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BACKGROUND: Since the discovery of hepatitis C virus (HCV) as a major cause of non-A non-B hepatitis, advances have been made in our understanding of the molecular biology of HCV and its relatedness to the flaviviruses and pestiviruses. The use of molecular techniques to construct an antibody assay has enabled the accumulation of information concerning the natural history and pathogenesis of HCV infection. OBJECTIVES: The objective was to review the literature to March 1994 on the structure, function and genetics of HCV and to correlate these findings with approaches to diagnosis that have contributed to our understanding of HCV infections. STUDY DESIGN: We reviewed the virological and medical literature from 1988 to March of 1994 with a focus on the stated objectives. RESULTS: Although the structure of HCV has been well-defined, our knowledge of the function of all the genes of HCV is incomplete. Structural core and envelope proteins as well as enzymes have been described. The 5' end of the polypeptide is most conserved. Genotyping of isolates varies according to the part of the gene examined. Several genotypes exist and tend to predominate in global populations. Antibodies to the various proteins can be measured by EIA assays and positive specimens often require confirmatory testing. Uniquely sensitive nucleic acid detection systems for RNA amplified by PCR have enabled a better understanding of the natural history, epidemiology and responses to treatment. CONCLUSIONS: Well-designed studies for the detection of nucleic acid, antibodies and antigens using a variety of viral gene products will provide even more information about HCV infections and help lead to treatment and prevention.

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This review article describes several applications of the widely used enzyme immunoassay (EIA) procedure. EIA methods have been adapted to solve problems in diagnostic virology where sensitivity, specificity, or practicability is required. Concurrent developments in hybridoma and conjugation methods have increased significantly the use of these assays. A general overview of EIA methods is given together with typical examples of their use in diagnostic medical virology; attention is drawn to possible pitfalls. Recent advances in recombinant DNA technology have made it possible to produce highly specific nucleic acid probes that have a sensitivity approximately 100 times greater than that of EIA. Some applications of these probes are described. Although the non-labelled nucleic acid probes for use in the field are not as refined as non-labelled immunoassays, their range of applications is expected to expand rapidly in the near future.

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Enzyme immunoassays represent in many cases the preferred procedure for the detection of antigens or corresponding antibodies. However, many of the current procedures are performed suboptimally. This article reviews the available designs, auxiliary recognition systems, production and purification of antibodies, conjugation procedures, solid-phase materials, recording and interpretation of results, and quality control and standardization of procedures to improve the reproducibility of tests.

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The periodate-mediated conjugation of horseradish peroxidase to antibody is one of the most popular methods to prepare conjugates for enzyme immunoassays of antigens or corresponding antibodies. A very simple method to obtain peroxidase, which is both about five times cheaper than the rather expensive commercial preparations and has a significant higher activity, is reported. Moreover, the conjugation method was critically investigated and considerably simplified. Conjugates thus obtained are about three times more active than the best obtained with the original method.

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A method for simultaneous peptide mapping of polypeptides contained in a mixture is presented. The polypeptides were first separated by conventional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The strip of gel containing these unstained polypeptide bands was subsequently embedded perpendicular to the direction of electrophoresis in the stacking gel of a second gel. The proteolytic enzymes, loaded on top of the second gel, were brought in contact with the substrates through moving boundary electrophoresis. The peptides thus generated were then resolved by electrophoresis in a gradient gel. A polychromatic silver staining method added an extra dimension to the identification and characterization of the peptides in the maps obtained in that specific peptides got specific colors. Moreover, the sensitivity of this method was illustrated by the demonstration that original quantities in the submicrogram range of nonradioactive proteins (exemplified here by the structural proteins of densonucleosis virus) largely sufficed for satisfactory maps. Other advantages of this procedure over current methods included (i) the elimination of the purification step (and consequently virtually no loss or contamination), (ii) that only the strict minimum of material (necessary for the ultimate visualization of the maps) had to be used, (iii) that no special two-dimensional electrophoresis equipment was needed, and (iv) the consistency, speed, and simplicity of the method.

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HSV and DNV viral antigens have been localized by electron microscopy using the protein A-gold technique. The labelling of HSV antigens was detected over the (naked and enveloped) viral particles as well as on the cytoplasm and the nucleoplasm. In contrast, DNV antigens were revealed only over clusters of viral particles in the nucleus. The high sensitivity of the technique and the good ultrastructural preservation allowed a very fine identification of the labelled structures. Thus, the protein A-gold technique can be applied generally for the ultrastructural detection and identification of viral antigens and might be useful for diagnostic purposes.

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The antigens of herpes simplex virus types 1 and 2 are detected and localized in vitro in Hep-2 cells by indirect and direct immunoperoxidase. The direct procedure is applied successfully for the diagnosis of genital disease on cytosmears of cervical cells of patients with herpes simplex type 2 infection. With other procedures, as anti-complement immunoperoxidase (ACIP) and anti-IgG immunoperoxidase (AIIP) the Epstein-Barr Nuclear Antigen (EBNA) was detected precisely in EB3, Jijoye and Raji lymphocytes using anti-EBNA antibodies from patients with nasopharyngeal carcinoma or Burkitt lymphoma, in dilution 1:10-1:5120. The indirect immunoperoxidase localized intracytoplasmic early antigen (EA) in 5% of EA--positive Raji cells induced by superinfection of EBV. The viral capsid antigen (VCA) is detected by this method in 5%-10% of virus producing cell lines Jijoye and P3HR-1. The standardized kits of immunoperoxidase procedures for diagnosis of intracellular viral antigens using monoclonal antibodies are under investigation.

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There exists a serious lack of rapid and sensitive methods to identify densonucleosis viruses and to discriminate among them. Two different enzyme-linked immunosorbent assays (ELISA) were adapted for this purpose, which were both significantly faster and more sensitive than currently used ELISA procedures. This increase in sensitivity was due to an improvement in the conjugation procedure of peroxidase to antibody, the establishment of the optimum conditions for the various incubations, an optimisation of the substrate (H2O2) concentration, and the use of a new H-donor. The speed of the assay could be considerably shortened by the use of polyethylene glycol-6000 (i.e. the total time of the assay needed for maximum sensitivity of the indirect assay was only 2 hours). The assays using peroxidase conjugates were found considerably more sensitive than those using alkaline phosphatase, which is very probably due to a more efficient and better controlled conjugation procedure for peroxidase. The virus could be detected in the pg to ng range in a large excess of nonspecific antigens and titers for the antisera usually exceeded 10(6). Small differences in the viruses could be detected. Several factors, which may influence the sensitivity and specificity of these densonucleosis virus assays, were further investigated.

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Densonucleosis virus cannot code for its four structural proteins if each of them has a unique sequence. The objective of the present investigation, therefore, was to establish whether: (i) the viral genome contains overlapping genes; (ii) the virus incorporates host proteins; or (iii) one of the structural proteins is a dimer. Two independent methods were employed for this purpose. First, the viral proteins, solubilized in sodium dodecyl sulfate, were purified after dansylation and were analyzed by peptide mapping, using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Second, an enzyme-linked immunosorbent assay was developed for a comparative analysis of the viral proteins solubilized by sodium dodecyl sulfate. It was demonstrated with both techniques that densonucleosis virus has four unique structural proteins, all with extensive sequence homologies. Moreover, all structural proteins contained intraprotein, but no interprotein, disulfide linkages. These results indicated similarities between densonucleosis virus and representatives of the two other genera of the Parvoviridae.

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