Comprehensive genome analysis of hepatitis B virus using nanopore sequencing technology in patients with previously resolved infection and spontaneous reactivation without drug exposure.

A 75-year-old Japanese woman experienced persistent fatigue and progressive jaundice for 6 weeks, and was subsequently diagnosed with acute liver failure. She had not received any immunosuppressive therapies and/or antineoplastic chemotherapy. Blood tests revealed elevated levels of HBsAg, HBV-DNA, and anti-HBc IgG, while anti-HBc IgM was negative.

Switching from combination therapy with entecavir hydrate plus tenofovir alafenamide fumarate to tenofovir alafenamide fumarate monotherapy in patients with chronic hepatitis B based on nucleotide sequences of hepatitis B virus pregenome RNA.

Aim: Patients with chronic hepatitis B virus (HBV) infection experiencing viral breakthrough (BTH) or partial response (PR) during lamivudine (LAM) or entecavir hydrate (ETV) administration often took ETV plus tenofovir alafenamide fumarate (TAF) due to the emergence of a drug-resistance mutation. However, in patients lacking drug-resistance mutation against TAF, sufficient antiviral effects may be achievable with TAF monotherapy. We assessed the drug-resistance profile through nucleotide sequences of HBV pregenome RNA, and subsequently changed to TAF monotherapy from ETV plus TAF.

Significance of portal venous blood flow as a factor to determine liver function in patients with decompensated cirrhosis due to hepatitis C virus infection following achievement of sustained viral response by sofosbuvir plus velpatasvir.

Aim: To determine the outcomes concerning portal venous blood flow and portosystemic shunts in patients with decompensated cirrhosis due to hepatitis C virus (HCV) infection who achieved sustained viral response (SVR) following antiviral therapy.

Methods: Portal hypertension-related events and liver function were evaluated in 24 patients achieving SVR following sofosbuvir plus velpatasvir therapy.

Results: Serum albumin level (median; g/dL) increased from 2.

Superiority of tenofovir alafenamide fumarate over entecavir for serum HBsAg level reduction in patients with chronic HBV infection: A 144-week outcome study after switching of the nucleos(t)ide analog.

Background: To evaluate the long-term efficacy of switching of the nucleos(t)ide analog used for treatment from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) in patients with chronic HBV infection.

Methods: A total of 103 patients with serum HBsAg levels of ≥100 IU/mL who had received ETV were enrolled. The nucleos(t)ide analog used for the treatment was switched from ETV to TAF, and the changes in serum HBsAg levels during the 144-week period before and after the drug switching were compared in 74 patients who had received ETV at least for 192 weeks.

Significance of furosemide in patients with cirrhosis treated with or without zinc acetate hydrate supplementation.

Background: The Japanese guidelines for the treatment of cirrhosis suggest zinc supplementation to prevent hepatic encephalopathy in patients with cirrhosis and zinc deficiency, although the factors that are associated with therapeutic efficacy remain unknown.

Method: A total of 159 patients with chronic liver diseases but without previous zinc supplementation were analyzed. Factors associated with serum zinc levels as well as the therapeutic efficacy of zinc supplementation were evaluated.

Furosemide as a factor to deteriorate therapeutic efficacy of tolvaptan in patients with decompensated cirrhosis.

Aim: To optimize the therapeutic strategy for patients with decompensated cirrhosis manifesting hepatic ascites and/or edema, factors affecting the outcome of patients receiving tolvaptan were evaluated.

Methods: The subjects were 165 patients receiving tolvaptan including 116 patients (70%) also treated with furosemide. The therapeutic efficacy of tolvaptan was defined as "effective" when a body weight reduction of 1.

Furosemide as a factor to deteriorate therapeutic efficacy of rifaximin in patients with decompensated cirrhosis.

Aim: To optimize the therapeutic strategy for cirrhotic patients manifesting hepatic encephalopathy, factors affecting the outcome of patients receiving rifaximin were evaluated.

Methods: The subjects were 95 patients receiving rifaximin. Serum ammonia levels were measured serially during rifaximin treatment.

Involvement of portosystemic shunts in impaired improvement of liver function after direct-acting antiviral therapies in cirrhotic patients with hepatitis C virus.

Aim: Factors responsible for impaired improvement of liver function despite sustained viral response after direct-acting antiviral agents therapies in cirrhotic patients with hepatitis C virus need to be elucidated.

Methods: Liver function and the extent of portosystemic shunting were evaluated for 79 patients with compensated cirrhosis, in whom sustained viral response had been achieved after direct-acting antiviral agents therapies for hepatitis C virus at least 3 years earlier.

Results: Portosystemic shunts were observed in 63 patients (80%).

Significance of switching of the nucleos(t)ide analog used to treat Japanese patients with chronic hepatitis B virus infection from entecavir to tenofovir alafenamide fumarate.

The significance of switching of the nucleos(t)ide analog used to treat patients with hepatitis B virus (HBV) from entecavir (ETV) to tenofovir alafenamide fumarate (TAF) is uncertain. The subjects of this study were 159 patients with HBV who received treatment with ETV followed by TAF. Among these patients, serial changes in the HBV marker levels were monitored in 92 patients in whom the serum HBsAg levels were ≥100 IU/mL during the 48-week period immediately before and after the switching.

A case of genotype-3b hepatitis C virus in which the whole genome was successfully analyzed using third-generation nanopore sequencing.

A 42-year-old Chinese man with chronic hepatitis C virus (HCV) infection visited our hospital for antiviral therapy. The subgenotype could not be determined using the HCV GENOTYPE Primer Kit (Institute of Immunology, Tokyo, Japan), which can identify genotype 3a HCV exclusively among genotype 3 HCV. Thus, the whole-genome sequence of HCV was analyzed using the MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK), a third-generation single-molecule sequencing platform.

NS5A-P32 deletion as a factor involved in virologic failure in patients receiving glecaprevir and pibrentasvir.

Background: This study sought to clarify the factors involved in virologic failure in patients with HCV receiving retreatment with glecaprevir/pibrentasvir (GLE/PIB) in real-world practice.

Methods: Forty-two patients who had previously received direct-acting antivirals (DAAs) therapies consisting of 35, 3, 3, and 1 patient(s) with genotype (GT)-1b, GT-2a, GT-2b, and GT-3b HCV, respectively, received GLE/PIB for 12 weeks. Resistance-associated substitutions (RASs) at baseline were evaluated, and the dynamics of NS5A-RASs were assessed by deep sequencing in patients showing virologic failure.

Serum asunaprevir concentrations showing correlation with the extent of liver fibrosis as a factor inducing liver injuries in patients with genotype-1b hepatitis C virus receiving daclatasvir plus asunaprevir therapy.

Aims: Liver injury can occur during antiviral therapies with direct-acting antivirals (DAAs), potentially necessitating discontinuation of the therapies, with consequent worsening of the sustained viral response (SVR) rates, in patients with hepatitis C virus (HCV). To clarify the mechanisms involved in serum transaminase level elevation, we performed a retrospective evaluation of the serum concentrations of daclatasvir and asunaprevir, both classified as DAAs, in patients receiving treatment with a combination of the two drugs.

Methods: Subjects were 278 Japanese patients with genotype-1b HCV who received daclatasvir plus asunaprevir therapy for more than 4 weeks.

Significance of NS5B Substitutions in Genotype 1b Hepatitis C Virus Evaluated by Bioinformatics Analysis.

To evaluate the effects of HCV NS5B amino acid substitutions on treatment outcome in Ledipasvir (LDV)/Sofosbuvir (SOF) for Japanese patients with genotype 1b HCV infection, NS5B sequences were examined in i) seven patients experiencing virologic failure after LDV/SOF in real-world practice, ii) 109 SOF-naïve patients, iii) 165 patients enrolled in Phase-3 LDV/SOF trial. A218S and C316N were detected in all patients with viral relapse; the percentages of these substitutions in SOF-naïve patients were 64.2% and 55.

Retreatment with sofosbuvir/ledipasvir with or without lead-in interferon-β injections in patients infected with genotype 1b hepatitis C virus after unsuccessful daclatasvir/asunaprevir therapy.

Aim: To improve the therapeutic efficacy of sofosbuvir/ledipasvir (SOF/LDV) for the retreatment of patients after daclatasvir/asunaprevir (DCV/ASV), a customized therapy with or without lead-in interferon (IFN)-β injections was formulated according to the types of resistance-associated substitutions (RAS) in the non-structural protein (NS)5A region of genotype 1b hepatitis C virus (HCV).

Methods: Thirty-three patients failing prior DCV/ASV received SOF/LDV for 12 weeks. Patients with HCV carrying unfavorable NS5A-RAS and/or those previously treated with simeprevir were given lead-in IFN-β injections twice a day for 2 weeks; sequential changes in the NS5A-RAS during the injection period were evaluated using deep sequencing.

Chronic hepatitis caused by hepatitis C virus showing a discrepancy between serogroup and genotype because of intergenotypic 2b/1b recombination: A pitfall in antiviral therapy with direct-acting antivirals.

A 40-year-old male patient with virologic relapse after daclatasvir plus asunaprevir therapy for a serogroup 1 hepatitis C virus (HCV) infection visited our hospital for retreatment. Virologic examinations revealed that a genotype 2b HCV strain carrying both NS3-S122N / D168A and NA5A-R30Q / L31M / Q54H / Y93H mutations had relapsed. The patient received sofosbuvir plus ribavirin therapy, but virologic relapse occurred once again.

"Reversi-type virologic failure" involved in the development of non-structural protein 5A resistance-associated variants (RAVs) in patients with genotype 1b hepatitis C carrying no signature RAVs at baseline.

Aims: The therapeutic efficacy of daclatasvir/asunaprevir was inferior in patients with non-structural protein 5A (NS5A)-R30Q mutant hepatitis C virus strains at baseline, compared with those with wild-type strains, even though the half maximal effective concentration of NS5A inhibitors was lower in mutant strains than in wild-type strains. In these patients, R30Q and Y93H mutant strains, which are highly resistant to NS5A inhibitors, emerged at virologic failure. The mechanisms involved in such virologic failure were examined.

Development of rare resistance-associated variants that are extremely tolerant against NS5A inhibitors during daclatasvir/asunaprevir therapy by a two-hit mechanism.

Aim: The virologic characteristics of resistance-associated variants (RAVs) developing in patients receiving dual oral therapy with daclatasvir/asunaprevir, including those with previous triple therapy with simeprevir, were evaluated.

Methods: A total of 206 patients with genotype-1b HCV infection, including 5 patients with previous simeprevir therapy, were treated with daclatasvir/asunaprevir for 24 weeks. Resistance-associated variants in the NS5A regions at baseline and during/after therapy were evaluated using cycling-probe real-time polymerase chain reaction combined with direct sequencing.

Significance of variants associated with resistance to NS5A inhibitors in Japanese patients with genotype 1b hepatitis C virus infection as evaluated using cycling-probe real-time PCR combined with direct sequencing.

Background: Dual oral therapy with daclatasvir plus asunaprevir yielded an SVR rate of 85% among patients with genotype 1b HCV. Treatment failure mainly occurred in patients with pre-existing HCV with NS5A-Y93H mutation. The significance of the mutation was evaluated.

A novel simple assay system to quantify the percent HCV-RNA levels of NS5A Y93H mutant strains and Y93 wild-type strains relative to the total HCV-RNA levels to determine the indication for antiviral therapy with NS5A inhibitors.

Aim: Oral treatment with asunaprevir and daclatasvir has been reported to yield a SVR ratio of 80% in patients with genotype 1b HCV infection, however, treatment failure has been reported, especially in patients with HCV strains showing the NS5A-Y93H mutation at baseline. An assay system to detect such strains was established to facilitate selection of appropriate candidates for this antiviral therapy.

Methods: Primer sets and 2 types of cycling probe mixtures were designed, and real-time PCR was performed with HCV-RNA purified from 332 patients with genotype 1b HCV infection, and the results were compared with those obtained by direct sequencing.

Novel hepatitis B virus strain developing due to recombination between genotypes H and B strains isolated from a Japanese patient.

Aim: In Japan, genotypes B and C are the predominant genotypes isolated from patients with chronic hepatitis B, while genotype A predominates in patients with acute hepatitis B. Globalization, however, appears to have changed the distribution of the hepatitis B virus (HBV) genotypes. Thus, the viral characteristics of HBV genotypes other than genotypes A, B and C were examined.

Purification, crystallization and preliminary X-ray analysis of a hexameric beta-glucosidase from wheat.

The wheat beta-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6xHis at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO4 and 150 mM NaCl using the hanging-drop vapour-diffusion method.