A random-primed complementary DNA library was constructed from plasma containing the uncharacterized non-A, non-B hepatitis (NANBH) agent and screened with serum from a patient diagnosed with NANBH. A complementary DNA clone was isolated that was shown to encode an antigen associated specifically with NANBH infections. This clone is not derived from host DNA but from an RNA molecule present in NANBH infections that consists of at least 10,000 nucleotides and that is positive-stranded with respect to the encoded NANBH antigen. These data indicate that this clone is derived from the genome of the NANBH agent and are consistent with the agent being similar to the togaviridae or flaviviridae. This molecular approach should be of great value in the isolation and characterization of other unidentified infectious agents.
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...
A specific assay has been developed for a blood-borne non-A, non-B hepatitis (NANBH) virus in which a polypeptide synthesized in recombinant yeast clones of the hepatitis C virus (HCV) is used to capture circulating viral antibodies. HCV antibodies were detected in six of seven human sera that were shown previously to transmit NANBH to chimpanzees. Assays of ten blood transfusions in the United States that resulted in chronic NANBH revealed that there was at least one positive blood donor in nine of these cases and that all ten recipients seroconverted during their illnesses. About 80 percent of chronic, post-transfusion NANBH (PT-NANBH) patients from Italy and Japan had circulating HCV antibody; a much lower frequency (15 percent) was observed in acute, resolving infections. In addition, 58 percent of NANBH patients from the United States with no identifiable source of parenteral exposure to the virus were also positive for HCV antibody. These data indicate that HCV is a major cause of NANBH throughout the world.
We measured antibody (anti-HCV) to hepatitis C virus, which causes non-A, non-B hepatitis, by radioimmunoassay in prospectively followed transfusion recipients and their donors. Of 15 patients with chronic non-A, non-B hepatitis documented by liver biopsy, all seroconverted for the antibody; of 5 with acute resolving non-A, non-B hepatitis, 3 (60 percent) seroconverted. The development of anti-HCV was delayed (mean delay, 21.9 weeks after transfusion, or 15 weeks after the onset of clinical hepatitis) and took approximately one year in one patient. Antibody has persisted in 14 of the 15 patients with chronic disease (mean follow-up, greater than or equal to 6.9 years; maximum, greater than or equal to 12), but has disappeared in the 3 with acute resolving disease after a mean of 4.1 years. Anti-HCV was detected in samples of donor serum given to 14 (88 percent) of the 16 anti-HCV-positive patients for whom all donor samples were available. Only 33 percent of the anti-HCV-positive donors tested had an elevated serum concentration of alanine aminotransferase; 54 percent were positive for antibody to the hepatitis B core antigen (anti-HBc). We conclude that hepatitis C virus is the predominant agent of transfusion-associated non-A, non-B hepatitis and that screening of donors for anti-HCV could prevent the majority of cases of the disease. "Surrogate" assays for anti-HBc and alanine aminotransferase would have detected approximately half the anti-HCV-positive donors involved in the transmission of hepatitis that we identified.
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