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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.

Molecular diagnostic test systems refer to techniques and laboratory protocols for detecting, identifying and amplifying nucleic acid sequences. By differentiating one sequence from another, even if they differ only by a single nucleotide base change, a wide range of clinically useful and scientifically valuable diagnostic information may be obtained. In the field of human diseases molecular diagnostics are applicable to infectious organisms, genetic and hereditary diseases, cell and tissue typing, and to genetic mutations, recombinants, duplications or rearrangements associated with various forms of cancer. Genomic matching between individuals has become practical and is used widely for parentage testing and for evidence in forensic and crime situations. In the future, individual DNA “fingerprints” may be deposited in data banks as sources of unique individual identification.

A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluated and compared with a standard solid-phase radioimmunoassay for anti-HAV, CBA was found to be sensitive and specific for the detection of anti-HAV, as demonstrated by the 98% concordance of CBA and solid-phase radioimmunoassay test results. The standard CBA test was modified for the differential detection of acute (immunoglobulin M) and convalescent (immunoglobulin G) anti-HAV by incorporation of a step in which immunoglobulin G anti-HAV was preferentially absorbed with S. aureus cells (protein A). The modified CBA test was shown to be capable of differentiating between acute- and convalescent-phase sera. The modified CBAM test was able to detect immunoglobulin M anti-HAV up to approximately 4 weeks after the onset of illness.

A codon-specific change in the function of transfer RNA from Escherichia coli was found in cells infected with phage Qβ. In a tRNA-dependent, cell-free system for protein synthesis, tRNA preparations from infected cells showed a markedly decreased ability to support translation of polycytidylic acid into polypeptide, as compared to tRNA from uninfected cells. However, tRNA from infected cells supported polyuridylic acid-directed phenylalanine incorporation equally as well as tRNA from uninfected cells. Ribosomal binding studies of prolyl-tRNA in response to polycytidylic acid showed that tRNA from infected cells had 2 to 3 times less ability to bind ribosomes. These results suggest that the decreased ability of tRNA from infected cells to support polycytidylic acid translation is related to the decrease in ribosomal binding of the prolyl-tRNA.

125I-labeIed specific immune globulin was used in a 2-step procedure to detect hepatitis B virus-associated antigen in blood serum or plasma. In step 1, undiluted sample was incubated overnight in polypropylene tubes coated with hyperimmune guinea pig antiserum. The serum sample was then removed and specific 125I-antibody was added and incubated for a period of 90 min. The presence of antigen in the original sample was then reflected by the count rate of the tube after the unreacted labeled antibody was removed. A typical dose response curve was observed for antigen in the concentration ranges of 0.005–0.32 μg/ml. Replicate analyses of normal sera and a low-titer positive sera revealed method variations which were distributed between ± 5 standard deviations from the replicate mean values. The radioimmunoassay was 500–1,000 times as sensitive as complement fixation for ad-subtype antigens. For ay-subtype antigens, the method was 128–512 times as sensitive as complement fixation. Assay values of 4,500 blood donor sera had a typical distribution, with most counts falling between +3 and –3 standard deviations relative to a negative control serum. However, 6% of the samples gave a count rate greater than 3 standard deviations from the negative control mean, and about 3.5% gave a count rate greater than 5 standard deviations above the negative control mean. The increased sensitivity of the method resulted in the detection of more positive sera in a variety of donor groups than other immunological methods. In two volunteer donor groups, 1.4 and 1.9% of the samples were positive. In two analyses of commercial paid donors, one group was 4.6% positive and one was 5.5% positive.

Overby , L. R. (University of Illinois, Urbana), G. H. Barlow , R. H. Doi, Monique Jacob, and S. Spiegelman . Comparison of two serologically distinct ribonucleic acid bacteriophages. I. Properties of the viral particle. J. Bacteriol. 91: 442–448. 1966.—Two ribonucleic acid (RNA) coliphages, MS-2 and Qβ, have been characterized physically and serologically. MS-2 has an S 20, w value of 79, a molecular weight of 3.6 × 10 6 , a density of 1.422, and p H 3.9 as its isoelectric point. Qβ has an S 20, w of 84, a molecular weight of 4.2 × 10 6 , a density of 1.439, and an isoelectric point at p H 5.3. One host ( Escherichia coli A-19) permits a distinction between the two on the basis of a marked difference in plaque size. They are distinct immunochemically, no serological cross-reaction being detectable.

Summary The inactivation of the bacteriophage, Q β, with specific antiserum was studied. The reaction exhibited a continual decrease in rate that was evident from the beginning. The same limiting value of the surviving fraction was reached by all concentrations of a serum. The deviation from first order kinetics was not attributable to dissociation or pre-existing phage heterogenity. The results support the idea of the development of resistant phages by interaction with antiserum components. The curve of neutralization could be approximated mathematically.

A two-step direct radioimmune test was used to detect hepatitis B virus associated antigen in human serum or plasma. The procedure involved the use of polypropylene tubes coated with hyperimmune guinea pig serum, and 125I-labeled specific hepatitis B virus antigen immune globulin. The technique appeared to be more sensitive than other immunologic assays, including indirect radioimmune methods based on competition of unlabeled antigen with 125I-labeled antigen. Various blood donor populations were tested for antigen by the direct radioimmune test, by complement fixation, agar gel diffusion and counterimmunoelectrophoresis. The increased sensitivity of the radioimmune procedure resulted in the detection of about 3 to 8 times as many positive sera as the above three immunologic methods. The percentages of positive sera by radioimmune assay were: about 1% in volunteer blood donors, about 2% in commercial blood donors and about 40% in hepatitis-implicated patients.

SummaryWith the availability of sensitive laboratory techniques for the serological detection of markers of hepatitis A and B infection, it is possible to reach an aetiological diagnosis with reasonable accuracy in cases of viral hepatitis occurring in residential institutions. The accuracy of diagnosis is increased if the immune status of all inmates to hepatitis A and B has been previously determined. Furthermore, considerable savings in the prophylactic use of human immune globulin can be effected if the type of viral hepatitis occurring can be diagnosed, and if the immune status of patients and staff to hepatitis A is known.

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.

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.

When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.

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.

Replication of herpes simplex virus in WI-38 cells was inhibited by phosphonoacetic acid, as measured by decreased virus cytopathogenic effect and incorporation of radiolabeled thymidine in virus-infected cells. The drug appeared to have no effect on adsorption, penetration, or release of the virus nor on the synthesis of ribonucleic acid or protein. It appeared to inhibit virus deoxyribonucleic acid synthesis.

PLASMA of hepatitis B antigen (HBsAg) carriers consists mostly of spherical particles of diameter 20 nm, together with a much smaller number of filamentous structures with the same diameter. In addition, larger spherical particles, 42 nm in diameter with a 28 nm core, first described by Dane et al.1, are routinely found in crude plasma or purified preparations of HBsAg. Dane particles also contain a primed DNA polymerase activity2 capable,of synthesising new DNA. Robinson et al.2 examined DNA from Dane particles by electron microscopy and found open, double-stranded circular configurations approximately 0.78 µm long. We have examined DNA isolated from Dane particles directly and also after formation of radioactive products by the endogenous DNA polymerase reaction. The DNA structures appeared in electron micrographs as double-stranded linear strands and closed circles, with examples of completely and partially open circles, and circles with attached linear segments in different lengths. The open circles with attached linear segments are suggestive of a replicative form, and this is consistent with a rolling circle model4 for replication. This model for replication is strengthened by the comparison of the types of DNA found in particles before and after reaction of the polymerase. The number of rolling circles and the length of the tails were greatly increased after DNA synthesis. This type of replication has not been reported for animal viruses.

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.

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.

Biotechnology is approaching its 15th anniversary. The importance of recombinant DNA technologies was recognized in 1974 when scientists recommended that ethical, environmental, technical and commercial issues surrounding genetic engineering should be assessed broadly before rapid exploitation of the impending breakthroughs. Most of these issues have been clarified and we have seen amazing progress in applying biotechnologies to problems in human health, to animal and plant life, and to areas of energy and the environment.

A 3-year longitudinal survey in a residential institution for the mentally retarded was carried out to study the epidemiology of hepatitis B virus (HBV) and to elucidate the different responses which Down's syndrome (DS) and non-Down's (ND) subjects have to HBV infection. Sensitive tests for the HBV surface antigen and antibody (HBsAg and anti-HBs), core antibody (anti-HBc) and `e' antigen and antibody (HBeAg and anti-HBe) were used. The HBsAg and anti-HBs content of positive sera was quantitated accurately.

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.

Viele Autoren haben uber histologische Leberbefunde asymptomatischer HBSAG-Trager berichtet. Zahlreiche Untersuchergruppen haben nur minimale histologische Leberveranderungen beschrieben [1–4], wahrend andere Autoren signifikante morphologische Alterationen fanden [5–8]. Diese widerspruchlichen Mitteilungen veranlasten uns, klinisch und biochemisch gesunde HBsAG-positive Blutspender in eine prospektive Studie aufzunehmen. Ziel der Untersuchung ist es, Aufschlus uber die Langzeitprognose des asymptomatischen HBsAG-Tragers zu gewinnen.

We compared the radioimmunoassay (RIA) and counterimmunoelectrophoretic (CIE) methods in detecting hepatitis B antigen (HBsAG) in 407 acute and 336 convalescent sera of adults with viral hepatitis. The CIE method demonstrated that 41% of acute and 28% of 14-to 17-day serum specimens were HBsAg-positive. The RIA method demonstrated seropositivity in 60% of acute and 56% of convalescent specimens (P less than .001). Eighty-four percent of coded specimens initially positive for HBsAg by RIA were found to have subtype antigenic determinants d or y; 92% of the HBsAg-negative controls were negative for subtype antigens, confirming the specificity of the RIA test. RIA subtyping data corroborated earlier work with immunodiffusion techniques.

Introduction Lupus erythematosus, because of its protean manifestations, has often posed a difficult diagnostic problem. In spite of many well-established criteria, specific diagnosis has at times been impossible and has often been made only by implication. Consequently, any additional diagnostic clue which helps to complete the diagnostic pattern is of definite value. Historical .—The finding of the L. E. cell by Hargraves 1 in 1948 was an important diagnostic milestone. Brunsting 2 in evaluating the lupus erythematosus clot test classifies lupus erythematosus patients into three groups: Patients demonstrating classical clinical features together with a positive demonstration of the L. E. cell. Patients with less classical clinical findings. In this group, which is the largest, the L. E. cell may be demonstrated only by repeated effort. Patients demonstrating an inconsistent clinical picture together with a finding of the L. E. cell after

Overby , L. R. (University of Illinois, Urbana), G. H. Barlow, R. H. Doi, Monique Jacob, and S. Spiegelman . Comparison of two serologically distinct ribonucleic acid bacteriophages. II. Properties of the nucleic acids and coat proteins. J. Bacteriol. 92: 739–745. 1966.—The ribonucleic acid (RNA) molecules and coat proteins of two RNA coliphages, MS-2 and Qβ, have been characterized. MS-2 RNA shows an S 20,w of 25.8 and a molecular weight by light scattering of 10 6 . The corresponding parameters for Qβ-RNA were 28.9 and 0.9 × 10 6 . A difference in base composition was reflected in the adenine-uracil ratio, which was 0.95 for MS-2 and 0.75 for Qβ. The two RNA preparations are readily separated by chromatography on columns of methylated albumin. Both gave identical bouyant densities in cesium sulfate of 1.64 g/ml. The coat protein subunits were of similar molecular weights: 15,500 (Qβ) and 14,000 (MS-2). They differed, however, in that the Qβ-protein lacked tryptophan and histidine, whereas the MS-2 protein lacked only histidine.

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.

Disodium phosphonoacetate when administered orally or topically to mice experimentally infected with herpes simplex virus was able to significantly reduce the mortality associated with the agent. In addition, this compound was able to reduce herpesvirus lesions on the corneas of infected rabbits.