 MU, s. c., every other day for 6 months.

Serum anti-HCV was tested by a second generation commercial EIA kit and assayed before and at the end of treatment.

Oligonucleotide primers for detection, genotyping and quantitation of HCV RNA were synthesized by a DNA synthesizer (Pharmacia, LKB, Uppsala, Sweden).

HCV RNA was extracted from 100 μl of serum, as described previously¹³⁾, and extracted RNA was precipitated with isopropanol and washed with ethanol. The HCV RNA pellet was dissolved in diethylpyrocarbonate-treated distilled water (DEPC-H20) and stored until use. Synthesis of cDNA from HCV RNA samples was carried out at 37°C for 1hour with 20 units of Moloney Murine Leukemia Virus reverse transcriptase (MMLV-RT, GIBCO BRL, Gaithersburg, MD. USA). The cDNA product was amplified on two stage PCR. The two sets of primer used for RT-PCR were derived from 5′-untranslated region (5′-UTR) of HCV genome¹⁴⁾. The sequences were 5′-CTG TGA GGA ACT ACT GTC TT-3′ (sense, nucleotides 28 to 47) and 5′-CTG TGA GGA ACT ACT GTC TT-3′ (antisense, nucleotides 229 to 248) as an outer primer set, and 5′-TTC ACG CAG AAA GCG TC TAG-3′ (sense, nucleotides 46 to 65) and 5′-GTT GAT CCA AGA AAG GAC CC-3′ (antisense, nucleotides 171 to 190) as an inner primer set.

After extraction of HCV RNA and synthesis of cDNA as described above, the double stage PCR were performed with the four genotype-specific oligonucleotide primers described by Enomoto et al¹⁵⁾. Kato et al.^16,17) Chayama et al¹⁸⁾ that were deduced from the nucleotide sequences in the NS5 region. The sequences of the universal primers for four genotypes used in the first round PCR were 5′-TGG GGA TCC CGT ATG ATA CCC GCT GCT TTGA-3′ (sense, corresponding to nucleotides 8230 to 8260 of HCV-J ¹⁶⁾) and 5′-GGC GGA ATT CCT GGT CAT AGC CTC CGT GAA-3′ (antisense, corresponding to nucleotides 8601 to 8630 of HCV-J ¹⁶⁾). The sequences of the primers in the second round PCR for HCV-US, -J, -K2a and -K2b, which are HCV genotypes 1a, 1b, 2a and 2b, respectively, according to the nomenclature of a recently proposed system ¹⁹⁾, were 5′-CGA CAT CCGT ACG GAG GAGG-3′ (sense) and 5′-CAG GCT GCC CGG GCC TTG AT-3′ (antisense) for HCV genotype 1a, 5′-TGA CAT CCG TGT TGA GGA GT-3′ (sense)and 5′-CGG GCC GCA CAG GCC TCC AA-3′ (antisense) for genotype 1b, 5′-TAT GTT CAA CAG CAA GGG CCA GA-3′ (sense) and 5′-CCT GGT CAT AGC CTC CGT GAA-3′ (antisense) for genotype 2a, and 5′-CCT GGT CAT AGC CTC CGT GAA-3′ (sense) and 5′-CCTGGTCATAGCCTCCGTGAA - 3′ (antisense) for genotype 2b.

10μl of synthesized cDNA was amplified on two stage PCR as described previously²⁰⁾. The first PCR was carried out for 35 cycles of three steps, 1 min at 94°C, 1 min at 40°C and 1min at 72°C, respectively, and then 1 μl of the first PCR products was put into the prepared 4 each genotype specific microtube. The second PCR was carried out for 30 cycles of three steps, 1 min at 94°C, 45 seconds at 55°C, and 1 min at 72°C. The amplified DNA fragments were electrophoresed in 2% agarose gels with ethidium bromide staining and were observed under UV light.

HCV RNA was quantified by a competitive RT-PCR assay using synthetic mutant HCV RNA as an internal template^21,22). A mutant RNA was synthesized by means of site-directed mutagenesis and in vitro transcription.

A cDNA included 5′-UTR of HCV genome from the serum of HCV carrier was cloned into an M13mp18 vector system. After the above the M13mp 18-5′UTR plasmid was digested with restriction endonucleases, BamHI and Hind III (NEB, USA), and cloned again into pGEM4-z vector (pGEM-5′UTR). A deletion mutant (pGEM-5′UTRdel) was made by PCR. A mutant containing 80 base pairs deletion (pGEM-5′UTRdel)was digested Hind III, which served as templates for T7 RNA polymerase, and then the DNA template was removed using DNase I (Promega, RQ1 RNase free DNase I). The above mutant RNA transcripts were purified by phenol-chloroform extraction and ethanol precipitation and used as internal templates in quantitative competitive PCR. HCV RNA extraction using 50μl of serum was performed as described above.

An equal amount of sample RNA was put into a set into a set of microtubes that already had serially 10-fold diluted mutant RNA.

The sequences of primer used in the first PCR were HCV QC1 5′-CCA CCA TAG ATC ACT CCC CTGT-3′ (corresponding position 28 to 48 of HCV-II) as a sense primer and HCVL70, 5′-TTG AGG TTT AGG ATT CGT GCT CAT-3′ (corresponding position 343 to 366 of HCV-II) as an antisense primer.

The first PCR was carried out for 40 cycles of steps for 1min at 95°C, 1min at 55°C and 2min at 72°C, and then the second PCR was carried out for 25 cycles of steps for 1min at 94°C, 45seconds at 55°C and 1min at 72°C.

Complete response (CR) was defined as a normalization of serum ALT levels at the end of therapy. A sustained response (SR) was defined as the presence of persistently normal serum ALT levels and negative seroconversion of HCV RNA during the entire follow up (for at least six months) after CR. Nonsustained response (NSR) was of serum ALT levels became elevated again during follow up after CR. Patients were defined as nonresponders (NR) if serum ALT levels were persistently abnormal during or after treatment.

The Chi-square test or Fisher’s exact test was used for comparison between group frequencies. Univariate and multivariate analysis were used to establish which factors contributed to a sustained response to IFN therapy. We used SPSS (version 6.0) program for statistical analysis of all variables.