Futoshi Nagasaki

Enhanced intracellular retention of a hepatitis B virus strain associated with fulminant hepatitis.

A plasmid carrying 1.3-fold HBV genome was constructed from a HBV strain that caused five consecutive cases of fulminant hepatitis (pBFH2), and HepG2 cells were transfected with pBFH2 or its variants. The pBFH2 construct with A1762T/G1764A, G1862T, and G1896A showed the largest amount of core particle-associated intracellular HBV DNA, but no significant increase of extracellular HBV DNA in comparison with the wild construct, suggesting that these mutations might work together for retention of the replicative intermediates in the cells. The retention might relate to the localization of hepatitis B core antigen (HBcAg) in the nucleus of HepG2, which was observed by confocal fluorescence microscopy. HBcAg immunohistochemical examination of liver tissue samples obtained from the consecutive fulminant hepatitis patients showed stronger staining in the nucleus than acute hepatitis patients. In conclusion, the fulminant HBV strain caused retention of the core particles and the core particle-associated HBV DNA in the cells.

Analysis of the entire nucleotide sequence of hepatitis B causing consecutive cases of fatal fulminant hepatitis in Miyagi Prefecture Japan.

We encountered five consecutive patients with fulminant hepatitis induced by acute hepatitis B virus (HBV) infection in 2000–2001 in Japan. They had not had previous contact each other, and were referred to us from different hospitals. Although a 69‐year‐old woman could be rescued by intensive internal treatment, the four patients died. We analyzed the partial (nt 278–646) and entire nucleotide sequences of the HBV obtained from them, and their divergences were 0–0.3% and 0–0.2%, respectively. The results suggested that they had been infected with the same HBV isolates. The isolates belonged to genotype B and subgenotype B2 on the phylogenetic tree analysis (AB302942–AB302946). As for the nucleotides sequences of them, previously reported mutations of G1896A, A1762T, and G1764A were present. Amino acid analysis revealed that previously reported Ile97Leu and Pro130Non‐Pro in the core region and Trp28Stop in the precore region were present. As for the entire nucleotide sequences among B2, AB302942 showed low divergences with AF121245 and AB073834 (1.7%), and X97850 from patients with fulminant hepatitis (3.2%). We compared the two consensus nucleotides derived from AB302942 and X97850 (fulminant hepatitis) versus AY121245 and AB073834 (non‐fulminant hepatitis), which revealed a difference in nt 1,504 located in the P and X region. Nucleotide 1,504 was C for isolates from fulminant hepatitis and G for non‐fulminant hepatitis, and it was recognized among most of the isolates belonging to B2 registered on GenBank. Further studies could disclose the mechanism of severe inflammation of liver that finally leads to fulminant hepatitis. J. Med. Virol. 80:967–973, 2008.

Analysis of the full-length genome of hepatitis B virus in the serum and cerebrospinal fluid of a patient with acute hepatitis B and transverse myelitis

Although many extrahepatic manifestations have been described in patients with acute or chronic hepatitis B, there are few reports about neurological disorders. We describe a 55-year-old man who contracted acute hepatitis B virus (HBV) infection and transverse myelitis. His neurological findings were gradually reduced along with the recovery from hepatitis. The cerebrospinal fluid (CSF) was revealed to be positive for HBsAg and HBV DNA. Full-length sequences of HBV in his serum and CSF were determined, and it was revealed that these two isolates had mutations at nucleotide (nt) 1762/1764 in the core promoter region and nt 1896 in the precore region. They were identical to each other except for two ambiguous codes at nt 2020 and 2631 in the CSF isolate. After cloning of the amplicons, substitutions at nt 2020 and 2631 were found in 6 (38%) of the 16 CSF clones. One clone of the 6 CSF clones had an additional substitution at nt 2119. These substitutions were not found in 16 serum clones. The presence of HBV clones unique to CSF suggests that HBV was a possible causative agent of the myelitis.

A case of severe recurrent hepatitis with common variable immunodeficiency

Severe hepatitis with an indistinct etiology manifested in a 16‐year‐old boy who had no particular history. The histological features of the liver and clinical course of the patient were similar to those of patients with autoimmune hepatitis characterized by interface hepatitis and severe lobular inflammation of the liver and recurrent exacerbations of hepatitis. We administered intravenous glycyrrhizin preparation daily or three times a week combined with the oral administration of ursodeoxycholic acid daily throughout the term after the initial onset of disease for the control of disease activity. The normalization of the concentration of alanine aminotransferase in serum was achieved in response to the therapy during the course. The serum concentration of immunoglobulins of the patient gradually decreased from the onset of the disease to an unacceptable level without globulin preparation during the following period of 17 months. Immunological tests revealed impairment of immunoglobulin production bythe B cell population of the patient, which led to the diagnosis of the patient as common variable immunodeficiency (CVID). The patient, with improved liver histology after 27 months from the onset of disease, benefited from the current combination therapy without severe infection through the avoidance of overimmunosuppression. CVID is defined as a heterogeneous syndrome characterized by various degrees of hypogammaglobulinemia without any specific predisposing causes, frequently associated with autoimmunity. Diagnostic criteria and therapeutic options of persistent hepatitis with CVID are to be established, as discussed in the current report.

A case of HIV co-infected with hepatitis B virus precore/core deletion mutant treated by entecavir.

We report a case of a HIV and hepatitis B virus (HBV)‐co‐infected patient to whom entecavir (ETV) was administered initially before the notification regarding the potential mutagenesis effect on HIV against the nucleoside analog. Since initial evaluations indicated the advanced stage of chronic hepatitis B and preserved numbers of peripheral CD4+ lymphocytes without the manifestation of immunodeficiency, priority was given to the management of HBV. We started HBV therapy with ETV at a dose of 0.5 mg daily without using any HIV drugs. The viral loads of both HBV and HIV‐1 decreased gradually during the 5 months following the initial administration of ETV. HBV was well controlled by the gradual replacement of ETV with highly‐active antiretroviral therapy against HIV with a regimen including atazanavir, emtricitabine, and tenofovir. HBV was genotyped as A2 with the quasispecies pool consisting of the −1G precore/core deletion mutant strain.

Analysis of the entire nucleotide sequence of hepatitis B virus: characteristics of HBeAg-positive asymptomatic carriers, HBeAb positive asymptomatic carriers and patients with liver cirrhosis.

Entire nucleotide sequences of the HBV genome typical for various stages of HBV carriers are currently unknown. Comparison between conserved sequences in HBeAg-positive asymptomatic carriers (HBeAg ASCs) and mutations characteristic for HBeAb-positive asymptomatic carriers (HBeAb ASCs) are of clinical importance. In this study, we determined the entire nucleotide sequences of the HBV genome of patients infected with genotype C (HBeAg ASCs, 11 cases; HBeAb ASCs, seven cases; patients with liver cirrhosis (LC), five cases). Mutations in the entire nucleotide sequences were found more frequently in HBeAb ASCs than in HBeAg ASCs. In the precore/core (preC/C) region, amino acid mutations were more frequent in HBeAb ASCs (3.03%) than in HBeAg ASCs (0.00%) and patients with LC (0.69%). It was suggested that the mutations in the preC/C region had a close relationship with clinical status of HBV carriers. Mutations of leucine to isoleucine at a.a. 100 of the core region and of threonine to serine at a.a. 340 of the polymerase region were found frequently in HBeAb ASCs. In patients with LC, it was suggested that defective interfering particles (DI particles) play a role in the progression of stages. We conclude that attention should be given to mutations at a.a. 340 of the polymerase protein in addition to core protein.

HBe seroconversion and HBs mutation among Japanese hepatitis B virus carriers

It has been reported in Germany that seroconversion to anti-HBe or anti-HBs is frequently associated with genotype changes of hepatitis B virus (HBV) from genotype A to genotype D. We previously reported that the HBeAg-negative state in Japan was significantly more common in patients infected with genotype B HBV than those infected with genotype C HBV. To determine whether the high prevalence of genotype B in the HBeAg-negative state is due to a change from genotype C to genotype B, 72 pairs of serum samples before and after HBe seroconversion were examined for nucleotide sequences in the S gene (amino acids 42-164) among Japanese HBV carriers. No one was identified to have undergone genotype change during HBe seroconversion. A total of 71 codon mutations were observed. Sixty-two of these 71 codon mutations (87.3%) were non-synonymous. Genotype B HBV had no mutational hot spots. In genotype C, there was a mutational hot spot at amino acid 126 of the S protein, and it was suggested that Thr126 before HBe seroconversion was more susceptible to becoming an asymptomatic carrier after HBe seroconversion than Ile126. In conclusion, genotype changes during HBe seroconversion were not found to be common in Japan.