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.

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Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral hepatitis genome

Background Because many persons with chronic hepatitis C virus (HCV) infection are asymptomatic, population-based serologic studies are needed to estimate the prevalence of the infection and to develop and evaluate prevention efforts. Methods We performed tests for antibody to HCV (anti-HCV) on serum samples from 21,241 persons six years old or older who participated in the third National Health and Nutrition Examination Survey, conducted during 1988 through 1994. We determined the prevalence of HCV RNA by means of nucleic acid amplification and the genotype by means of sequencing. Results The overall prevalence of anti-HCV was 1.8 percent, corresponding to an estimated 3.9 million persons nationwide (95 percent confidence interval, 3.1 million to 4.8 million) with HCV infection. Sixty-five percent of the persons with HCV infection were 30 to 49 years old. Seventy-four percent were positive for HCV RNA, indicating that an estimated 2.7 million persons in the United States (95 percent confidence interval, ...

https://www.nejm.org/doi/pdf/10.1056/NEJM199908193410802

The Prevalence of Hepatitis C Virus Infection in the United States, 1988 through 1994

Hepatitis C virus (HCV) infects an estimated 170 million persons worldwide and thus represents a viral pandemic, one that is five times as widespread as infection with the human immunodeficiency virus type 1 (HIV-1). The institution of blood-screening measures in developed countries has decreased the risk of transfusion-associated hepatitis to a negligible level, but new cases continue to occur mainly as a result of injection-drug use and, to a lesser degree, through other means of percutaneous or mucous-membrane exposure. Progression to chronic disease occurs in the majority of HCV-infected persons, and infection with the virus has become the main indication . . .

https://www.nejm.org/doi/pdf/10.1056/NEJM200107053450107?listPDF=true

Hepatitis C Virus Infection

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 frame that could encode a viral polyprotein precursor of 3011 amino acids. While there as little overall amino acid and nucleotide sequence homology with other viruses, the 5' HCV nucleotide sequence upstream of this large open reading frame has substantial similarity 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 of a 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.

https://www.pnas.org/content/pnas/88/6/2451.full.pdf

Genetic organization and diversity of the hepatitis C virus.

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, ≥6.9 years; maximum, ≥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 ami...

Detection of antibody to hepatitis C virus in prospectively followed transfusion recipients with acute and chronic non-A, non-B hepatitis

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.

https://science.sciencemag.org/content/sci/244/4902/362.full.pdf

An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis

Hepatitis C virus (HCV) infection can be detected by using immunoassay techniques that measure reactivity to viral protein antigens. In this study seven discreet proteins derived from HCV genomic coding sequences have been expressed, purified and characterized. Six proteins represent the structural regions of the core (C22-3), the envelope (E1 and E2), and the non-structural regions NS3 (C33C), NS3–NS4 (C100-3) and NS5. The seventh, C25, is a chimeric fusion protein containing C33C, C100-3 and C22-3 regions. Using these recombinant proteins, multi-antigen radioimmunoassays and enzyme immunoassays (EIA) were designed. The fusion protein, C25, was demonstrated to be an improved antigen for serodiagnosis of HCV antibody. Use of the C25 protein accelerated HCV antibody detection by 3–46 weeks in non-A, non-B hepatitis seroconversion cases and significantly increased the rate of detection in a paid donor population by 20%. The C25 assay also demonstrated excellent specificity in 2446 randomly selected low prevalence samples. The repeated reactive rate in this group of samples was 0.5%.



Samples from volunteer blood donors pre-selected for repeat reactivity with the first generation C100-3-based HCV antibody tests (n= 175) were tested using the C25 assay. The C25 assay detected 37.7% samples as reactive and 53.1% samples as non-reactive. This result was in agreement with all other supplementary tests that include RIBATM, multi-antigen assay, Abbott neutralization and peptide assay. The other 9.2% samples were classified as ‘indeterminant’ because these samples were only partially in agreement with some of the above supplementary tests.



The C25 enzyme-linked immunosorbent assay (ELISA), with its improved assay sensitivity, can identify additional HCV antibody reactive cases in both hepatocellular carcinoma and cryptogenic cirrhosis patients. The C25 and multi-antigen EIA assays were used to investigate the vertical transmission of HCV. It was observed that these improved assays are able to detect antibodies in a vertical transmission case. The C25 ELISA was also compared with a synthetic peptide assay. The C25 assay was found to be superior to the peptide assay that performed poorly in the detection of the C33C only reactive samples.

Use of recombinant HCV antigen in the serodiagnosis of hepatitis C virus infection: Significant improvement in HCV antibody detection as compared with the first generation HCV C100-3 ELISA and the synthetic peptide EIA tests

K. Berger

Papers

An assay for circulating antibodies to a major etiologic virus of human non-A, non-B hepatitis

Use of recombinant HCV antigen in the serodiagnosis of hepatitis C virus infection: Significant improvement in HCV antibody detection as compared with the first generation HCV C100-3 ELISA and the synthetic peptide EIA tests