J Gen Virol

Wild-type levels of pregenomic RNA and replication but reduced pre-C RNA and e-antigen synthesis of hepatitis B virus with C(1653) --> T, A(1762) --> T and G(1764) --> A mutations in the core promoter.

Gunther S. Piwon N. Will H.
Heinrich-Pette-Institut fur Experimentelle Virologie und Immunologie an der Universitat Hamburg, Federal Republic of Germany. will@hpi.uni-hamburg.de
Hepatitis B virus (HBV) isolates with A-1762 to T and G-1764 to A mutations in the core promoter have been associated with active hepatitis, severe liver disease following liver transplantation, hepatocellular carcinoma and acute fulminant courses--in the latter case combined with a C-1653 to T mutation. In this study, a mutant core promoter region containing the T-1653, T-1762 and A-1764 mutations was placed into the context of a wild-type HBV genome and analysed by transfection. The mutations reduced the level of pre-C mRNA (by 55%) and e-antigen secretion. In contrast, no significant effects on the levels of pregenome/C and pre-S/S mRNAs, intracellular core, polymerase, and pre-S /S2 proteins and secreted S-antigen were observed. The amount of progeny virus DNA in the cells and in the culture medium was increased marginally, if at all.

The sequence and phylogenetic analysis of a novel hepatitis E virus isolated from a patient with acute hepatitis reported in the United States.

Year 1998
Schlauder GG. Dawson GJ. Erker JC. Kwo PY. Knigge MF. Smalley DL. Rosenblatt JE. Desai SM. Mushahwar IK.
Abbott Laboratories, Virus Discovery Group, Experimental Biology Research, North Chicago, IL 60064, USA. george.schlauder@add.ssw.abbott.com
A variant of hepatitis E virus (HEV), designated HEV US-1, was identified in a hepatitis patient in the United States (US); the patient had no history of travel to areas where HEV is endemic. Nucleotide sequences were obtained from the 5' end of open reading frame (ORF) 1 (1418 nt), the 3' end of ORF1 (1359 nt), the entire ORF2 and ORF3 regions, and the 3'-untranslated region (2127 nt). The HEV US-1 strain is significantly divergent from other human HEV isolates with nucleotide identities ranging from 76.8 to 77.5%. Phylogenetic analyses indicate that HEV US-1 and a recently discovered HEV variant from swine may represent separate isolates of a new strain of HEV, significantly divergent from the Mexican and Burmese strains. Synthetic peptides derived from the carboxyl amino acids of ORF2 and ORF3 were shown to be useful for detecting exposure to HEV. In addition, IgM class antibodies directed against HEV US-1 synthetic peptides were detected in the US patient infected with HEV US-1, but were absent using synthetic peptides from the Burmese or Mexican strains of HEV. A preferential reactivity to HEV US-1 specific peptides has lead to the identification of a second isolate of this virus also from a patient with acute hepatitis from the US. The discovery of these HEV variants may be important in understanding the worldwide distribution of HEV infection.

Extensive analysis of duplicated-inverted hepatitis B virus integrations in human hepatocellular carcinoma.

Year 1998
Pineau P. Marchio A. Mattei MG. Kim WH. Youn JK. Tiollais P. Dejean A.
Unite de Recombinaison et Expression Genetique, INSERM U163, Institut Pasteur, Paris, France.
Hepatitis B virus (HBV) DNA is found chromosomally integrated into the genome of the majority of hepatocellular carcinomas (HCC) arising in chronic HBV carriers suggesting that, in some instances, viral sequences may be directly responsible for oncogenic conversion. In an attempt to clarify the oncogenic potential of integrated HBV sequences, we performed an extensive analysis of two single integrations present in HCC which developed in non-cirrhotic livers from HBsAg-positive Korean patients. In both cases, integrated viral sequences were characterized by a duplicated-inverted configuration involving the flanking cellular sequences, a pattern consistently found in many amplicons isolated from mammalian cells. Integration sites are characterized by an AT-rich content and the presence of topoisomerase I and II cleavage target sequences as well as other recombination-prone motifs. The chromosomal locations of the integration sites were determined as 8q13 and 10q22 in the human genome, two regions known to harbour genes involved in tumorigenesis. The cis-activating potential of the integrations in their original configuration was also investigated in a transient transfection assay in HepG2 cells. Integrated sequences, rather than activating heterologous promoters, show either no activity or a weak tendency to inhibit activation of neighbouring reporter genes. The implications of our findings for the understanding of primary liver cancer development are discussed.

Low level or absent in vivo replication of hepatitis C virus and hepatitis G virus/GB virus C in peripheral blood mononuclear cells.

Year 1998
Mellor J. Haydon G. Blair C. Livingstone W. Simmonds P.
Department of Medical Microbiology, University of Edinburgh, UK.
To investigate which subsets of peripheral blood mononuclear cells (PBMCs) are susceptible to infection with hepatitis C virus (HCV) and hepatitis G virus (HGV) or GB virus C (GBV-C), a PCR-based assay using tagged primers in the core region (HCV) and NS3 region (HGV/GBV-C) for the specific detection of negative strand (replicating) viral RNA sequences was developed. In liver biopsy samples both positive and negative strands of HCV RNA were detected, at levels ranging from 3 to 11 x 10(6) RNA copies per 10(6) cells and 3.7-4.2 x 10(3) copies per 10(6) cells respectively, while lower frequencies of positive strands of GBV-C/HGV RNA were detected (from 13 biopsies, the highest frequency was 7.3 x 10(3) per 10(6) cells). In no samples were negative RNA strands detected. To investigate extra-hepatic replication of HCV and GBV-C/HGV, CD4+, CD8+ and B lymphocytes, monocytes and putative dendritic cell populations were separated from PBMCs from ten study subjects. Detection of positive strand HCV RNA was largely confined to B lymphocytes (at levels of up to 5 x 10(3) copies per 10(6) cells), while detection of negative strands was confined to a single subset (dendritic cells) of one of the study individuals. Similarly, GBV-C/HGV was detected at low levels in only twelve of twenty PBMC samples, while negative strands were uniformly absent. The low levels of HCV and GBV-C/HGV RNA in PBMCs suggest that these cells are at most a minor reservoir for virus replication. The absence of detectable replication of GBV-C/HGV suggests that the actual site of GBV-C/HGV replication remains to be discovered.

Herpes simplex virus hepatitis in macrophage-depleted mice: the role of massive, apoptotic cell death in pathogenesis.

Year 1998
Irie H. Koyama H. Kubo H. Fukuda A. Aita K. Koike T. Yoshimura A. Yoshida T. Shiga J. Hill T.
Department of Pathology, Teikyo University School of Medicine, Tokyo, Japan. h-irie@rb3.so-net.or.jp
Infection with herpes simplex virus or hepatitis viruses can lead to fulminant hepatitis, but there is controversy about the underlying conditions needed for such disease. To investigate how the impairment of host defences might be involved, macrophages were depleted by administration of silica to mice before intravenous injection with herpes simplex virus type 1 (HSV-1). Such mice died rapidly and their livers were yellowish and shrunken (acute yellow atrophy), and occasionally grossly haemorrhagic. Small foci of apoptotic cells developed in the liver lobules; these rapidly became confluent and zonal over time. The overall lesion pattern was similar to massive hepatic necrosis, and there was extensive HSV replication in the liver lesions. In the liver, DNA fragmentation characteristic of apoptosis followed the time course of HSV-1 propagation. These findings suggest that one of the underlying conditions for fulminant viral hepatitis may be inadequate macrophage response, and that the massive hepatic damage, often defined as cell necrosis, may actually be apoptosis of liver cells subsequent to virus infection.

Infection of a chimpanzee with hepatitis C virus grown in cell culture.

Year 1998
Shimizu YK. Igarashi H. Kiyohara T. Shapiro M. Wong DC. Purcell RH. Yoshikura H.
Department of Hepatitis Virology (SKBB), Faculty of Medicine, University of Tokyo, Japan. yks@m.u-tokyo.ac.jp
Culture supernatant harvested from Daudi cells, a lymphoplastoid cell line, after 58 days of infection with the H77 strain of hepatitis C virus (HCV), was inoculated into a chimpanzee. HCV RNA, as detected by RT-PCR, first appeared in the serum and liver 5 and 6 weeks, respectively, after inoculation. Peripheral blood mononuclear cells (PBMC) collected on week 7 were also positive for HCV RNA. The major sequences of hypervariable region 1 (HVR1) of the viral genome recovered from the inoculated chimpanzee were the ones which were the majority in the original H77 inoculum and not those which were in the majority in the culture supernatant. Only the sequence recovered from PBMC was the same as the major one found in the cell culture.