Original Paper

Intervirology 2005;48:133137 DOI: 10.1159/000081740

Received: March 29, 2004 Accepted after revision: May 17, 2004

Hepatitis B Virus Genotypes in Korea: An Endemic Area of Hepatitis B Virus Infection

Byung-Cheol Song Xiu Ji Cui Heung Up Kim
Department of Internal Medicine, College of Medicine, Cheju National University, Jeju, Korea

Key Words Hepatitis B virus Genotype Chronic hepatitis B Liver cirrhosis Hepatocellular carcinoma

types have clinical relevance, distribution of HBV genotype in each area should be monitored when management for chronic HBV infection is planned.
Copyright 2005 S. Karger AG, Basel

Abstract Objectives: It has been reported that distribution of hepatitis B virus (HBV) genotypes shows geographic difference and are associated with clinical outcomes of HBV infection, including response to antiviral therapy and progression of chronic liver diseases. In this study, we analyzed the distribution of HBV genotypes according to the various clinical outcomes of chronic HBV infection in Korea, which is one of the most endemic areas of HBV infection. Methods: A total of 200 patients with chronic HBV infection were enrolled. Clinical diagnoses of the 200 patients with chronic liver diseases were as follows: hepatitis B e antigen (HBeAg)-positive healthy carrier (dened as HBeAg+, anti-HBe, HBV DNA+ by hybridization, normal transaminase; n = 40); inactive HBsAg carrier (n = 40); chronic hepatitis B (n = 40); liver cirrhosis (n = 40); hepatocellular carcinoma (n = 40). HBV genotypes were determined by nested polymerase chain reaction using genotype-specic primers. Results: All patients except 2 (inactive HBsAg carriers) were positive for nested PCR and they have genotype C regardless of clinical outcomes. Conclusions: HBV genotype was genotype C regardless of various clinical outcomes of chronic HBV infection in Korea. Considering that HBV geno-

Introduction

Hepatitis B virus (HBV) infection is associated with various clinical manifestations such as acute hepatitis, asymptomatic inactive hepatitis B surface antigen (HBsAg) carrier, chronic hepatitis, liver cirrhosis, and eventually hepatocellular carcinoma [1, 2]. It has been suggested that these clinical outcomes might be affected by host factor, viral factor and environmental factor [317]. Among these, recently, it has been suggested that HBV genotype may affect the clinical outcomes of HBV infection [12 17]. HBV genotypes are divided into 8 genotypes (AH) [1821], based on intergroup divergence of 8% or greater of the entire genome sequence and shows characteristic geographic distribution [2229]: genotype A is predominantly found in North America, Northwest Europe, India, and Central Africa; genotypes B and C are mostly found in Asia; genotype D is commonly found in the Mediterranean area and India; genotype E is predominantly found in Africa; genotype F is found in American natives, Polynesia, and Central and South America, and genotype G has been reported in the USA and France.

2005 S. Karger AG, Basel 03005526/05/04830133$22.00/0 Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com Accessible online at: www.karger.com/int

Byung-Cheol Song, MD, PhD Department of Internal Medicine, College of Medicine Cheju National University, Cheju National University Hospital 154 Samdo 2-dong, Jeju 690-716 (Korea) Tel. +82 64 7501253, Fax +82 64 7578276, E-Mail drsong@cheju.ac.kr

And recently reported, genotype H is found in Central America [21]. Some studies found that HBV genotype B is associated with a higher rate of spontaneous hepatitis B e antigen (HBeAg) seroconversion (dened as undetectable HBeAg/ HBV DNA, and acquisition of anti-HBe) compared with genotype C [1417]. Patients with genotype C showed poor response to interferon- therapy [30, 31] and higher relapse rate after lamivudine-induced HBeAg loss (dened as undetectable HBeAg/HBV DNA) [32]. In addition, prevalence of HBV genotype C is more frequent in patients with liver cirrhosis and hepatocellular carcinoma [13, 15], indicating that HBV genotypes themselves may have clinical relevance. Two studies reported that over 95% of chronic HBV carriers were genotype C in Korea [27, 28]. However, in these studies, all studied patients were selected from those who had high viremia by hybridization method. And these studies excluded the patients with asymptomatic inactive HBsAg carrier, liver cirrhosis and hepatocellular carcinoma. Therefore, these studies did not reect the true distribution of HBV genotypes in Korea. Recently, we also reported that all patients with chronic HBV carrier had genotype C in this area [33]. However, in our previous study, the sample size was small and we used the restriction fragment length polymorphism, which was not sensitive in patients with inactive HBsAg carriers because of low viral load. Therefore, many patients with low viral load, including inactive HBsAg carriers, was not fully evaluated in our previous study. Therefore, the aim of this study was to determine the distribution of HBV genotypes according to the various clinical outcomes of chronic HBV infection in Korea, in which the prevalence of HBV infection currently ranges from 5 to 7% [34, 35] and HBV infection is a leading cause of mortality from chronic liver diseases.

tests, and liver biopsy in some cases. The denitions of chronic liver diseases was as follows [2, 36]: (1) HBeAg-positive healthy carrier, which is usually encountered in the period of immune tolerance phase, was dened by detectable HBsAg, HBeAg and HBV DNA (by hybridization) in serum, undetectable anti-HBeAg, and normal serum alanine aminotransferase (ALT); (2) inactive HBsAg carrier was dened as detectable HBsAg and anti-HBe in serum, undetectable HBeAg, undetectable levels of HBV DNA by hybridization, normal serum ALT, and without any evidence of liver cirrhosis and hepatocellular carcinoma; (3) chronic hepatitis B is dened as persistent elevation or uctuation of serum ALT over 6 months without any evidence of any other etiology of chronic liver disease; (4) liver cirrhosis was diagnosed on the following criteria: having clinically relevant portal hypertension (esophageal varices and/or ascites, splenomegaly with platelet count !100,000/mm3) [37] and imaging features suggestive of liver cirrhosis on ultrasonography [38], and (5) hepatocellular carcinoma was diagnosed either histologically or radiologically on the basis of hypervascular liver masses with serum -fetoprotein levels exceeding 400 ng/ml [39]. To allocate the same number of patients in each clinical disease, 40 consecutive patients were enrolled in each chronic liver disease. Patients were excluded if they had any of the following: acute hepatitis B, concomitant hepatitis C or D virus infection, any history of antiviral therapy, history of immunosuppressive therapy, and history of heavy alcohol drinking. The serum samples were collected with written informed consent and the study protocol was approved by the Ethics Committees of our institution. Serologic Testing HBsAg, HBeAg and anti-HBe were assayed using commercially available enzyme immuoassay kits from Abbott Laboratories (North Chicago, Ill., USA). HBV DNA levels were measured by the Digene Hybrid Capture Assay (Digene Corp., Gaithersburg, Md., USA). Genotyping of HBV Nucleic acids were extracted from 100 l serum that had been stored at 80 using a High Pure Viral Nucleic Acid Kit (Roche, Penzberg, Germany). HBV DNA was amplied by nested polymerase chain reaction (PCR) using the universal primers for the outer primers, followed by two different mixtures containing typespecic inner primers (table 1) as described earlier by Naito et al. [40], which were designed on the basis of the conserved nature of nucleotide sequences in regions of the pre-S1 through S genes, and were designed on the basis of the differences in the sizes of the genotype-specic bands. We undertook all necessary precautions to prevent cross-contamination, and negative controls were included at each step.

Patients and Methods

Patients The study patients with chronic HBV infection were enrolled between May 2001 and December 2002 at Cheju National University Hospital. This study included new patients as well as patients attending for a follow-up visit. They were followed at 3- to 6-month intervals. At every visit, physical examinations, tests for serum biochemistry, -fetoprotein and ultrasonography were performed. Computed tomography or magnetic resonance imaging and liver biopsy were performed, if clinically needed. For the analysis of HBV genotypes in various clinical outcomes of chronic HBV infection, we classied the chronic liver diseases into ve categories on the basis of laboratory tests, radiological

Results

During the study period, a total of 542 patients with HBsAg-positive patients visited out hospital. Clinical diagnoses of these patients were as follows: acute hepatitis B (n = 2; 0.4%), HBeAg-positive healthy carrier (n = 81;

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Table 1. Primer sequences used for HBV genotyping by nested PCR

Primer Outer primer Sense Antisense Inner primer Mix 1 Sense Antisense

Sequences

5 -TCA CCA TAT TCT TGG GAA CAA GA-3 (nt. 2,8152,837) 5 -CGA ACC ACT GAA CAA ATG GC-3 (nt. 685704)

Type A specic Type B specic Type C specic Type D specic Type E specic Type F specic

5 5 5 5 5 5 5 5

-GGC TCC AGT TCC GGA ACA GT-3 (nt. 6786) -CTC GCG GAG ATT GAC GAG ATG T-3 (nt. 113134) -CAG GTT GGT GAG TGA CTG GAG A-3 (nt. 324345) -GGT CCT AGG AAT CCT GAT GTT G-3 (nt. 165186) -GCC AAC AAG GTA GGA GCT-3 (nt. 2,9792,996) -CAC CAG AAA TCC AGA TTG GGA CCA-3 (nt. 2,9552,978) -GCT ACG GTG GGT TAC CA-3 (nt. 3,0313,051) -GGA GGC GGA TCT GCT GGC AA-3 (nt. 3,0783,097)

Mix 2 Sense

Antisense

Table 2. Baseline characteristics of the study patients

Variables

HBeAg-positive healthy carrier 29.3811.4 22/18 21.9812.8

Chronic hepatitis B 135.7812.8 29/11 384.38541.4

Inactive HBsAg carrier 50.3817.1 17/23 26.288.4

Liver cirrhosis 52.0810.7 26/14 64.9838.9

Hepatocellular carcinoma 56.6811.1 32/8 82.9866.9

Age, years Males/females ALT, IU/l

14.9%), inactive HBsAg carrier (n = 227; 41.9%), chronic hepatitis B (n = 102; 18.8%), liver cirrhosis (n = 76; 14%), and hepatocellular carcinoma (n = 54; 10%). Among them, 40 consecutive patients at each clinical disease were studied. As a result, a total of 200 patients were studied. The baseline demographic characteristics of the 200 patients are summarized in table 2. PCR was positive in 198 of the 200 patients, except 2 who were inactive HBsAg carriers. Interestingly, all patients who were positive for nested PCR using genotypespecic primer showed typical size of genotype C regardless of various clinical outcomes of chronic HBV infection in this area.

Discussion

In this study, we found that HBV genotype was genotype C regardless of various clinical outcomes in Korea. Even though there is geographic distribution of HBV ge-

notypes [2229], this result is very unique because HBV genotype was homogeneous in all patients with various clinical manifestations. This result may relate to the anthropologic characteristics of the monoracial Korean population. In addition, in Korea, most of HBV infection is considered to be vertically transmitted. Therefore, it is suggested that the same HBV genotype may be conserved in the same population for a long duration. Recently, several studies reported that HBV genotype B is associated with a higher rate of spontaneous HBeAg seroconversion compared with genotype C [1417]. In addition, HBV genotype C is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma [13, 15]. Considering the previous reports, HBV genotype C might show poor clinical outcomes in patients with chronic hepatitis B. So far, Korean patients with chronic hepatitis B have shown unique features even when compared with Asian patients in terms of durability of antiviral therapy [41 43]. We reported that around 50% of the patients with

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HBeAg loss had relapse after lamivudine or interferontherapy [42, 43], indicating that durability of antiviral therapy is not maintained in this area. However, we did not know the exact reasons of high relapse in our previous reports. Recently, Chien et al. [32] reported that HBV genotype is a major determinant for durability for lamivudine therapy. Therefore, our previous observation of high relapse after antiviral therapy may be partly associated with the homogeneous distribution of HBV genotype C in this area.

In conclusion, HBV genotype in Korea was genotype C regardless of various clinical outcomes of chronic HBV infection. Considering that HBV genotypes have clinical relevance, distribution of HBV genotype in each area should be monitored when management for chronic HBV infection is planned.

Acknowledgement
This work was supported by a grant (No. R05-2002-001062-0) from the Korean Science and Engineering Foundation.

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