Sera or liver tissues were obtained from 70 patients with chronic liver diseases who consisted of 37 sporadic chronic hepatitis (CH), 12 hepatocellular carcinoma (HCC), 16 post-transfusion NANB hepatitis (PT-NANBH), 4 blood donors negative for HBsAg and 1 healthy family member of a patient with sporadic CH. All patients showed positivity in the second generation anti-HCV enzyme immunoassay (Anti-HCV-II EIA; Abbott Laboratories, Chicago, IL., U.S.A.). Frozen liver specimens, biopsied, were available in 8 sporadic CH and 12 HCC.

RNA from sera or liver tissues was extracted by acid-guanidinium-phenol method as described previously¹⁹⁾. Briefly, 200μl of serum was mixed with 200 ul of solution-D (4 M guanidinium thiocyanate, 100 mM Tris-HCl, pH 7.5; 0.5% N-lauroylsarcosine; 0.1 M beta-mercaptoethanol) and 2 M sodium acetate (pH 4.5). A piece of frozen liver tissue biopsied was homogenized in 400 ul of solution-D by Dounce’s homogenizer, and 2 M sodium acetate (pH 4.5) was added. After phenol/chloroform extraction and then isopropanol precipitation, the pellet was dissolved in 20 μl of 1% DEPC-treated distilled water.

An aliquot (4 ul) of RNA solution was mixed with 10 μl mixture containing 10 pmole of the outer antisense primer (#167 R, Table 1), 50 mM tris-HCl (pH 7.5), 75 mM KCl, 3 mM MgCl₂, 0.5 mM of each dNTP, then heat treated for 5 min at 70°C. 20 units of Moloney Murine Leukemia Virus Reverse Transcriptase (MMLV-RT; BRL, Gaithersburg, Md, U.S.A.) was added and incubated at the mixture for 60 min at 37°C, then 10 min at 90°C

Oligonucleotides were synthesized by DNA synthesizer (Pharmacia LKB Gene Assembler Plus, Uppsala, Sweden).

Oligonucleotide primers deduced from the NS5 region of the HCV genome were five sets, including an outer or universal primer set (401 bp) and four type-specific primer sets. The sequences of primers used as outer set in the first round PCR was #166 and #167R, which correspond to positions nucleotide (nt) 8230 to 8260 and the complementary sequence to nt 8601 to 8630 of HCV-J, respectively³⁾.

To determine the genotype of HCV-J, -US, -K1 and -K2 in the second round PCRs, the primers which were designed by Enomoto et al.,⁷⁾ and Kato et al.,¹⁴⁾, #192 (nt 8291 to 8310, sense) and #193R (nt 8487–8506, antisense) for HCV-J, #194 (nt 8291 to 8310, sense) and #195R (nt 8487 to 8506, antisense) for HCV-US, #173 (nt 8281 to 8301, sense) and # 174R (nt 8462 to 8481, antisense) for HCV-K1, #151 (nt 8283 to 8302, sense) and #153R (nt 8460 to 8479, antisense) were used (Table 1). The expected sizes of PCR products for each type, HCV-US, HCV-J, HCV-K1 and HCV-K2 were 216 bp, 216 bp, 201 bp and 197 bp, respectively, and they were respectively corresponding to type I, -II, -II, and -III on the proposed phylogenic classification of HCV^7,12,14)(Fig. 1).

In the first PCR, 50 μl mixture, containing 10 mM tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01% gelatin, 2.5 units of recombinant Taq. DNA polymerase (Pharmacia, Biotech. U.S.A.), 200μM each dNTP, 0.1 μM of each outer primer and 10 μl of cDNA was amplified by Thermocycler (Perkin-Elmer-Cetus Co., LA, U.S.A.) for 35 cycles. Each cycle included denaturation at 94°C for 1 min, primer annealing at 40°C for 1 min and primer extension at 72°C for 1 min. Then, with 1 μl of the first PCR products in the prepared 4-each type specific microtube, the second PCR was carried out for 30 cycles with denaturation for 1 min at 94°C, primer annealing for 45 seconds at 55°C and primer extension for 1 min at 72°C. The products of the second PCR were subjected to electrophoresis on 2% agarose gel (BRL, Gaithersburg, Md, U.S.A.), stained with ethidium bromide and observed under U.V. light.

37 patients with sporadic CH and 4 blood donors all showed positivity in RT-nested PCR, while 9 (56.2%) of 16 cases with post-transfusion hepatitis were positive. Among 12 patients with HCC, 5 cases (62.5%) of 8 sera, 10 cases (83.3%) of 12 frozen tumorous tissue specimens and 5 cases (71.4%) of 7 non-tumorous tissue specimens were positive. A healthy person who was positive for anti-HCV and normal in ALT level showed negativity in RT-nested PCR.

The strategy for gonotyping HCV is illustrated in Fig. 2. Type specific PCR products, obtained through the procedure of RT-nested PCR, were visualized after they were electrophoresed and stained with ethidium bromide (Fig. 3).

Among a total 70 individuals, available 43 serum samples were typed by RT-nested PCR. The prevalence of type II HCV was 75%, type III HCV was 25% and there was no co-infection with type II and -III HCV. 24 cases (64.8%) of 37 sporadic CH were type II HCV, while 13 cases (35.2%) were type III HCV.

All cases of 6 HCC and 9 PT-NANBH showed only type II HCV and 3 cases (75.0%) of 4 blood donors were type II HCV, while 1 case (25.0%) was type III HCV (Table 2).

In frozen liver tissues, type II HCV was 63.0%, type III HCV was 3.7% and co-infection with type II and-III HCV were shown in 18.5%(Table 3, Fig. 4).

Among 27 cases which were obtained by liver biopsy, 6(75.0%) of 8 sporadic CH, 7(58.3%) of 12 tumorous tissues and 4(57.1%) of 7 non-tumorous tissues of HCC. Co-infection with type II and -III HCV was 25.0% in sporadic CH, 16.7% in tumorous tissues of HCC and 14.3% in non-tumorous tissues of HCC, respectively.

8 liver tissue specimens were obtained in sporadic chronic patients. In sera, all 8 cases showed type II HCV, while in tissues, 6 cases were type II and 2 cases were co-infection with type II and -III HCV. Typing results in sera were relatively well correlated with those in tissues (75%), but the positivity in type III could not be observed (Table 4).

In sera, 6 cases which were positive in RT-nested PCR among 12 HCC patients, were all type II HCV, while, in tuomorus liver tissues, type II HCV was 7 cases, type III HCV was 1 case, co-infections with type II and -III HCV were 2 cases and, in non-tumorous tissues, type II was 4 cases and co-infections were 1 case (Table 5).