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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.

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.

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.

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.

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.

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.

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.

PLASMA of hepatitis B patients and antigen carriers contains a high concentration of spherical and filamentous particles (HBsAg), about 20 nm in diameter and without evidence of nucleic acid. There are also a relatively few spherical particles, 42 nm in diameter with a 28 nm core, first described by Dane et al.1. Dane particles contain a unique deoxyribonucleic acid2 and a DNA polymerase3 capable of synthesising DNA so they are considered to be a leading candidate for a human hepatitis B virus. Double stranded DNA of unique base composition has been isolated from the naked intranuclear particle of hepatocytes of human hepatitis liver5. The large quantity of HBsAg, resembling defective virus, in the plasma of carriers prompted us to investigate a possibility that there may exist free or ‘unassembled’ DNA corresponding to the empty particles in these plasmas. The presence of both free DNA and empty particles could result from faulty assembly of a virus. We report here the presence of free DNA in HBsAg positive plasma and this free DNA showed homology with radiolabelled Dane particle DNA by molecular hybridisation.