Non-A, non-B hepatitis: ultrastructural evidence for two agents in experimentally infected chimpanzees.

Two different ultrastructural alterations were observed in liver cells of chimpanzees inoculated with plasma derived from two different patients with non-A, non-B hepatitis. During the acute phase of illness in one group of four chimpanzees, peculiar tubular structures, composed of two unit membranes with electron-opaque material in between, were observed in the cytoplasm of hepatocytes. In contrast, these structures were never detected in the liver cells of the second group of five chimpanzees that received the second inoculum, However, nuclear changes, usually associated with aggregates of 20- to 27-nanometer particles, were found in hepatocytes of the latter animals. Although these particles resembled viruses, they were not as uniform as small virus particles often appear. In five other chimpanzees inoculated with non-A, non-B hepatitis material not known to be related to the first two inocula, cytoplasmic structures were found in four, and nuclear structures were found in the remaining one. Thus, all 14 chimpanzees inoculated with transmissible non-A, non-B hepatitis agents could be classified as having either nuclear or cytoplasmic changes. These observations add support to epidemiologic data suggesting that there may be more than one agent of non-A, non-B hepatitis.

Cell culture systems for the detection of HCV infection.

In spite of the recent progress in molecular biology of the hepatitis C virus (HCV) genome, the biological characteristics of this virus remain poorly known. This is primarily because biological assays for HCV have been limitted to the experimental inoculation of chimpanzees. It is imperative to develop either a less expensive animal model or a cell-culture system for propagating HCV. Several studies, including ours, have provided evidence for replication of the HCV genome in cell cultures. Although the reported systems are not yet fully satisfactory for wide application to in vitro studies of HCV, they are useful at least for examining the infectivity of HCV materials.

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

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.

Multicycle infection of hepatitis C virus in cell culture and inhibition by alpha and beta interferons.

We previously reported that a clone of a human lymphocytic cell line, HPBMa clone 10-2, supports the genome replication of hepatitis C virus (HCV). In the present study, the multicycle transmission of HCV from infected cells to new cells was demonstrated by coculture with drug-resistant cells as recipients; i.e., the system was proved productive for infectious HCV. Inhibition of viral replication by alpha and beta interferons was observed.

Cytoplasmic antigen in hepatocytes of chimpanzees infected with non-A, non-B hepatitis virus or hepatitis delta virus: relationship to interferon.

We previously described a cytoplasmic antigen, detected by monoclonal antibodies, in hepatocytes of chimpanzees experimentally infected with the parenterally transmitted form of non-A, non-B hepatitis virus or with the hepatitis delta virus. The expression of this antigen appears to be a host-specified response to infection with these two hepatitis viruses but not with hepatitis A virus, hepatitis B virus or enterically transmitted non-A, non-B hepatitis virus. To determine whether this antigen, found in parallel with the hepatocyte cytoplasmic structures described previously, is associated with interferon, as suggested by others, we studied by immunofluorescence liver biopsies from chimpanzees treated with an interferon inducer or exogenous interferon for the presence of the antigen. In two hepatitis B virus carrier chimpanzees and one normal chimpanzee treated with the interferon inducer polyinosinic-polyribocytidylic acid-poly-l-lysine carboxymethylcellulose, the antigen became detectable in hepatocytes within 2 weeks of initiation of the treatment, remained detectable throughout the treatment and disappeared within 4 weeks after treatment was terminated. Electron microscopy revealed that the biopsies positive for the antigen exhibited the hepatocyte cytoplasmic changes; convoluted membranes and microtubular aggregates, identical to those described originally for chimpanzees infected with non-A, non-B hepatitis virus. The antigen was not detected in any of the biopsies from a control chimpanzee that received only the carboxymethylcellulose used to stabilize the interferon inducer. In addition, liver biopsies obtained from a hepatitis B virus carrier chimpanzee during treatment with exogenous human leukocyte interferon were found to be positive for the antigen as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Correlation between the infectivity of hepatitis C virus in vivo and its infectivity in vitro.

A murine retrovirus-infected human T-cell line, HPB-Ma, supported replication of hepatitis C virus (HCV) at least as well as the previously reported Molt4-Ma cells. Cloning of HPB-Ma cells revealed a clonal variation of cellular susceptibility to HCV infection. Using one of the sensitive clones, we tested HCV inocula from different sources for their infectivity titer in cell culture. The in vitro titers obtained correlated with the reported infectivity titers of the inocula in chimpanzees. Thus, the system appears to be useful for estimating the in vivo infectivity of HCV.

Monoclonal antibodies to the hypervariable region 1 of hepatitis C virus capture virus and inhibit virus adsorption to susceptible cells in vitro.

To analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate-specific and epitope position-independent manner.

Characterization of long-term cultures of hepatitis C virus.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.

Replication of hepatitis C virus.

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.

Solubilization of envelopes of HVJ (Sendai virus) with alkali-emasol treatment and reassembly of envelope particles with removal of the detergent.

The envelopes of HVJ (Sendai virus) virions were solubilized with alkali-Emasol treatment. The solubilized envelope subunit(s) associated with hemagglutination-inhibiting antibody blocking, neuraminidase, and low hemagglutinating (HA) activities had a sedimentation coefficient of 8.8S. Envelope fragment-like structures were assembled from the solubilized subunits after Emasol was removed by gel filtration. These reassembled envelope particles with HA activity had cell-fusion activity as well as hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reassembled particles revealed that they mainly consisted of two kinds of polypeptides.

Cell type-specific enhancement of hepatitis C virus internal ribosome entry site-directed translation due to 5' nontranslated region substitutions selected during passage of virus in lymphoblastoid cells.

Low-level replication of hepatitis C virus (HCV) in cultured lymphoblastoid cells inoculated with H77 serum inoculum led to the appearance of new virus variants containing identical substitutions at three sites within the viral 5' nontranslated RNA (5'NTR): G(107)-->A, C(204)-->A, and G(243)-->A (N. Nakajima, M. Hijikata, H. Yoshikura, and Y. K. Shimizu, J. Virol. 70:3325-3329, 1996). These results suggest that virus with this 5'NTR sequence may have a greater capacity for replication in such cells, possibly due to more efficient cap-independent translation, since these nucleotide substitutions reside within the viral internal ribosome entry site (IRES). To test this hypothesis, we examined the translation of dicistronic RNAs containing upstream and downstream reporter sequences (Renilla and firefly luciferases, respectively) separated by IRES sequences containing different combinations of these substitutions. The activity of the IRES was assessed by determining the relative firefly and Renilla luciferase activities expressed in transfected cells. Compared with the IRES present in the dominant H77 quasispecies, an IRES containing all three nucleotide substitutions had significantly greater translational activity in three of five human lymphoblastoid cell lines (Raji, Bjab, and Molt4 but not Jurkat or HPBMa10-2 cells). In contrast, these substitutions did not enhance IRES activity in cell lines derived from monocytes or granulocytes (HL-60, KG-1, or THP-1) or hepatocytes (Huh-7) or in cell-free translation assays carried out with rabbit reticulocyte lysates. Each of the three substitutions was required for maximally increased translational activity in the lymphoblastoid cells. The 2- to 2.5-fold increase in translation observed with the modified IRES sequence may facilitate the replication of HCV, possibly accounting for differences in quasispecies variants recovered from liver tissue and peripheral blood mononuclear cells of the same patient.

Organ specificity of the antigens reacting with the 48-1 and S-1 antibodies in chimpanzees infected with hepatitis C virus.

48-1 and S-1 antibodies produced by lymphoblastoid cells transformed with Epstein-Barr virus were reported to be associated with infection by not only the hepatitis non-A, non-B (NANB) virus but also hepatitis delta virus. Appearance of the antigens reacting with these antibodies in the liver of chimpanzees was recently found to be a host response to alpha-interferon induced by infections of both viruses. To investigate organ specificity of these antigens, various organs obtained from chimpanzees with hepatitis C (NANB) were examined. In addition to the liver, the adrenals and spleen were found to be positive by immunofluorescence. The positive reactions of these three organs were also confirmed by radioimmunoassay. By electron microscopy, microtubular aggregates similar to those observed in the liver were detected in the adrenals, but not in the spleen. The results suggested that these antigens existed in the liver, adrenal, and probably spleen of chimpanzees infected with hepatitis C.

Hepatitis C virus: detection of intracellular virus particles by electron microscopy.

We previously demonstrated that a human T-cell line, HPBMa10-2 derived from HPBALL, was capable of supporting a productive infection of hepatitis C virus (HCV). We subsequently found Daudi cells, a human B-cell line, to be susceptible to HCV infection. Employing these cell lines infected with HCV as well as liver obtained during the acute phase of hepatitis C from a chimpanzee, we observed intracellular HCV particles by electron microscopy (EM). We detected viruslike particles with a diameter of approximately 50 nm in the cytoplasmic vesicles. In Daudi cells harvested 15 days after virus inoculation, the appearance of cytoplasmic tubular structures which we originally described for chimpanzee hepatocytes in association with HCV infection was noted. Immunoperoxidase EM using antibodies against HCV core and envelope demonstrated that the particles contained the viral antigens.

Replication of GB virus C (hepatitis G virus) in interferon-resistant Daudi cells.

We previously reported that Daudi cells, a Burkitt's lymphoma cell line, were capable of supporting productive infection of hepatitis C virus (HCV). During continual cultivation after HCV infection, the culture became resistant to interferons (IFNs). This resistant cell line, coded as H-903, was used as host cells for replication of GB virus C (GBV-C), also known as hepatitis G virus. GBV-C RNA was detected in the culture by reverse transcription-PCR for more than 130 days after inoculation, while it was detected for 44 days but not later in the parental IFN-sensitive Daudi cells. Productive infection of GBV-C in the H-903 system was confirmed by serially inoculating supernatants from infected cultures into uninfected cells. The viral E2 antigen was detected by immunofluorescence in the cells inoculated with the fifth passage of GBV-C. The presumed capsid-coding region of the viral genome in the inoculum, in the serially passaged virus, or in the virus produced by a long-term culture was only 16 amino acids long, suggesting that the GBV-C with a short core sequence was replication competent.

Evidence for in vitro replication of hepatitis C virus genome in a human T-cell line.

A human T-cell line, MOLT-4, either uninfected or infected with murine retroviruses, was tested for its susceptibility to hepatitis C virus (HCV) infection. The cell cultures were inoculated with a serum containing HCV and then examined for the presence of viral sequences by cDNA/PCR. In murine retrovirus-infected MOLT-4 (MOLT-4 Ma) cells, intracellular minus-strand viral RNA, a putative replication intermediate, was first detected 3 days after inoculation, and the maximum signal was seen on day 7. When the cells were continuously subcultured in fresh medium, HCV sequences were intermittently detected in cells over a period of 3 weeks. In MOLT-4 cells free of retroviruses, replication of minus-strand HCV RNA appeared less efficient than in MOLT-4 Ma cells. The presence of minus-strand viral RNA in MOLT-4 Ma cells inoculated with HCV was confirmed by in situ hybridization with a strand-specific RNA probe. Immunofluorescence tests with antibodies specific for HCV core and NS4 antigens showed that MOLT-4 Ma cells were positive for viral antigen 7 days after inoculation. Thus, it appears likely that the HCV genome can replicate in the human T-cell line MOLT-4.

Further studies of 48-1 antigen in serial liver biopsies from chimpanzees infected with hepatitis delta virus.

The 48-1 antibody, initially reported to react specifically with non-A, non-B infected liver tissue, has been found to react also with liver specimens from chimpanzees infected with hepatitis delta virus (HDV). To clarify further the relation between HDV and appearance of the antigen reacting with the 48-1 antibody (48-1 Ag), immunoperoxidase studies were carried out on serial liver specimens from chimpanzees infected with HDV. Immunohistochemical and serological findings suggested that the appearance of 48-1 Ag paralleled that of HDV. Double immunoperoxidase staining revealed HDAg in the nucleus and 48-1 Ag in the cytoplasm of the same hepatocytes as well as in different hepatocytes separately. The course of appearance of microtubular aggregates paralleled that of 48-1 Ag. The present results suggested that expression of 48-1 Ag was related to infection with HDV, probably because expression of this antigen is induced from the host genome.

Neutralizing antibodies against hepatitis C virus and the emergence of neutralization escape mutant viruses.

We developed an in vitro assay for antibodies to hepatitis C virus (HCV) that bind to virions and prevent initiation of the replication cycle in susceptible cells in vitro. These antibodies therefore appear to be capable of neutralizing the virus. Using this assay and a standard inoculum of HCV of known infectivity, we have measured the antibody in serial serum samples obtained from the same chronically infected patient over 14 years following onset of his hepatitis. Such antibody was found in sera collected within 5 years of onset of hepatitis but not in later sera. In double immunoprecipitation experiments with anti-human immunoglobulin, the same sera that contained neutralizing antibody were found to contain antibody that bound to HCV to form antigen-antibody complexes immunoprecipitable with anti-human globulin. Similarly, plasma collected from this patient in 1990, 13 years after onset of hepatitis, and which contained HCV that had diverged genetically from the 1977 strain, did not contain antibody capable of neutralizing either the 1977 or the 1990 strain of HCV. However, plasma collected a year later (1991, 14 years after onset of hepatitis) contained neutralizing antibody to the 1990, but not the 1977, strain of HCV. These results suggest that HCV does induce antivirion antibody, as measured by blocking of initiation of the replication cycle of virus in cells and by the formation of immunoprecipitable antigen-antibody complexes but that these antibodies are isolate specific and change over time. Thus, these antivirion antibodies function as neutralizing antibodies and are probably in vitro correlates of the attempt of the host to contain the emergence of neutralization-resistant variants of HCV over time.