Motokazu Mukaide

Hepatitis B virus genotype assignment using restriction fragment length polymorphism patterns.

Hepatitis B virus (HBV) is classified into genotypes A–F, which is important for clinical and etiological investigations. To establish a simple genotyping method, 68 full‐genomic sequences and 106 S gene sequences were analyzed by the molecular evolutionary method. HBV genotyping with the S gene sequence is consistent with genetic analysis using the full‐genomic sequence. After alignment of the S sequences, genotype specific regions are identified and digested by the restriction enzymes, HphI, NciI, AlwI, EarI, and NlaIV. This HBV genotyping system using restriction fragment length polymorphism (RFLP) was confirmed to be correct when the PCR products of the S gene in 23 isolates collected from various countries were digested with this method. A restriction site for EarI in genotype B was absent in spite of its presence in all the other genotypes and genotype C has no restriction site for AlwI. Only genotype E is digested with NciI, while only genotype F has a restriction site for HphI. Genotype A can be distinguished by a single restriction enzyme site for NlaIV, while genotype D digestion with this enzyme results in two products that migrates at 265 and 186 bp. This simple and accurate HBV genotyping system using RFLP is considered to be useful for research on HBV.

New genotypes of TT virus (TTV) and a genotyping assay based on restriction fragment length polymorphism

A phylogenetic analysis, using the open reading frame 1 sequence of 93 TT viruses (TTV) obtained from various geographical areas, indicated that the virus could be classified into six different genotypes including three hitherto unreported genotypes. The high reliability of the six clusters was confirmed by bootstrap analysis. On the basis of these sequence data, a new simple genotyping assay based on a restriction fragment length polymorphism of TTV was developed. Using the enzymes NdeI and PstI, followed by cleavage with NlaIII or MseI, it was possible to distinguish between the six TTV genotypes. This system will provide the framework for future detailed epidemiological and clinical investigations.

The rs8099917 Polymorphism, When Determined by a Suitable Genotyping Method, Is a Better Predictor for Response to Pegylated Alpha Interferon/Ribavirin Therapy in Japanese Patients than Other Single Nucleotide Polymorphisms Associated with Interleukin-28B

ABSTRACT We focused on determining the most accurate and convenient genotyping methods and most appropriate single nucleotide polymorphism (SNP) among four such polymorphisms associated with interleukin-28B (IL-28B) in order to design tailor-made therapy for patients with chronic hepatitis C virus (HCV) patients. First, five different methods (direct sequencing, high-resolution melting analysis [HRM], hybridization probe [HP], the InvaderPlus assay [Invader], and the TaqMan SNP genotyping assay [TaqMan]) were developed for genotyping four SNPs (rs11881222, rs8103142, rs8099917, and rs12979860) associated with IL-28B, and their accuracies were compared for 292 Japanese patients. Next, the four SNPs associated with IL-28B were genotyped by Invader for 416 additional Japanese patients, and the response to pegylated interferon/ribavirin (PEG-IFN/RBV) treatment was evaluated when the four SNPs were not in linkage disequilibrium (LD). HRM failed to genotype one of the four SNPs in five patients. In 2 of 287 patients, the results of genotyping rs8099917 by direct sequencing differed from the results of the other three methods. The HP, TaqMan, and Invader methods were accurate for determination of the SNPs associated with IL-28B. In 10 of the 708 (1.4%) patients, the four SNPs were not in LD. Eight of nine (88.9%) patients whose rs8099917 was homozygous for the major allele were virological responders, even though one or more of the other SNPs were heterozygous. The HP, TaqMan, and Invader methods were suitable to determine the SNPs associated with IL-28B. The rs8099917 polymorphism should be the best predictor for the response to the PEG-IFN/RBV treatment among Japanese chronic hepatitis C patients.

Three different GB virus C/hepatitis G virus genotypes: Phylogenetic analysis and a genotyping assay based on restriction fragment length polymorphism

The 5′‐untranslated region (5′‐UTR) sequences of 33 GB virus C/hepatitis G virus (GBV‐C/HGV) obtained from different geographic areas were determined through reverse‐transcription polymerase chain reaction and dideoxy chain termination sequencing, the alignment of sequences, the estimation of the number of nucleotide substitution per site, and construction of phylogenetic trees. The 5′‐UTR of GBV‐HGV was found to be heterogeneous, with 70.9–99.5% homology. Three distinct phylogenetic branches were observed consistently in all phylogenetic trees. GBV‐C is the prototype for one, HGV for another, and there is a new branch which consisted of GBV‐C/HGV isolates from Asia. Genotype‐specific restriction sites for the restriction enzymes, ScrFI and BsmFI, were identified, and a simple restriction fragment polymorphism analysis was developed for genotyping. These data provide evidence that GBV‐C/HGV consists of three different genotypes. Our simple genotyping assay will also provide a tool for epidemiological studies of GBV‐C/HGV infection.

High prevalance of TT virus infection in Japanese patients with liver diseases and in blood donors

BACKGROUND/AIMS Although a novel DNA virus, TT virus (TTV), has been isolated from a patient with cryptogenic post-transfusion hepatitis, its pathogenic role remains unclear. To elucidate its prevalence and clinical impact in patients with liver diseases, the presence of TTV DNA was assessed in patients with liver diseases and blood donors (BDs) in Japan using two primer sets, one conventional and the other new and highly sensitive. METHODS We studied 261 samples, 72 with chronic hepatitis associated hepatitis C virus (HCV-CH), 57 with hepatocellular carcinoma associated HCV (HCV-HCC), 12 with HCC without either HCV or hepatitis B virus (NBNC-HCC), and 120 of BDs. RESULTS Using two primer sets, TTV DNA was detected in 68 (94.4%), 53 (93.0%), 12 (100%), and 98 (81.7%) HCV-CH, HCV-HCC, NBNC-HCC, and BDs, respectively. The prevalence was not significantly different between HCV-CH and HCV-HCC, or between HCV-HCC and NBNC-HCC. Comparison between patients with and without TTV revealed no significant differences in backgrounds or biochemical findings. Histopathological findings in patients with HCV-CH, and number, maximum diameter, and histological differentiation of HCC also did not demonstrate any relation to TTV infection. TTV strains can be divided into five groups using phylogenetic analysis, but no disease-specific group appears to exist. CONCLUSIONS Our data suggest that: 1) TTV is very prevalent among patients with liver diseases and even among BDs in Japan, 2) TTV infection does not impact on liver damage with HCV infection, and 3) TTV infection also does not affect the development or progression of HCC.

Influence of Hepatitis B Virus X and Core Promoter Mutations on Hepatocellular Carcinoma among Patients Infected with Subgenotype C2

ABSTRACT Hepatitis B virus (HBV) genotypes/subgenotypes and their related mutations in the HBV genome have been reported to be associated with hepatocellular carcinoma (HCC). To determine the HCC-associated mutations of the HBV genome in the entire X, core promoter, and precore/core regions, a cross-sectional control study was conducted comparing 80 Japanese patients infected with HBV C2 and suffering from HCC with 80 age-, sex-, and hepatitis B e antigen (HBeAg) status-matched patients without HCC (non-HCC group). Each HBeAg-positive group (31 with HCC; 29 without HCC) and HBeAg-negative group (49 with HCC; 51 without HCC) was also matched with respect to age and sex. The C1479, T1485, H1499, A1613, T1653, V1753, T1762/A1764, and A1896 mutations were frequent in this population. The prevalences of the T1653 mutation in the box α region and the V1753 and T1762/A1764 mutations in the basal core promoter region were significantly higher in the HCC group than in the non-HCC group (56% versus 30%, 50% versus 24%, and 91% versus 73% [P = 0.0013, P = 0.0010, and P = 0.0035, respectively]). The platelet count was significantly lower for the HCC group than for the non-HCC group (10.7 × 104 ± 5.1 × 104 versus 17.3 × 104 ± 5.1 × 104 platelets/mm3 [P < 0.0001]). Regardless of HBeAg status, the prevalence of the T1653 mutation was higher in the HCC group (52% versus 24% [P = 0.036] for HBeAg-positive patients and 59% versus 33% [P = 0.029] for HBeAg-negative patients). In the multivariate analysis, the presence of T1653, the presence of V1753, and a platelet count of ≤10 × 104/mm3 were independent predictive factors for HCC (odds ratios [95% confidence intervals], 4.37 [1.53 to 12.48], 7.98 [2.54 to 25.10], and 24.39 [8.11 to 73.33], respectively). Regardless of HBeAg status, the T1653 mutation increases the risk of HCC in Japanese patients with HBV/C2.

Impact of HIV Type 1 Protease, Reverse Transcriptase, Cleavage Site, and p6 Mutations on the Virological Response to Quadruple Therapy with Saquinavir, Ritonavir, and Two Nucleoside Analogs

Genotype alterations of HIV-1 protease, reverse transcriptase, cleavage sites p7/p1 and p1/p6, as well as p6(gag) and transframe protein p6* were studied in an observational cohort of 42 individuals who received antiretroviral therapy consisting of saquinavir, ritonavir, and two nucleoside analogs. In a multivariate logistic regression analysis, the prior protease inhibitor experience (odds ratio, 6.20; 95% CI, 1.22-31.38) and the presence of primary protease mutations (odds ratio, 9.99; 95% CI, 1.05-94.72) were independently associated with virological failure. Moreover, a trend was observed in that individuals with N-terminal amino acid insertions in the proline-rich motif of the p6(gag) protein were less likely to experience virological failure (OR, 0.17; 95% CI, 0.02-1.35; p = 0.09). In contrast, the presence of secondary protease, reverse transcriptase, or cleavage site mutations was not independently associated with treatment failure. However, mutations at cleavage site p7/p1 (p = 0.01) and C-terminal p6* mutations (p = 0.02) were both associated with primary protease mutations. In conclusion, the presence of primary protease mutations was the most important predictor of the subsequent virological response. Moreover, there is some evidence that insertions in the proline-rich area of the p6(gag) protein may affect the virological response. The relationship between mutations of cleavage sites or C-terminal p6* residues and protease mutations suggests that these alterations may serve a compensatory role, increasing viral fitness.

Prevalence and molecular epidemiology of GB virus C/hepatitis G virus infection in Mongolia

We studied the prevalence of GB virus C/hepatitis G virus (GBV‐C/HGV) infection among 112 patients with liver disease and 121 blood donors in Ulaanbaatar, Mongolia. Reverse transcription and polymerase chain reaction were employed to detect GBV‐C/HGV RNA using the specific primers derived from the 5′‐untranslated region (5′‐UTR) of the GBV‐C/HGV genome. Nucleotide sequences of all positive samples for GBV‐C/HGV RNA were determined. The sequences were analyzed by a molecular evolutionary method. Twenty‐five (10.7%) of 233 people were positive for GBV‐C/HGV RNA. Eight (6.6%), 11 (9.1%), and 30 (24.8%) blood donors were positive for GBV‐C/HGV RNA, HBsAg, and anti‐HCV, respectively, although 17 (15.2%), 65 (58.0%), and 64 (54.5%) patients with liver disease were positive for each viral marker. The prevalences of GBV‐C/HGV RNA, HBV, and HCV in the patients were significantly higher than those in blood donors (P < 0.05). There was no significant difference in the prevalence of anti‐HCV among people with and without GBV‐C/HGV RNA, while the prevalence of HBsAg among people with GBV‐C/HGV RNA was significantly higher than among those without GBV‐C/HGV RNA (P < 0.05). The molecular evolutionary tree showed that GBV‐C/HGV was a heterogeneous virus and all strains could be divided into 2 types. One is the same phylogenetic type as HGV, and the other is a new type that is different from GBV‐C and HGV. J. Med. Virol. 52:143–148, 1997.

Mechanism of Entecavir Resistance of Hepatitis B Virus with Viral Breakthrough as Determined by Long-Term Clinical Assessment and Molecular Docking Simulation

ABSTRACT The mechanism by which entecavir resistance (ETVr) substitutions of hepatitis B virus (HBV) can induce breakthrough (BT) during ETV therapy is largely unknown. We conducted a cross-sectional study of 49 lamivudine (LVD)-refractory patients and 59 naïve patients with chronic hepatitis B. BT was observed in 26.8% of the LVD-refractory group during weeks 60 to 144 of ETV therapy. A line probe assay revealed ETVr substitutions only in the LVD-refractory group, i.e., in 4.9% of patients at baseline, increasing to 14.6%, 24.4%, and 44.8% at weeks 48, 96, and 144, respectively. Multivariate logistic regression analysis adjusted for age, gender, HBV DNA levels, and LVD resistance (LVDr) (L180M and M204V, but not M204I) indicated that T184 substitutions and S202G (not S202C) were a significant factor for BT (adjusted odds ratio [OR], 141.12, and 95% confidence interval [CI], 6.94 to 2,870.20; OR, 201.25, and 95% CI, 11.22 to 3608.65, respectively). Modeling of HBV reverse transcriptase (RT) by docking simulation indicated that a combination of LVDr and ETVr (T184L or S202G) was characterized by a change in the direction of the D205 residue and steric conflict in the binding pocket of ETV triphosphate (ETV-TP), by significantly longer minimal distances (2.2 Å and 2.1 Å), and by higher potential energy (−117 and −99.8 Kcal/mol) for ETV-TP compared with the wild type (1.3 Å; −178 Kcal/mol) and LVDr substitutions (1.5 Å; −141 Kcal/mol). Our data suggest that the low binding affinity of ETV-TP for the HBV RT, involving conformational change of the binding pocket of HBV RT by L180M, M204V plus T184L, and S202G, could induce BT.

Development of real-time detection direct test for hepatitis B virus and comparison with two commercial tests using the WHO international standard

Aims:  A highly reproducible and sensitive hepatitis B virus real‐time detection direct (HBV RTD‐direct) test using DNA extraction by magnetic beads coated with polyclonal anti‐HBsAg, followed by the real‐time detection polymerase chain reaction (PCR) method, was developed for the detection of HBV DNA.