Ho-Suk Mun

Molecular Epidemiology of Hepatitis B Virus (HBV) Genotypes and Serotypes in Patients with Chronic HBV Infection in Korea

Objectives: Although hepatitis B virus (HBV) is endemic to Korea, no large-scale survey of HBV genotypes and serotypes based on sequence analysis has been performed. Methods: In the present study, we genotyped and serotyped HBV strains from 209 patients in two Korean regions, Seoul (107 patients) and Jeju (102 patients), an island off the southeastern Korean coast. Analyses were conducted using the direct sequencing method targeting the partial surface (S) gene (541 bp). Results: Phylogenetic analysis showed that all HBV strains from the 209 patients belonged to genotype C2 (100%). Of the 209 patients, 193 (92.3%), 12 (5.7%) and 1 (0.5%) were found to have the adr, adw and ayr serotypes, respectively. The other three strains (1.5%) showed unique serotype and were not typeable by sequence analysis. No HBV strains characteristic of Jeju island were observed. Conclusions: The extraordinary predominance of genotype C2 in chronic Korean patients, which is known to be associated with more severe liver disease than genotype B, suggests that the clinical manifestations of Korean HBV chronic patients are likely to differ from those found in other Asian countries, especially in Japan and Taiwan, where genotypes B and C coexist.

The prevalence of hepatitis B virus preS deletions occurring naturally in Korean patients infected chronically with genotype C

Although Korea is one of the endemic areas for hepatitis B virus infection (HBV), the prevalence of deletions in HBV preS region occurring naturally have not been determined. In the present study, the prevalence of preS deletions was determined in terms of clinical state and HBeAg serostatus in 120 patients with different clinical features [59 HBeAg positive, 61 HBeAg negative; 38 asymptomatic carriers, 21 patients with chronic hepatitis, 21 patients with liver cirrhosis, 40 patients with hepatocellular carcinoma (HCC)]. A total of 37 strains (30.8%) harbored deletions in the preS region. Overall, the frequencies of preS deletions tended to increase gradually according to the degree of the clinical severity of liver disease. The prevalence of preS1 deletions in HCC patients tended to be higher than in patients with liver cirrhosis (32.5% vs. 19%). The prevalence of preS2 deletions in HBeAg negative patients was significantly higher than in HBeAg positive patients (23% vs. 6.8%). The type of deletion encountered most frequently was one disrupting the preS1 start codon [14/37 strains (37.8%)], which showed a very high prevalence in HCC patients (9/40, 22.5%; HCC vs. asymptomatic carriers, P = 0.048). These results suggest that there might be the discrepancy between preS1 and preS2 mutations in the mechanism of enhancing the progression of chronic liver disease, especially the development of HCC and to maintain tolerance during the stage of immune tolerance. Specific deletion of the type disrupting preS1 start codon may play important roles in hepatocarcinogenesis, at least in Korean patients with chronic HBV infection. J. Med. Virol. 80: 1189–1194, 2008.

Novel F141L Pre-S2 Mutation in Hepatitis B Virus Increases the Risk of Hepatocellular Carcinoma in Patients with Chronic Genotype C Infections

ABSTRACT Several lines of evidence have suggested that some naturally occurring mutations of hepatitis B virus (HBV) play a critical role in hepatocellular carcinoma (HCC). Here, we describe a novel HCC-related pre-S2 mutation, F141L. To prove the relationship between the F141L mutation and HCC, molecular epidemiology studies using MboII PCR restriction analysis (PRA) were performed, and the molecular mechanism was investigated through construction of a stable hepatocyte cell line expressing the large surface HB protein (LHB) with the F141L mutation (F141L-LHB). Application of MboII PRA to samples from 241 Korean patients with chronic liver diseases of different clinical stages confirmed that F141L mutants were significantly related to HCC, even in comparison to liver cirrhosis (HCC, 26.3% of patients, or 26/99; liver cirrhosis, 3.8% of patients, or 2/52; P = 0.001). By studying stable cell lines, we found that F141L-LHBs could induce cell cycle progression by downregulating the p53 and p21 pathways and upregulating CDK4 and cyclin A. Furthermore, we found that in a colony-forming assay, the colony-forming rates in cell lines expressing F141L-LHBs were about twice as high as those of the wild type. In conclusion, our results suggest that F141L-LHBs may contribute importantly to the pathogenesis of HCC by inducing cell proliferation and transformation. So, the F141L mutation examined in this study could serve as a diagnostic marker for the prognosis of HCC.

Mycobacterium senuense sp. nov., a slowly growing, non-chromogenic species closely related to the Mycobacterium terrae complex.

A previously undescribed, slowly growing, non-chromogenic mycobacterium, isolated from a Korean patient with a symptomatic pulmonary infection, is described as representing a novel species. Its 16S rRNA gene sequence was unique and phylogenetic analysis based on 16S rRNA gene sequences showed that this organism belonged to the Mycobacterium terrae subclade. Phenotypically, the strain was generally similar to M. terrae and Mycobacterium nonchromogenicum, but its growth rate was slower than those of other M. terrae complex strains. A unique mycolic acid profile and phylogenetic analysis based on two different alternative chronometer molecules, hsp65 and rpoB, confirm the taxonomic status of this strain as a representative of a novel species. The name Mycobacterium senuense sp. nov. is proposed, with the type strain 05-832(T) (=DSM 44999(T) =KCTC 19147(T)).

Naturally occurring hepatitis B virus X deletions and insertions among Korean chronic patients.

Deletions and insertions in the hepatitis B virus (HBV) X region have been associated with severe forms of liver disease, including hepatocellular carcinoma (HCC). However, the molecular epidemiologic features of this virus have been described rarely. Deletions and insertions in the X region were determined by direct sequencing in a Korean cohort of 267 patients with different clinical statuses. Deletions and insertions were observed in two sets of six patients each (2.2%, 6/267). The prevalence of deletions or insertions was significantly higher in patients with severe liver disease, HCC, or cirrhosis of the liver (7.2%, 10/132) compared to patients who were carriers or had chronic hepatitis (1.5%, 2/135) (P = 0.017). All deletions in six strains were concentrated at the C terminal end of HBx, encompassing the 113th to 154th codons. A total of four novel types of insertions (PKLL, GM, FFN, and tt) were observed in six patients. Of particular interest, all six strains with insertions were accompanied by double mutations in the basal core promoter (BCP). In conclusion, these results suggest that deletions or insertions in the X region may contribute to disease progression in Korean patients with genotype C infection. J. Med. Virol. 83:65–70, 2011.

Differentiation of Mycobacterial Species by hsp65 Duplex PCR Followed by Duplex-PCR-Based Restriction Analysis and Direct Sequencing

ABSTRACT Here we describe a novel duplex PCR method which can differentiate Mycobacterium tuberculosis and nontuberculosis mycobacteria (NTM) strains by amplifying hsp65 DNAs of different sizes (195 and 515 bp, respectively). The devised technique was applied to 54 reference and 170 clinical isolates and differentiated all strains into their respective groups with 100% sensitivity and specificity. Furthermore, a duplex PCR-restriction analysis (duplex PRA) and a direct sequencing protocol were developed to differentiate NTM strains at the species and subspecies levels based on previously reported hsp65 DNA sequences (H. Kim et al., Int. J. Syst. Evol. Microbiol. 55:1649-1656, 2005) and then applied to 105 NTM clinical isolates. All NTM isolates were clearly differentiated at the species and subspecies levels by subsequent procedures (PRA or direct sequencing) targeting 515-bp NTM duplex PCR amplicons. Our results suggest that novel duplex PCR-based methods are sensitive and specific for identifying mycobacterial culture isolates at the species level.

Characterization of two hepatitis B virus populations in a single Korean hepatocellular carcinoma patient with an HBeAg-negative serostatus: a novel X-Gene-deleted strain with inverted duplication sequences of upstream enhancer site II.

Objectives: The aim of the study was to elucidate mutation patterns related to hepatocarcinogenesis in a Korean hepatocellular carcinoma (HCC) patient. Methods: We analyzed full genome sequences of 6 hepatitis B virus (HBV) clones from an HCC patient. Results: This patient harbored 2 HBV populations with genomes of different lengths (3,221 and 2,212 bp). In addition, we found 2 characteristic features not described so far in the full-genome sequence of deleted strains. First, 3 large deletion events (847, 144 and 48 bp) and a premature termination of the 182th codon of the surface antigen could lead to truncated or possibly nonfunctional forms of all HBV proteins. Second, these showed a novel mutation type not reported to date, which is a complex of an inverted duplication of 36-bp sequences containing an upstream enhancer site II (UEII), a remote insertion, and a large deletion event of the X region by homologous recombination. Conclusion: The fact that UEII is a binding site of liver-specific nuclear factor, which is expressed only in highly differentiated liver cells such as cancerous HepG2, strongly suggests a relationship between this novel mutation and hepatocarcinogenesis in this patient.

Direct Application of AvaII PCR Restriction Fragment Length Polymorphism Analysis (AvaII PRA) Targeting 644 bp Heat Shock Protein 65 (hsp65) Gene to Sputum Samples

To evaluate the usefulness of the AvaII PRA method targeting 644‐bp hsp65 gene for the direct detection of pathogenic mycobacteria from clinical specimens, we applied this method to 40 sputum samples and compared the results to those obtained by IS6110 PCR. Although this method showed a sensitivity slightly lower than IS6110 PCR (97.5% vs. 100%), it detected infections of M. avium complex (MAC) in two patients, which was not possible by IS6110 PCR. We conclude that AvaII PRA is a highly effective method for directly detecting pathogenic mycobacteria in primary clinical specimens.

Pulmonary disease caused by Mycobacterium xenopi: the first case in Korea

Mycobacterium xenopi is a nontuberculous mycobacterium (NTM) that rarely causes pulmonary disease in Asia. Here we describe the first case of M. xenopi pulmonary disease in Korea. A 66-year-old man was admitted to our hospital with a 2-month history of productive cough and hemoptysis. His past medical history included pulmonary tuberculosis 44 years earlier, leading to a right upper lobectomy. Chest X-ray upon admission revealed cavitary consolidation involving the entire right lung. Numerous acid-fast bacilli were seen in his initial sputum, and M. xenopi was subsequently identified in more than five sputum cultures, using molecular methods. Despite treatment with clarithromycin, rifampicin, ethambutol, and streptomycin, the infiltrative shadow revealed on chest X-ray increased in size. The patients condition worsened, and a right completion pneumonectomy was performed. The patient consequently died of respiratory failure on postoperative day 47, secondary to the development of a late bronchopleural fistula. This case serves as a reminder to clinicians that the incidence of NTM infection is increasing in Korea and that unusual NTM are capable of causing disease in non-immunocompromised patients.