Self-assembly of Q-beta and MS2 phage particles: possible function of initiation complexes.

Four kinds of particles were reconstituted with RNA and protein from the genetically unrelated bacteriophages Qbeta and MS2, namely, two homologous and two heterologous, with respect to RNA and protein. However, once Qbeta RNA (or MS2 RNA) reacted with a few molecules of either Qbeta or MS2 protein to form a nucleoprotein complex (initiation complex), it formed a phagelike particle only with subsequent addition of the same protein.

Hepatitis B virus antigen: validation and immunologic characterization of low-titer serums with 125 I-antibody.

Radioimmunnoassay was used to determine the serologic subspecificities of 85 blood donor serums positive for hepatitis B virus-associated antigen. There was complete agreement with results obtained by immunoprecipitatiotn of 43 serums. The remaining 42 serums were negative by immunoprecipitation but followed type-specific immunology by radioimmunoassay, and this served as a validation for authentic hepatitis B virus antigen.

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.

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.

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.

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.

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.

Antiviral potential of phosphonoacetic acid.

Phosphonoacetate has been found to inhibit specifically the replication of herpes-viruses. A partial inhibition of vaccinia virus represents the only activity outside the herpesvirus class. The drug was found to be a specific inhibitor of the virus-induced DNA polymerases. Normal cellular polymerases were relatively insensitive to phosphonoacetate, resulting in low cellular toxicity. Our working hypothesis is that the drug binds to the enzyme and that initiation of polynucleotide synthesis occurs in the presence of the drug and the required template, substrates, and cations. However, addition of deoxynucleosides to the elongating nascent chain is prevented by the enzyme-bound drug. Kinetic analyses indicated that phosphonoacetate did not interfere with the binding of DNA template to polymerase; and it did not compete with nucleotide substrate binding. The highly specific inhibitory effects of phosphonoacetate allowed for the selection of partially resistant strains of HSV. Resistance of virus to the drug in cell culture was directly correlated with the same relative resistance of the corresponding cell-free DNA polymerases. Phosphonoacetate was also effective therapeutically in herpesvirus skin and ocular infections in animals. Intraperitoneal administration of the drug reduced death and severity of disease in experimental encephalitis in hamsters. High specificity, low toxicity, and reproducible efficacy in lower animals suggested that phosphonoacetate could be a useful new antiviral drug. Sensitivity to phosphonoacetate also is a useful research tool as a genetic marker for herpesviruses.

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.

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.

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.

Inhibition of DNA polymerase from herpes simplex virus-infected wi-38 cells by phosphonoacetic Acid.

Infection of Wi-38 cells with herpes simplex virus induced an elevated DNA polymerase activity which had many biochemical properties different from normal cell DNA polymerase. Phosphonoacetic acid specifically inhibited the virus-induced DNA polymerase as compared to the normal WI-38 cell DNA polymerase. The compound did not appear to inhibit enzyme activity by interacting with the DNA primer.

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.

Post-transfusion hepatitis: the role of hepatitis B antibody.

Aliquots from units of blood previously transfused as part of a prospective post-transfusion hepatitis (PTH) study were rescreened for the presence of hepatitis B antibody (anti-HBS) to determine the effect of transfusion of such material. Anti-HBS was more commin in commercial blood. Infusion of anti-HBS was not associated with an increased or decreased risk of PTH, hepatitis B, or hepatitis B (HB) exposure. Receipt of anti-HBS did not modify the hepatitis which occurred. Receipt of large amounts of anti-HBS may be associated with an increased incidence of HB events. Preexisting anti-HBS was not only not protective against PTH, but more PTH (67% versus 40%) and hepatitis B (47% versus 12%) were seen in those patients with it.

Prevalence of hepatitis B e antigen and its antibody as detected by radioimmunoassays.

A solid-phase radioimmunoassay (RIA) for the detection of hepatitis B e antigen (HBeAg) and antibody (anti-HBe) was developed. The RIA was approximately 1,000-fold more sensitive than rheophoresis for HBeAg, and approximately 6,000-fold more sensitive than rheophoresis for anti-HBe. Generally, less than one-fifth of hepatitis B antigen (HBsAg)-positive sera from blood donors were positive for either HBeAg or anti-HBe by rheophoresis; in contrast, more than 90% of the samples were positive by the RIA method. The ratio of HBeAg to anti-HBe among HBsAg carriers varied in different geographic localities. Also, the presence of HBeAg correlated directly with the titer of HBsAg and the presence of Dane core particles. Anti-HBe was associated with lower titers of serum HBsAg.

Characteristics of herpes simplex virus resistance to disodium phosphonoacetate.

Herpes simplex virus (HSV), which was partially resistant to the inhibitory effect of disodium phosphonoacetate (PAA), could be recovered following four virus passages in the presence of 100 microgram/ml PAA. Resistant strains were isolated from both HSV type 1 and HSV type 2. Virus resistance to PAA was not complete, and in most isolations a significant proportion of the virus stock remained susceptible to the drug. Resistance was shown to be heritable and persisted through virus passage and cloning experiments. PAA inhibited the replication of virus-specific DNA in sensitive strains of HSV but not in resistant strains of HSV. In vitro experiments directly demonstrated that PAA inhibited the activity of the virus-specific DNA polymerase 10 times more effectively in PAA-susceptible HSV than in PAA-resistant HSV. The treatment of HSV-infected mice with high levels of PAA did not induce the formation of resistant virus strains.

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.

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.

Antigenic subgroups of hepatitis B (surface) antigen in Berlin (West) and their epidemiological aspects.

Sera of 334 hepatitis B patients and 118 sera of HBsAg carriers were tested for the distribution of the subgroups ad and ay by means of rheophoresis technique or solid phase radiommunoassay. The distribution of subgroups revealed a typical pattern. The ad-antigen determinant was more frequent in hepatitis B patients, blood donors and hemodialysis patients whereas the ay determinant was detected in drug users, guest workers and juveniles at a considerably higher percentage. The difference in distribution is discussed as an epidemiological phenomenon of hepatitis B virus, dependent on environmental factors characteristic for certain groups of the population. The routine testing for HGsAg subgroups is recommended as a valuable epidemiologic tool.

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.