Hepatitis C: looking at a virus that hasn't been seen.

Hepatitis C (HCV) is the first virus to be discovered by molecular cloning without direct use of biological or biophysical methods. HCV was first recognised in 1974 as non-A, non-B (NANB) hepatitis resulting from blood transfusions. It took almost 15 years to identify it successfully--by detecting a clone in a library of cDNA prepared from the nucleic acids extracted from plasma known to be infectious for chimpanzees. The clone was derived from a positive-sense RNA of about 10,000 nucleotides, with a long open reading frame encoding for a polyprotein of about 3000 amino acids. The structure of the RNA and the encoded polyprotein had properties similar to known flaviviruses and pestiviruses. Functions of viral proteins produced by proteolytic cleavage of the polyprotein are estimated by analogy with known viruses of similar genomic organisation. Each of the HCV proteins has been produced in recombinant organisms and used as an antigen in immunoassays to investigate serological responses during the course of infection. Seroconversions to both structural and non-structural antigens are observed during the course of disease but typical diagnostic serological patterns have not yet evolved. Immunoassays for HCV antibodies reacting with recombinant antigens are used widely for screening blood donations and for studying the epidemiology of HCV infection. Comparisons of the nucleic acid sequences from different isolates of HCV have shown considerable variability throughout the genome. The importance of this genomic heterogeneity will be a challenging problem for the future.

Hepatitis C virus: the major causative agent of viral non-A, non-B hepatitis.

A 'blind' recombinant immunoscreening approach, of general application to studies of infectious diseases, was used to clone and identify the genome of the previously uncharacterized non-A, non-B hepatitis (NANB) virus. This agent is a positive-stranded RNA virus that appears to be distantly related to the flaviviridae family. A recombinant viral antigen (C100-3) was used to develop a capture assay for circulating antibody. Data obtained using this assay indicate that this agent, termed the hepatitis C virus (HCV), is the major cause of post-transfusion, community-acquired and cryptogenic, NANB. Anti-C100-3 antibody appears to be directed towards dominant, non-structural viral epitopes. It is a non-neutralising antibody that develops generally late in infection and is a particularly good marker of chronic, persistent viraemia. Many asymptomatic but infectious blood donors can now be detected using this antibody assay. HCV is associated with the development of hepatocellular carcinoma and possibly, other liver diseases.

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

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.

Science and technology in mitigating AIDS.

The workshop emphasized contributions of genetic engineering in providing reagents and techniques for diagnosing and monitoring HIV infections. Despite a variety of tests for specific antibodies, virus antigens and nucleic acids no consistent serum profile has emerged that correlates well with virus life cycle or clinical course. HIV infections are a great deal more complex than hepatitis B infections where diagnosis and prognosis are very accurately based on serologic profiles. HIV generates at least 15 virus proteins all of which are immunogenic. The workshop emphasized studies on immunogenicity of the proteins. Full understanding of the virus life cycle and the clinical course of disease may require in-depth studies of the production and immunogenicity of the virus-directed catalytic and regulatory gene products. Fortunately, all of these reagents can be produced through biotechnology. The new techniques described in the workshop will allow expanded studies to proceed rapidly.

A specific immune response to purified HA antigen (HAAg) demonstrated by leukocyte migration inhibition in patients recovering from viral hepatitis A.

The mechanism responsible for liver cell necrosis in patients with hepatitis A is not known. Since the type of hepatic lesions and the clinical presentation of acute hepatitis B are similar and are probably related to the cell-mediated immune response to a viral antigen located in the liver cell, it is possible that a similar mechanism is involved in hepatitis A. In the present paper, immune reactivity to hepatitis A antigen (HAAg) was measured in 13 patients at the time of recovery from hepatitis A, by using the leukocyte migration inhibition test (LMIT) under agarose with purified HAAg as antigen. Eleven normal subjects without history of hepatitis and 4 patients convalescent from hepatitis B were used as controls. Inhibition of leukocyte migration by HAAg was found in 11 of the 13 patients, with an average migration index (MI) of 77.0% (SEM 3.5). No such inhibition was found in any of the controls: MI = 100.8% (SEM 1.0), P less than 0.0001. These findings show that, like for HBsAg in hepatitis B, an immune response specific for HAAg can be demonstrated by the LMIT after HAV infection. This response could perhaps be related to the liver injury associated to hepatitis A.

Challenges and opportunities in biotechnology.

The biotechnology industry is thriving, and many predicted accomplishments have actually occurred during the last decade. Cloning and expression of genetic information is now simple and routine. Initial commercial products have been realized, but there is much yet to be accomplished in evaluating the clinical significance of many other gene products made available by biotechnology resources. During the next decade, human health care and the pharmaceutical industry should be affected substantially by first- and second-generation recombinant DNA products. Recombinant vaccines, blood coagulation factors, and known biological modulators produced by rDNA technologies should be widely used. Further opportunities will be realized with increasing discoveries of new bioactive molecules and identification of NANB hepatitis and AIDS infectious agents. Full exploitation of health care products will depend on innovative new delivery systems or the ability to reconstruct mammalian and plant genes, providing for in-situ delivery of the necessary gene products.

Hepatitis B surface antigen (HBsAg) subtype-specific antibodies in persons vaccinated against hepatitis B.

One hundred sixty-three persons immunised against hepatitis B with a vaccine containing HBsAg either of adw or ayw subtype were examined for antibodies against the a, d, and y determinants of HBsAg. Sera were tested for antibodies against HBsAg adw and HBsAg ayw separately by a solid-phase radioimmunoassay using polystyrene beads coated with HBsAg of either adw or ayw subtype, and the relative amounts of antibodies against the single determinants were calculated. After the third immunisation, all vaccinees had antibodies against the common determinant a. A quantitative evaluation showed that on average about 50% of HBsAg-specific antibodies were directed against the a determinant, and about 50% against d or y, respectively. However, as only anti-a is protective against cross-infection with other HBsAg subtypes, the degree of immunity of a person vaccinated against hepatitis B should be evaluated by the determination of antibodies to a rather than antibodies against total HBsAg.

Non-A, non-B hepatitis: a prospective study of a hemodialysis outbreak with evaluation of a serologic marker in patients and staff.

An outbreak of non-A, non-B hepatitis (NANBH) in a hemodialysis unit was prospectively studied and the clinical, biochemical, and serologic events were correlated with an experimental immunodiffusion assay for serum antigen and antibody. One hundred sixteen subjects (76 dialysis patients and 40 staff members) were studied over an 8-month period. Hepatitis was defined as two consecutive SGPT levels greater than two times the upper limit of normal occurring in two separate samples drawn greater than 7 days apart in the absence of other likely causes of liver disease. Weekly serum specimens were obtained and tested for SGPT, SGOT, alkaline phosphatase, bilirubin HBsAg, anti-HBc, anti-HBs, total anti-HAV, and anti-HAV IgM by commercial reagents, and for antigen and antibody by agar gel diffusion using reference reagents previously obtained from well-documented posttransfusion NANBH patients. Clinical evaluations were performed three times per week. Thirty patients and none of the staff developed NANBH. The NANBH patients were asymptomatic, except for two patients with jaundice. Fifteen of the 30 patients were positive for antigen which was detectable in at least one serum collected during the acute phase. Six patients and 10 staff without clinical NANBH or abnormal serology had antigen. Antigenemia was also observed in three patients with acute hepatitis B, with chronic hepatitis B in one patient and with alcoholic hepatitis in one patient. Thus, an antigen was detected in a high proportion of patients during the acute phase of NANBH, and it was also found in exposed patients who had other liver diseases.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-hepatitis B core immunoglobulin M in the serologic evaluation of hepatitis B virus infection and simultaneous infection with type B, delta agent, and non-A, non-B viruses.

The clinical value of an enzyme-linked immunosorbent assay for the detection of immunoglobulin M (IgM) antibody to hepatitis B core antigen (anti-HBc IgM) was evaluated by testing serum samples from the following groups of patients: (a) 27 individuals who had been diagnosed as having acute hepatitis B virus (HBV) infection, (b) 29 hepatitis B surface antigen (HBsAg) carriers, (c) 6 subjects with acute non-B hepatitis, and (d) 10 HBsAg-negative but anti-HBc-positive subjects who were suspected of being index cases for the intimate transmission of HBV. Whereas 24 of the 27 individuals with presumed acute HBV infection exhibited anti-HBc IgM, only 2 of 29 HBsAg carriers were found to be positive. Hepatitis B surface antigen persisted during an 8-mo observation period in 3 anti-HBc IgM-negative subjects with acute HBsAg-positive hepatitis. Before anti-HBc IgM testing, it was considered that these cases had evolved to the HBsAg carrier state. However, the regular demonstration of anti-HBc IgM in acute type B hepatitis, as well as the failure to detect this antibody in the majority of HBsAg carriers, led to reclassification of these cases as probable instances of acute non-A, non-B or delta-agent hepatitis superimposed on the HBsAg carrier state. Through additional testing, the diagnosis of non-A, non-B (NANB) infection was confirmed in 2 of these cases, and delta-agent infection was identified in the third. None of the non-B hepatitis cases exhibited anti-HBc IgM. However, 5 of the 10 suspected type B index cases were anti-HBc IgM-positive, indicating that they were very recently infected and most likely had infected their cohabiting sexual partners. The results from this study indicate that testing for anti-HBc IgM may improve serodiagnostic accuracy when acute NANB and delta-agent hepatitis occur in previously unrecognized HBsAg carriers. Moreover, it may be a useful test in defining potential high risk sources of exposure to HBV.

Serodiagnosis of recent hepatitis B infection by IgM class anti-HBc.

The time sequence, relative reactivity, and persistence of anti-HBc IgM were assessed in patients with HBsAg-positive viral hepatitis. A solid-phase immunoassay was developed using the IgM capture procedure with anti-mu-coated polystyrene beads. HBcAg was purified from serum Dane particles and used as a probe with 125I-labeled anti-HBc IgG. This immunoassay exhibited a pronounced prozoning phenomenon, and relative titers of sera differed widely depending upon the dilution of serum tested. When all sera were tested at 1:5,000 dilution, results were comparable in different patient groups. Anti-HBc IgM persisted at detectable levels for up to 2 years, and it was necessary to establish relative titers to discriminate current from remote infections. A cut-off assay value was established, and in 12 cases of acute hepatitis B virus (HBV) infection, antibody exceeded this value for about 6 months after onset of HBs antigenemia. A similar profile of anti-HBc IgM persistence was observed in seven patients who developed an HBsAg chronic carrier state. Long-term viral replication did not sustain elevated IgM class-specific antibody levels. The studies suggest that anti-HBc IgM analyses may be useful for differentiating recent and current HBV infections from remote infections, eliminating HBV as the agent for non-A, non-B hepatitis in asymptomatic HBsAg carriers, and detecting HBV as the etiologic agent during silent (HBsAg negative) infections.

Reexamination of hepatitis B virus subtypes and e-antigen expression by radioimmunoassays.

Radioimmunoassay methods were used to determine both the hepatitis B virus (HBV) subtype and hepatitis B e antigen (HBeAg) and antibody (anti-HBe) status of a group of hepatitis B surface antigen (HBsAg)-positive blood donors. The study involved sera containing HBV of the three major occidental subtypes, adw2, ayw3, and ayw2. The previously reported association of the y-type virus with HBeAg and the d types with anti-HBe was again observed. However, when the two y subgroups, ayw2 and ayw3, were considered individually, it was evident that the ayw3 specimens alone accounted for the association with HBeAg while the ayw2 sera were strongly associated with anti-HBe. The study also indicated that the prevalence of HBeAg declined and that of anti-HBe increased progressively with advancing age. On the average, ayw2 donors were significantly older than the adw2 donors, and donors from both of these groups were older than the ayw3 donors. It is postulated that the observed age differences account, at least in part, for the differing prevalence of e markers in the three HBV subtype groups, and that these age differences, in turn, may reflect a tendency for infections with the ayw2 viral strain to persist longer than adw2 infections, and both of these longer than ayw3 infections. Alternately, the three subtypes may represent epidemiologic shifts from remote ayw2 and adw2 infections to more recent ayw3 infections.

Interpretation of various serological profiles of hepatitis B virus infection.

Serial serum specimens from 149 patients with clinically diagnosed hepatitis were tested for five hepatitis B serological markers: hepatitis B surface antigen and its antibody (anti-HBs); hepatitis B e-antigen and its antibody (anti-HBe); and antibody to hepatitis B core antigen (anti-HBc). The times of appearance, disappearance, and persistence of these markers were used to differentiate various serological profiles obtained from the study. Four distinctive profiles were found to be associated with acute hepatitis B followed by recovery, and three with chronic hepatitis. These serologic profiles were assessed as diagnostic and prognostic guides for clinical management of the disease.

Radioimmunoassay for detection of hepatitis B e antigen and its antibody. Results of clinical evaluation.

The performance of a solid phase radioimmunoassay (ABBOTT-HBe) for the detection of hepatitis B e antigen (HBeAg) and its antibody anti-HBe was evaluated in clinical studies. The reagents and procedure were found to be reproducible by seven investigators, and lab-to-lab variations were minimal. In HBsAg positive sera, ABBOTT-HBe detected HBeAg or anti-HBe in 90% of the specimens compared to 50% tested by immunodiffusion. During the early acute stage of viral infection, serum HBeAg coincided with the rise and decline of HBsAg and seroconversion to anti-HBe occurred most often prior to loss of HBsAg. Patients with clinical evidence of chronic liver disease showed persistence of HBsAg and HBeAg. Vertical transmission studies of hepatitis B virus infection from mother to newborns, showed that mothers whose sera were positive for both HBsAg and HBeAg resulted in a greater incidence of transmission of hepatitis B virus to their offspring than mothers whose sera were HBsAg positive but HBeAg negative.

An enzyme immunoassay for hepatitis B e-antigen and antibody.

A solid-phase enzyme-linked immunoassay for the detection of hepatitis B e-antigen (HBeAg) and antibody (anti-HBe) was developed and compared with rheophoresis and radioimmunoassay (RIA). The enzyme-immunoassay (EIA) was similar to RIA in sensitivity and was approximately 1000-fold more sensitive than rheophoresis for HBeAg, and approximately 6000-fold more sensitive than rheophoresis for anti-HBe.

Diagnosis of acute hepatitis A by HAVAB-M, a direct radioimmunoassay for IgM anti-HAV.

A three-step solid-phase radioimmunoassay, HAVAB-M, was developed for use in clinical labs as an aid to diagnosing hepatitis A. Polystyrene beads were coated with anti-human IgM (mu-chain specific) and were incubated successively with serum specimen, HAV, and anti-HAV 125I. HAVAB-M was used to assay sera from patients with hepatitis A and was found to have high sensitivity for the IgM antibody to HAV. The antibody was detectable within a few days of onset of symptoms of hepatitis, and it reached maximum concentrations within one to three weeks. The test was designed so that most patients' sera would become negative approximately three months after onset. HAVAB-M was shown to be specific for IgM antibody, with virtually no detection of IgG anti-HAV. The test showed no interference fro rheumatoid factor and no cross-reactivity with sera from patients with hepatitis B or other infectious diseases.

Properties of soluble DNA polymerase from sera of hepatitis B virus carriers.

A soluble DNA polymerase was purified 8,000-fold from hepatitis B surface antigen positive serum. The molecular weight of the enzyme by gel filtration was about 1.60 X 10(5), the sedimentation coefficient was 5.5S, the apparent Km for dTTP was 4 micrometer, the optimum pH in the presence of Mg2+ was 9.2, and the pl was 4.7. The enzyme was found in HBsAg-positive sera and required an external primer for activity. The properties of the DNA polymerase were different from hepatitis B virus particle enzyme and from vertebrate and bacterial DNA polymerases. The prevalence of this enzyme did not correlate with HBeAg or particle DNA polymerase in HBsAg-positive sera.

Non-A/non-B hepatitis in experimentally infected chimpanzees: cross-challenge and electron microscopic studies.

Inoculation of eight chimpanzees with factor VIII, factor IX, or "H" strain plasma resulted in enzymatic and histopathologic evidence of non-A/non-B hepatitis in all eight animals. Challenge of two chimpanzees convalescent from factor VIII-induced disease with either factor IX or "H" strain plasma resulted in non-A/non-B hepatitis only in the animal inoculated with factor IX materials. Reciprocal cross-challenge of a chimpanzee convalescent from factor IX-induced disease with factor VIII also produced unequivocal enzymatic and histopathologic evidence of non-A/non-B hepatitis. Cross-challenge of a chimpanzee convalescent from "H" strain-induced non-A/non-B hepatitis with factor VII did not cause a second bout of non-A/non-B hepatitis. These findings suggest the factor VIII materials and "H" strain plasma used in these studies share a common etiologic agent (or agents), but that factor VIII and factor IX may contain two distinct agents. Electron microscopic (EM) examination of thin-sectioned, acute-phase liver biopsies from all but one of the chimpanzees receiving the primary inocula revealed the presence of abnormal hepatocyte cytoplasmic structures previously shown to be associated with non-A/non-B hepatitis. Crystalline structure containing 25 to 30 nm particles were visualized by EM in the cytoplasm of endothelial or Kupffer cells in acute-phase liver biopsies obtained from three chimpanzees inoculated with either factor VIII materials or "H" strain plasma.