A total of 1,014 patients infected with HCV (554 males and 460 females; mean age, 45 years) were randomly recruited from 28 hospitals in varying regions in China (Table I). The registration ID of the present study is NCT01293279 (www.clinicaltrials.gov). The study was approved by the ethical committees of all the hospitals that patients were recruited from in the present study. Written informed consent was obtained from each patient. All the patients had been confirmed as positive for anti-HCV antibody and serum-HCV RNA. All the hospitals were divided territorially into North, Northeast, Southwest, South, Central, Northwest and East China (Fig. 1) (19). The study included 92 patients infected with HCV in North China, 91 in the Southwest, 104 in the South, 224 in Central China, 162 in the Northwest, 252 in the East and 89 in the Northeast. All the patients with HCV infection did not receive any treatment, and the age, gender and regions were taken into consideration.

Genomic DNA and total RNA from liver biopsies were extracted using the Qiagen RNeasy Mini kit (Qiagen, Inc., Valencia, CA, USA) (20).

HCV genotyping was conducted using the INNO-LiPA HCV II assay (Innogenetics, Zwijnaarde, Belgium), which is based on hybridization of probes with the 5′ non-coding region of HCV. According to the HCV gene sequences published in GenBank (AF333324, D16435, KF035127 and KC844040), the HCV 5′ non-coding region (nt299-1) was selected, and one pair of specific primers were designed for nested polymerase chain reaction (PCR). The sequences of the PCR primers were as follows: Sense: −299 5′-CCCTGTGAGGAACTWCTGTCTTCACGC-3′ (−299~ −273); and antisense: 5′-GGTGCACGGTCTACGAGACCT-3′ (−1~−21) (W is A or T). The probes used for the detection of the different serum types are also listed as follows: Genotype 1: −170 AATTGCCAGGACGACC; genotype 2: −83 TAGCGTTGGGTTGCGA; genotype 3: −170 AATCGCTGGGGTGACC; and genotype 6: −81 AGTAGCGTTGGGTTGC. The HCV 5′ non-coding region of the HCV genome was amplified using nested PCR. Briefly, 5 μl cDNA was amplified for >40 cycles in a total volume of 50 μl, each consisting of 1 min at 95°C, 1 min at 55°C and 1 min at 72°C. From the second round of amplification, 1 μl product was amplified again with the same primers for another 40 cycles. The nested PCR products were subjected to electrophoresis in a 2% agarose gel. The probes specific for the different types of HCV were added with a poly(dT) tail at their 3′ end. Primer (20 pmol) was incubated for 1 h at 37°C in 25 μl buffer (3.2 mM-dTTP, 25 mM-Tris-HC1 pH 7.5, 0.1 M-sodium cacodylate, 1 mM-CoCl₂, 0.1 M-dithiothreitol and 60 U terminal deoxynucleotidyl transferase). The reaction was stopped by adding 2.5 μl EDTA (0.5 M, pH 8.0) and diluted with 20X SSC. This solution was applied on a nitrocellulose membrane, and was fixed to the membrane by baking at 80°C for 2 h, and then was hybridized with equal volumes of the nested PCR products.

Serum was obtained from the HCV-infected patients, and the total RNA was isolated using an RNeasy Mini kit and RNase-Free DNase Set (Qiagen, Hilden, Germany). The HCV RNA content was detected by Abbott RealTime HCV (Abbott Laboratories, Des Plaines, IL, USA).

Genotyping for the IL-28B gene was performed by the iPLEX system [MassARRAY^® Real-Time Transactional Memory (RTTM) software for SNP genotyping; iPLEX Gold; Sequenom, San Diego, CA, USA]. The DNA was blind coded and tested using a 384-SpectroCHIP^® microarray (Sequenom). A matrix-assisted laser desorption/ionization time-of-flight mass spectrometer was used for data acquisitions from the SpectroCHIP microarray. The results were analyzed using Sequenom MassARRAY RTTM software. The region on around the IL-28B gene on chromosome 19 contains multiple SNPs in linkage disequilibrium. The present study focused on the genotyping of thirteen SNPs associated with IL-28B: rs8013142, rs28416813, rs10853728, rs7248668, rs8105790, rs11881222, rs12979860, rs12980275, rs4803219, rs8099917, rs8109886, rs4803223 and rs10853727.

The Hardy-Weinberg disequilibrium test was performed for each SNP genotype associated with IL-28B. The gene frequencies were compared between groups using the χ² test with Yates correction or Fisher’s exact test. Group means were presented as the mean ± standard deviation, and were compared by analysis of variance and Student’s t-test. The serum HCV RNA levels were expressed as the original values. The HCV RNA levels were divided into high, medium and low level groups. The statistical analyses were performed with the SPSS 12.0 statistical package (SPSS, Inc., Chicago, IL, USA). All statistical analyses were based on two-sided hypothesis tests, with P<0.05 used to indicate a statistically significant difference.

A total of 1,014 HCV-infected patients were randomly recruited from 28 hospitals in the 7 regions of China for the present study (Fig. 1 and Table I). The overall gender ratio of male to female was 1.204 (554/460). The HCV-infected population was divided according to their age into four groups: The old-aged adults (>60 years old), the middle-aged adults (45–59 years old), the young adults (18–44 years old) and the young (<18 years old). HCV genotypes 1, 2, 3 and 6 were included in the study. Furthermore, HCV RNA expression was detected and divided into high (RNA≥1×10⁷), medium (1×10⁵≤RNA<1×10⁷) and low (RNA<1×10⁵) expression.

The HCV genotypes in Chinese patients were investigated. The data revealed that HCV genotype 1 was distributed more extensively compared with other genotypes in all regions, with the exception of South and Northwest China (P<0.01). In South China, the distribution of HCV genotypes 1 and 3 was higher compared with other genotypes, with percentages of 38 and 37%, respectively. The percentages of HCV genotypes 1 and 2 in the Northwest region were 45.6 and 41%, respectively. In all regions, with the exception of Southwest (15%) and South (17%) China, the percentage of genotype 6 was recorded to be the lowest, (Fig. 2).

In all the recruited HCV-infected patients, the old-aged adults, middle-aged adults, young adults and the young accounted for 14.8 (150/1,014), 37.7 (382/1,014), 47.3 (480/1,014) and 0.2% (2/1,014) of cases, respectively. The patients with compensated and decompensated cirrhosis accounted for 8.8 (89/1,014) and 5.3% (54/1,014) of the cases, respectively (Fig. 3). The old-aged, middle-aged and young adults accounted for 27, 60 and 13% of cases, respectively, in the compensated cirrhosis group, and for 28, 57 and 15% of cases, respectively, in the decompensated cirrhosis group (Fig. 3). The distribution of the young in the purely HCV-infected patients was significantly higher compared with that in the compensated and decompensated cirrhosis groups. However, in the compensated and decompensated cirrhosis groups, the distribution rate of the middle-aged adults was higher than the other groups (P<0.01).

In the middle-aged adults group, the number of male patients was higher compared with the number of females in North and South China, which was the opposite of the results found in the other regions. In particular, in Eastern China, the number of female patients was significantly higher compared with the number of males (P<0.01). However, in the young adult group, there were far more males compared with females in the majority of the regions, with the exception of North China (P<0.01) (Table II).

In all regions, no gender difference was found in HCV genotype 1 (P<0.05). However, in HCV genotype 2, there were significantly less males compared with females in North and Southwest China (P<0.01). In HCV genotype 3, the males were extensively distributed in all the regions, with the exception of the Northeast. In HCV genotype 6, the infected rate in males was significantly higher than that in females in South China (P<0.01) (Table III).

The HCV RNA in all the genotypes was highly expressed, with the exception of genotype 1b+2. There was no difference in HCV RNA expression among HCV genotypes 1, 2 and 3. However, the number of patients with high RNA expression in genotype 6 was significantly higher compared with that in the other genotypes (P<0.01). In genotype 1b+2 patients, the number of patients with high RNA expression was lower compared with the patients with medium and low levels of RNA expression, but no statistical significance was found due to the small number of specimens (Fig. 4).

A total of 13 SNPs associated with IL-28B (rs8013142, rs28416813, rs10853728, rs7248668, rs8105790, rs11881222, rs12979860, rs12980275, rs4803219, rs8099917, rs8109886, rs4803223 and rs10853727) were investigated in 1,014 HCV-infected patients in the present study. The distribution of host genotypes in each SNP were detected (Table IV). The data show that the percentage of genotype C/T in rs8013142 (98.13%), G/C in rs28416813 (77.71%), C/C in rs10853728, rs12979860, rs4803129 and rs8109886 (66.07, 84.71, 84.32 and 81.56%, respectively), G/G in rs7248668 (85.21%), T/T in rs8105790, rs8099917 and rs10853727 (71.40, 85.40 and 98.52%, respectively), and A/A in rs11881222, rs12980275 and rs4803223 (83.73, 81.36 and 84.32%, respectively) were significantly higher compared with the other two allelic genes. Notably, there were significant differences in the genotype percentages of all SNPs between HCV genotypes 1 and 2 in the majority of SNPs (with the exception of rs8013142, rs8099917, rs8109886 and rs10853727). Genotypes rs28416813 (C/C), rs4803219 (T/T) and rs10853727 (C/C) were not found in any of the recruited HCV patients (Table IV).

Overall, no geographical differences in SNPs existed in the Chinese HCV-infected population. However, SPSS analysis revealed that several correlations exist between the SNPs genotypes and HCV (P<0.01).