Objective　To determine the concordance in various hepatitis C (HCV) genotyping methods and to investigate the distribution of HCV genotypes in Guizhou area of Southwest China.
　　Methods　Serum samples from 206 patients (100 with chronic hepatitis and 106 with hemopathy) were detected for antibody of HCV by second generation enzyme-labelled immunosorbent assay (ELISA). Thirty-five anti-HCV positive samples were detected for HCV RNA by RT-polymerase chain reaction (PCR) and 30 HCV RNA positive samples were determined for their genotypes by three various genotyping methods ［PCR with type-specific primers at the core region (primer-set), slot-blot hybridization with type-specific probes at NS5B region (blotting) and the restric fragment length polymorphism analysis of PCR products of 5'NC region (RFLP)］. Ten samples with the known genotype were analysed by the direct sequencing.
　　Results　Of 30 samples with positive HCV RNA, the types of 22 could be classified by three methods, and the genotypes determined by various methods had complete concordance. The types of 6 samples could be classified by two methods and 5 had agreement subtypes. The types of two samples could be classified only by RFLP. Overall, 27(90.0%) had subtype 1b infection and 3(10.0%) had subtype 2a infection. The nucleotide sequence of 8 samples with subtype 1b and one with subtype 2a were analysed by the direct sequencing. The subtypes determined by sequence analysis were in complete concordance with those decided by various genotyping methods.
　　Conclusions　Subtype 1b is the predominent HCV genotype in Guizhou area, while subtype 2a is less common. There was a good concordance with the genotyping results obtained by various HCV genotyping methods.

Chin Med J 1998; 111(2):128-132

　　Hepatitis C virus (HCV) is the major etiology of parenteral non-A, non-B hepatitis. Since the convenient methods for detection of HCV antibody (anti-HCV) and HCV RNA were established, the epidemiology of HCV infection is investigated around the world. Mean- while, the basic and clinic studies on HCV have also made rapid progress.

　　HCV has been classified into at least 9 major genotypes and over 30 subtypes based on analysis of complete or partial HCV genomic sequences.1　It has been found that the genotypes and subtypes differ in their geographical distribution, the severity of disease and in the responsiveness to interferon treatment.2-4　Therefore, it is important to make sure of HCV genotypes.

　　There are several methods for HCV genotyping besides nucleotide sequence analysis. These include polymerase chain reaction (PCR) with type-specific primers deduced from C region, restriction fragment length polymorphism (RFLP) analysis of PCR products from 5'NC region and hybridization with type-specific probes deduced from NS5B region. Each method is dependent on a region of HCV genome. Whether the genotyping result based on different region can be compared is still unknown. The purpose of the present study is to determine the concordance in various genotyping methods and to investigate the distributions of HCV genotypes in Guizhou area, southwest China.

METHODS

Primer synthesis
　　Primers were synthesized by a DNA synthesizer Model 391 (Applied, systems, Tokyo, Japan). The sequence of primers used in this study are as follows: H66 (sense), 5'-CCCATGGGCTTCTCGTATGATACCCG-
CTGCTT-TGA-3'; H67 (antisence), 5'-GGTGGGG-
CGGAATACCTGGTCATAGCCTCCGTGAA-3'; H68 (sence), 5'-TGCGGTTATTGCCGTTGTCGCGCCA-
GCGG-3'; H69 (antisence), 5'-GGCAGAATACCTAGTCATGGCCTCTGTGAA-3'.

Patients
　　Serum samples were collected from 206 individuales with a high risk of exposure to HCV at Affiliated Hospital of Guiyang Medical University, Guiyang, China from June 1993 to May 1994. They included 100 patients with chronic hepatitis, and 106 blood recipients with hemopathy. Chronic hepatitis patients were clinically diagnosed. The diagnosis of hemopathy was based on the clinic symptoms and the detection of peripheral blood and bone marrow extraction. A second generation enzyme-labelled immunosorbent assay (ELISA) was used to detect HCV induced antibody, 35 anti-HCV positive samples were stored at -20℃, and then brought to Kanazawa Medical University, Japan for further analysis.

Detection of HCV RNA
　　HCV RNA was detected by reverse transcription-nested PCR with the primers derived from 5'non-cording region (5'NCR). The primary cDNA synthesis was carried out by the random primer (hexamers) and the nested PCR was done by outer primers (H70, H71)5　and inner primers (H72, H73)5　respectively. The amplified HCV cDNA has 256 base paired (bp).

HCV genotyping
　　Samples with positive HCV RNA were subjected to a genotype analysis by three methods. The first method is the slot-blot hybridization with type-specific cDNA probes. This was done as described by Enomoto (blotting).6　The second method is PCR described by Okamoto (primer-set).7　The third method is restriction fragment length polymorphism of PCR products of the 5'non-coding region based on the procedure by Davidson with a slight modification.8

Direct sequencing of PCR product
　　To investigate the sequence of the HCV infection and confirm the validity of genotyping methods used in present study, the direct sequencing of PCR product in NS5B region (the same region as that in blotting) was performed in eight patients infected with 1b and one patient with 2a, determined by the method described adove. PCR product was purified with low melting agarose gel followed by prep-A-gene extraction and was subjected to cycle sequencing reaction in the presence of (r-32P) ATP labelled inner primer either H68 or H69 with Takara cycle sequence kit. The cycle was done according to manufactural protocol. The products of sequencing reaction were subjected to electrophoresis in 6% PAGE gel followed by autoradiograph at -80℃ for 12 hours. The direct sequencing of PCR product in 5'non-coding region (the same region as that in detection of HCV RNA) was also done by the same procedure as described above in one patient who had a mixed infection with subtype 1b togather 2a, since the amplification of cDNA with specific primers from NS5B region was not successful.

Nomenclature system
　　A new nomenclature system for HCV genotype proposed by Simmonds et al was used in this study. According to this system, 1a, 1b, 2a, 2b corresponded to PT,k1, k2a, k2b named by Enomoto and Ⅰ、Ⅱ、Ⅲ、Ⅳ designated by Okamoto, respectively.

RESULTS

　　The results of anti-HCV and HCV RNA of 206 serum samples are shown in Table 1. Thirty-five serum samples were anti-HCV-positive and only 30 samples were positive for HCV RNA by the nested PCR at 5'-non coding region. The 30 patients consisted of 18 patients with chronic hepatitis and 12 blood recipients with hemopathy.

Table 1. HCV antibody and HCV RNA in patients studied

Diagnosis Number

of cases

 Anti-HCV
(positive)		 HCV RNA
(positive)		 HCV genotype
1a 1b 2a 2b 3a 4a
Chronic liver disease 100 21 18 0 17 1 0 0 0
Posttranfusion with 　 　 　 　 　 　 　 　 　
　hemopathy 106 14 12 0 10 2 0 0 0
Total 206 35 30 0 27 3 0 0 0
　　The HCV genotypes in the 30 patients were examined with blotting, primer-set and RFLP methods. Twenty-two samples were classified by the three methods. Twenty samples had subtype 1b infection and 2 had subtype 2a infection. The genotypes decided by the three methods were exactly the same. Six samples were classified only by two methods, and 5 samples had subtype 1b infection. The typing results based on two methods also were concordant. One sample had a mixed infection (HCV 1b together with 2a) base on primer-set. However, it had HCV 2a infection determined by the RFLP and further was identified by the direct sequence. Two samples that were unclassified by either primer-set or blotting method were classified by RFLP analysis. Overall, HCV could not be amplified by RT-PCR in 13.3%, 13.3% and 6.7% of serum samples based on the NS5B region, core region and 5'NC region of HCV genome respectively (Table 2).

Table 2. The comparison of genotyping results based on various systems

　 System Assigned*
genotype
Okamoto Enomoto Simmonds Enomoto
Region of HCV studied Core NS5B 5'NC NS5B 　
Method PCR Blotting RFLP Sequencing 　
No.of patients 30 30 30 10 30

The typing

 　 　 　 　 　
　1b 23 24 25 　8 27
　2a 　2 　2 　3

　　　2**

 　3
　1b+2a 　1 　0

0

 　 　
Undetermined 　4 　4

2

 　 　
　　* based on all data； ** genotype of 1 sample was bsed on the sequence of 5'NC region.

　　The nucleotide sequence of PCR product from 10 patients was determined by direct sequencing (Fig.). For NS5B region, the sequence homology within 8 patients was

Fig. The nucleotide sequence of the 10 patients by direct sequencing of PCR product (A: NS5A region and B:5'NC region). The horizonal lines are identical with most top sequence.
between 94.5%-98.8%. When the represent sequence (HC-G1) was comparied with the published sequences, nucleotide and deduced amino acid sequences were almost identical to of HCVJ. The nucleotide sequence of HC-G4 was quite different from these of 8 patients and had a high homology with HCVJ6 (Table 3). For 5'NCR, the sequence identity between HCV G14 and HCVJ6 was 96.4%.

Table 3. The sequence homology between Guizhou isolate
and HCV types

HCV type NS5B region homology (%)
Nucleotide 　 Amino acid
HC-G1 HC-G4 HC-G1 HC-G4
HCVI 81.2 67.9 　 90.9 60.8
HCVJ 95.1 67.3 　 94.5 56.4
HCVJ6 67.9 89.7 　 69.1 80.0
HCVJ8 66.1 78.8 　 65.5 72.7


　　 For the analysis of distribution of HCV genotypes in these 30 patients, the sequence data were considered the definitive genotype for 10 samples with segnence data. For 20 samples without sequence data, 12 samples were classified for their genotypes based on three methods, 6 samples were classified for their genotype based on two methods and two samples were classified by the RFLP. Overall, subtype 1b was common in Guizhou area (27/30, 90.0%), subtype 2a was less common (3/30, 10.0%), and the other HCV genotypes were not fonud in this study.

DISCUSSION

　　HCV has been classified into multiple, distinct genotypes on the basis of nucleotied sequence homology. The distributions of HCV genotypes varies in different geographical areas. In America and Europe, subtype 1a and 1b are predominent. While in most Asian countries, subtype 1b is predominent. In China, the genotype distributions of HCV were different in different areas. In northern area, the subtype 2a was detected in 46%-70% of HCV infection patients. While more than 90% patients from southern area had subtype 1b infection.2 The results in present study indicate that HCV 1b is the most common genotype, HCV 2a being less common, and that the other HCV genotypes are not accounted in Guizhou area of Southwest China.

　　HCV genotype is essential for diagnostic and epidemiological studies as well as predication of response to interferon therapy. A definite determination of genotype can be obtained by sequenec analysis in an individual region of HCV. However, sequence analysis needs special condition and technology, without which, this method is very laborious for routine diagnostic use or large-scale prevalence studied. Alternative molecular biology methods, including primer-set, blotting and RFLP, have been developed and proved to be technically easier than sequence. The base of each method is dependent on some nucleotide variance in a conservative region. The templet region belongs to C region, NS5B region or 5'NCR in primer-set, blotting and RFLP, respectively. The comparison of HCV genotypes based on different methods will provide a foundation for selection of genotyping method. This study demonstrated that there was always a proportion of cases which cannot be genotyped by whatever methods. This may result from the failure of genome amplification due to the mismatching between the templet and primers. However, in cases that can be determined for their genotype by three methods, the genotyping results are completely concordent. These data indicate that there is a very good concordance in these genotyping methods.

　　Although the primer-set was thought to be simpler and was commonly used, this method leads to inconsistent result with a high frequency of mixed genotype infections, most of which appeared to be subtype 1b together with another subtypes.9　This method can only detect 4～5 (1a, 1b, 2a, 2b) subtypes of HCV but can not detect many other new subtypes. In 5'NCR, sequences that are characterized by certain genotypes are included. However, a nucleotide variation may result in the deletion of a restriction enzyme-cut site. Some subtypes, for example, HCV 1a and HCV 1b, sometimes can not be accurately distinguished by RFLP analysis of 5'NCR. The blotting method can accurately distinguish many of the genotypes (1a, 1b, 2a, 2b, 3a, 4a) and can also predict some other new genotypes by the preparation of the new-specific probes. Therefore, the blotting is a more useful genotyping method.

　　Department of First Biochemistry (Yamada N and Date T), Division of Gastroenterology, Department of Internal Medicine (Takada A and Tsutsumi M), Kanazawa Medical University, Uchinada, lshikawa, 920-02 Japan.
　　Department of Infectious Diseases, the Affiliated Hospital of Guiyang Medical College, 550001, China (Ding JJ)

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