The nucleotide sequence of the RNA genome of the human hepatitis C virus (HCV) has been determined from overlapping cDNA clones. The sequence (9379 nucleotides) has a single large open reading frme that could encode a viral polyprotein precursor of 3011 amino acids. While there is little overall amino acid and nucleotide sequence homology with other viruses, the 5' HCV nucleotide sequence upstream ofthis large open reading frame has substantial simiarit to the 5' termini of pestiviral genomes. The polyprotein also has significant sequence similarity to helicases encoded by animal pestiviruses, plant potyviruses, and human flaviviruses, and it contains sequence motifs widely conserved among viral replicases and trypsin-like proteases. A basic, presumed nucleocapsid domain is located at the N terminus upstream ofa region containing numerous potential N-linked glycosylation sites. These HCV domains are located in the same relative position as observed in the pestiviruses and flaviviruses and the hydrophobic profiles of all three viral polyproteins are similar. These combined data indicate that HCV is an unusual virus that is most related to the pestiviruses. Significant genome diversity is apparent within the putative 5' structural gene region of different HCV isolates, suggesting the presence of closely related but distinct viral genotypes.A recombinant immunoscreening approach has recently been used to isolate a cDNA clone (5-1-1) from the genome of an infectious human hepatitis agent that is immunologically unrelated to the hepatitis A and B viruses (1). Clone 5-1-1 and overlapping clones hybridized to a single-stranded RNA molecule that was present in infectious plasma and that encodes immunological epitopes that cross-react in non-A, non-B hepatitis (NANBH) cases from around the world (1, 2).Termed the hepatitis C virus (HCV), this agent appears to be the major cause of posttransfusion and sporadic NANBH worldwide and plays a major role in the development of chronic liver disease including hepatocellular carcinoma (ref.2; for review, see ref.3). We now report the nucleotide sequence of the HCV genome and we discuss its genetic organization and diversity. § MATERIALS AND METHODSThe nucleotide sequence was deduced from a large series of overlapping cDNA clones (150-800 base pairs) derived from the same random-primed Agtll cDNA library used to isolate clone 5-1-1 (1). The source of virus was a plasma pool derived from a chimpanzee with a chronic NANBH infection. This animal represented the second passage of the agent contaminating a human factor VIII concentrate (4). Based initially on the sequence of clone 5-1-1, 32P-labeled synthetic oligonucleotides (30-mers) were used as hybridization probes (5) to isolate cDNA clones overlapping with both termini of clone 5-1-1. Each successive cycle of this repetitive "walking" process usually involved the sequencing of at least 6 different cDNAs from each end. Each Agtll cDNA clone was sequenced on both strands after subcloning the EcoRI cDNA insert into bacterioph...

Hepatitis

No abstract

Broad, multispecific CD4؉ and CD8 ؉ T-cell responses to the hepatitis C virus (HCV), as well as viruscross-neutralizing antibodies, are associated with recovery from acute infection and may also be associated in chronic HCV patients with a favorable response to antiviral treatment. and CD8؉ T-cell responses to E1E2 and NS345 was obtained by first priming with Th1-adjuvanted proteins and then boosting with chimeric, defective alphaviruses expressing these HCV genes. In addition, this prime/ boost regimen resulted in the induction of anti-E1E2 antibodies capable of cross-neutralizing heterologous HCV isolates in vitro. This vaccine formulation and regimen may therefore be optimal in humans for protection against this highly heterogeneous global pathogen.

The inhibitory receptor programmed death-1 (PD-1) is present on CD8؉ T cells in chronic hepatitis C virus (HCV), but expression patterns in spontaneously resolving infections are incompletely characterized. Here we report that PD-1 was usually absent on memory CD8؉ T cells from chimpanzees with resolved infections, but sustained low-level expression was sometimes observed in the absence of apparent virus replication. PD-1-positive memory T cells expanded and displayed antiviral activity upon reinfection with HCV, indicating conserved function. This animal model should facilitate studies of whether PD-1 differentially influences effector and memory T-cell function in resolved versus persistent human infections.The hepatitis C virus (HCV) is a major etiological agent of hepatic cirrhosis and hepatocellular carcinoma (3). Although the mechanism(s) facilitating chronic infection is not fully understood, HCV-specific cytotoxic T lymphocytes (CTLs) that persist during the chronic phase of infection have an immature phenotype (1), impaired proliferative capacity, and reduced cytotoxic function and cytokine secretion (9,16,19,20). It is therefore probable that the dysfunction of HCV-specific CD8 ϩ T cells plays a role in HCV persistence.Much recent interest has centered upon the inhibitory T-cell receptor programmed death-1 (PD-1) as a marker and mediator of impaired CTL function in persistent viral infections. Studies in lymphocytic choriomeningitis virus (LCMV)-infected mice have demonstrated that in persistent infection virus-specific T cells express PD-1, while in resolved infection, LCMV-specific cells lose expression of this molecule (2). In addition, the in vivo blockade of PD-1 interaction with one of its ligands, programmed death-ligand 1 (PD-L1), improved proliferation and cytokine secretion by LCMV-specific CD8 ϩ T cells and reduced viral titers in persistently infected mice (2). More recently, studies have shown that in human immunodeficiency virus (HIV)-infected individuals, HIV-specific T cells express 12,17) and that the levels of expression of this molecule correlate with viremia and with CD4 ϩ T-cell count (4, 17). HCV-specific CD8 ϩ T cells from the blood and liver of humans with chronic hepatitis C also express PD-1, and the in vitro blockade of PD-1-PD-L1 interaction enhanced antigendriven proliferation and cytokine secretion (8,11,13,18). Furthermore, in one study of acute human infection, the loss of PD-1 expression by HCV-specific CD8 ϩ T cells was shown to correlate with viral clearance (18). These data support the hypothesis that the PD-1-positive phenotype is associated with the impairment of virus-specific CD8 ϩ T-cell function, and as such, PD-1 expression is a correlate of the outcome of HCV infection.The goal of this study was to further evaluate the relationship between PD-1 expression and virus persistence, using the only animal model of human HCV infection. We analyzed the phenotype of HCV-specific CD8 ϩ T cells in chimpanzees that had spontaneously resolved HCV infection 5 to 7 years pr...