Detection of cytomegalovirus antibodies by an enzyme-linked immunosorbent assay using recombinant polypeptides of the large phosphorylated tegument protein pp150.

Parts of the large phosphorylated tegument protein, pp150, of human cytomegalovirus (HCMV) were expressed in bacteria. The resulting fusion proteins were tested in a Western blot (immunoblot) assay for reactivity with a monoclonal antibody against pp150, with a polyspecific rabbit antiserum, and with human reconvalescent-phase sera. Those fusion proteins that performed well in the Western blot assay were used as antigens in enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against HCMV. Five different recombinant beta-galactosidase fusion proteins were evaluated by ELISA using 62 seropositive and 38 seronegative human serum samples. Of all the proteins tested, one peptide representing 162 amino acids of pp150 was superior to the others with regard to sensitivity and specificity. All sera known to be positive for antibodies against HCMV were identified by combining the results of the ELISAs with the different pp150 fusion proteins. Therefore, it appears that peptides from a single protein of HCMV might be sufficient to identify HCMV-seropositive individuals by recombinant ELISA.

[Genotoxicity of a standardized Hypericum extract]. / Genotoxizität eines standardisierten Hypericum-Extraktes.

St. John's wort (Hypercum perforatum) contains hypericin and hypericin-like substances as well as flavonoids, of which particularly Quercetin has generated a wide-spread controversial discussion with respect to mutagenic action. The genotoxicity of a standardized aqueous ethanolic Hypericum extract (Hypericum extract Steigerwald, Psychotonin M) was verified in different in-vivo and in-vitro testsystems with mammalian cells. The in-vitro investigations were performed with the HGPRT (hypoxanthine guanidine phosphoribosyl transferase)-test, UDS (unscheduled DNA synthesis)-test and with the cell transformation test using Syrian hamster embryo cells. Both the in-vitro tests as well as the in-vivo tests--fur spot test of the mouse and the chromosome aberration test with the bone marrow cells of the chinese hamster--were negative, giving completely no indication of a mutagenic potential of Hypericum extract. These investigations lend support to the view that results from bacterial short-term tests are of very limited transferability to human.

Prokaryotic expression of immunogenic polypeptides of the large phosphoprotein (pp150) of human cytomegalovirus.

The large phosphorylated matrix protein pp150 of human cytomegalovirus (HCMV) is the polypeptide most frequently reactive in immunoblotting analyses with human antisera when compared with other viral proteins. Several defined regions of pp150 were expressed as beta-galactosidase fusion proteins and these were tested for their immunoreactivity with human sera and their immunogenicity. One antigenic region could be expressed in large amounts and was found to carry immunodominant epitopes, as shown by immunoblotting and ELISA. A rabbit antiserum raised against recombinant pp150 antigens produced in bacteria proved to be useful for immunofluorescence and immunohistochemistry studies of HCMV-infected cells and tissues. The results suggest that this anti-pp150 serum will help to elucidate the process of virus assembly and antigen detection in infected cells.

Detection of human cytomegalovirus DNA and viral antigens in tissues of different manifestations of CMV infection.

Biopsy and autopsy specimens from 22 patients with cytomegalovirus (CMV) infections were investigated by means of in situ hybridization (ISH) to detect viral DNA and by immunohistochemistry (IHC) to visualize viral proteins. Both methods proved to be valuable tools for histopathology. ISH sometimes recognized cells that did not show typical CMV inclusions. An antiserum against the full spectrum of viral proteins (non-infectious enveloped particles) detected most cytomegalic cells in disseminated and organ-limited infections. An antiserum against a recombinant polypeptide (XP1) was particularly useful in connatal CMV infections and organ-limited infections. We have demonstrated that IHC and ISH studies in parallel are the best approach to the detection of CMV infections in pathological specimens.

The two major structural phosphoproteins (pp65 and pp150) of human cytomegalovirus and their antigenic properties.

Human cytomegalovirus (HCMV) purified from cell culture contains two dominant structural phosphoproteins with apparent molecular weights of 65,000 and 150,000, designated as pp65 and pp150 respectively. The humoral immune response of infected individuals against pp65 is relatively weak and is not always detectable by Western blot analyses. This report shows that recent clinical isolates of HCMV do not necessarily have pp65 as a prominent constituent, suggesting that the low immune reaction is due to variable expression of the pp65 in natural infections. However, the HCMV strains tested in this study produced the large structural phosphoprotein (pp150) in about equal amounts. The pp150 is remarkably immunogenic, if compared with all other virion constituents; serum pools and individual sera from HCMV-infected patients recognized this particular protein intensively in immunoblot assays. Thus, phosphoprotein pp150 seems to be the primary polypeptide candidate for expression cloning in order to develop reagents for novel ways of HCMV diagnosis.

Map position and nucleotide sequence of the gene for the large structural phosphoprotein of human cytomegalovirus.

Human cytomegalovirus particles contain a phosphoprotein of 150,000 (pp150) apparent molecular weight in their matrix; the protein appears particularly reactive in Western blot analyses with human antisera. The gene for pp150 was mapped by screening a bacteriophage lambda gt11 cDNA expression library with monospecific rabbit antisera. Subsequent hybridization of cDNA with cosmid and plasmid clones containing the human cytomegalovirus strain AD169 genome mapped the gene to HindIII fragments J and N. The gene is transcribed into a late 6.2-kilobase RNA. The nucleotide sequence of this region was determined, and a transcription initiation site and two polyadenylation sites of an abundant transcript were located by primer extension and nuclease protection experiments. The reading frame for pp150, deduced from computer analyses, gives rise to a polypeptide of 1,048 amino acids in length; protein secondary structure analysis revealed multiple beta-pleated sheets in hydrophilic clusters, providing a possible explanation for the immunogenic properties of the polypeptide.