Minako Hijikata

Selective transmission of hepatitis C virus in vivo and in vitro

SummaryA human plasma containing quasi-species of hepatitis C virus (HCV) was inoculated to a chimpanzee and to human lymphocytic cell lines, HPB-Ma clone 10-2, AD HPB, and Daudi, which support replication of HCV. Among six different hypervariable region (HVR) sequences detected in the inoculum, the same two were recovered both in vivo and in vitro.

Replication of hepatitis C virus

SUMMARY. The mode of replication of the hepatitis C virus (HCV) remains poorly understood. Attempts to produce a tissue culture model containing replicating HCV have been largely unsuccessful. Recent studies on sera from patients chronically infected with HCV have shown that viral particles may be found in high‐or low‐density fractions. High‐density fractions contain non‐infectious virions whilst infectious particles can be derived from low‐density material. Using appropriate infectious fractions we have successfully infected a number of human cell lines allowing studies of HCV replication to be initiated.

Detection of a 5′ end subgenome of hepatitis C virus terminating at nucleotide 384 in patients’ plasma and liver tissues

Summary.u2002 Quadri and Negro [Dig Liver Dis 2001; 33: 480] reported greater distribution of 5′ end genomic RNA of hepatitis C virus (HCV) over its 3′ end in the liver of patients with recurrent hepatitis C after liver transplantation. We not only confirmed their results by quantifying the 5′ end subgenomes in various specimens by using dilution and real‐time polymerase chain reaction methods, but also discovered that such subgenomes terminated at nucleotide (nt) 384 of the viral genome or in its immediate upstream. The subgenomes in the plasma uniformly, with a few exceptions, ended at this position, while those in the liver more heterogeneously at various points upstream of nt 384. Subgenome populations ending some points in the downstream of nt 384 were not detected. The amount of the 5′ end subgenome, while fluctuating during the clinical course of the patients, exceeded that of the longer sized HCV genomes which included the intact genome, and, when the relative ratio of the 5′ end subgenome increased, the amount of longer sized HCV RNA tended to decrease, suggesting a suppressive effect of the 5′ end subgenome on viral replication.

Isolation of human monoclonal antibodies to the envelope e2 protein of hepatitis C virus and their characterization.

We isolated and characterized two human monoclonal antibodies to the envelope E2 protein of hepatitis C virus (HCV). Lymphoblastoid cell lines stably producing antibodies were obtained by immortalizing peripheral blood mononuclear cells of a patient with chronic hepatitis C using Epstein-Barr virus. Screening for antibody-positive clones was carried out by immunofluorescence with Huh7 cells expressing the E2 protein of HCV strain H (genotype 1a) isolated from the same patient. Isotype of resulting antibodies, #37 and #55, was IgG1/kappa and IgG1/lambda, respectively. Epitope mapping revealed that #37 and #55 recognize conformational epitopes spanning amino acids 429 to 652 and 508 to 607, respectively. By immunofluorescence using virus-infected Huh7.5 cells as targets both antibodies were reactive with all of the nine different HCV genotypes/subtypes tested. The antibodies showed a different pattern of immuno-staining; while #37 gave granular reactions mostly located in the periphery of the nucleus, #55 gave diffuse staining throughout the cytoplasm. Both antibodies were shown by immuno-gold electron microscopy to bind to intact viral particles. In a neutralization assay (focus-forming unit reduction using chimeric infectious HCV containing structural proteins derived from genotypes 1a, 1b, 2a, 2b, 3a, 4a, 5a, 6a, and 7a), #55 inhibited the infection of all HCV genotypes tested but genotype 7a to a lesser extent. #37 did not neutralize any of these viruses. As a broadly cross-neutralizing human antibody, #55 may be useful for passive immunotherapy of HCV infection.

Useful Markers for Predicting in Vivo Infectivity of Hepatitis C Virus

Aiming to find useful markers for predicting the in vivo infectivity of hepatitis C virus (HCV), we investigated differences between inocula with high and low in vivo infectivity. Measurement of the genomic RNA titer by reverse transcriptase-polymerase chain reaction (RT-PCR) alone was not sufficient. However, combining RT-PCR with differential flotation in 1.063 g/ml solution, immunoprecipitation with anti-human immunoglobulin, as well as adsorption to HPB Ma cells (recently shown to support replication of HCV), appeared to have a high predictive value for estimating whether a sample will be highly infectious in vivo. Furthermore, rough estimation of the in vivo infectivity titer of a sample can be obtained from its in vitro infectivity titer measured in HPB Ma culture.

C型肝炎ウイルスの in vitro 感染実験系

我々は先に, ヒトT細胞株 Molt 4・MaやHPB・Ma細胞がC型肝炎ウイルス (HCV) に対して感受性を持つことを見出した。この系を用いた実験で, これまでに次の事が明らかになった。(1) HPB・Maを用いて測定したHCV材料の in vitro 感染価は, in vivo 感染価 (チンパンジー感染価) と相関する。(2) HCV粒子の細胞への吸着効率は感染価と相関する。吸着効率の悪い材料ではHCV粒子は抗体と結合した免疫複合体を形成している。(3) 吸着阻止を指標とする抗体測定系を作り, HCV持続感染患者における抗体出現を調べた所, 持続感染においても吸着阻止抗体 (即ち感染防御抗体) は産生されるが, それは isolate-specific である。(4) in vitro においてもインターフェロン (IFN) のαやβによってHCV増殖は抑制される。