Integration of Proviral DNA in Chicken Cells Infected with Schmidt-Ruppin Rous Sarcoma Virus Is Not Enhanced by DNA Repair

The effect DNA repair might have on the integration of exogenous proviral DNA into host cell DNA was investigated by comparing the efficiency of proviral DNA integration in normal chicken embryonic fibroblasts and in chicken embryonic fibroblasts treated with UV or 4-nitroquinoline-1-oxide. The cells were treated with UV or 4-nitroquinoline-1-oxide at various time intervals ranging from 6 h before to 24 h after infection with Schmidt-Ruppin strain A of Rous sarcoma virus. The chicken embryonic fibroblasts were subsequently cultured for 18 to 21 days to ensure maximal integration and elimination of nonintegrated exogenous proviral DNA before DNA was extracted. Integration of proviral DNA into the cellular genome was quantitated by hybridization of denatured cellular DNA on filters with an excess of (3)H-labeled 35S viral RNA. The copy number of the integrated proviruses in normal cells and in infected cells was also determined from the kinetics of liquid RNA-DNA hybridization in DNA excess. Both RNA excess and DNA excess methods of hybridization indicate that two to three copies of the endogenous provirus appear to be present per haploid normal chicken cell genome and that two to three copies of the provirus of Schmidt-Ruppin strain A of Rous sarcoma virus become integrated per haploid cell genome after infection. The copy number of viral genome equivalents integrated per cell treated with UV or 4-nitroquinoline-1-oxide at different time intervals before or after infection did not differ from the copy number in untreated but infected cells. This finding supports our previous report that the integration of oncornavirus proviral DNA is restricted to specific sites in the host cell DNA and suggests a specific mechanism for integration.     

Anti-idiotype antibodies that mimic gp86 of human cytomegalovirus inhibit viral fusion but not attachment.

Human cytomegalovirus (CMV) infects cells by sequential processes involving attachment, fusion with the cell membrane, and penetration of the capsid. We used two monoclonal anti-idiotype that mimic one of the CMV envelope glycoproteins, gp86, to study its role in the early phases of CMV infection. Neither of two such antibodies inhibited virus binding to human embryonic lung (HEL) fibroblasts; however, both antibodies inhibited the fusion of CMV with HEL cells, as measured by an assay in which viral envelope is labeled with a fluorescent amphiphile (octadecyl rhodamine B chloride, or R18), resulting in increased fluorescence during fusion of virus with the cell membrane. Because these anti-idiotype antibodies were shown previously to bind to specific receptors on HEL cell membranes, these findings suggest that both gp86 and its cell membrane receptor may function in the fusion of human CMV with HEL cells.     

Rapid diagnosis of cytomegalovirus in immunocompromised patients using monoclonal antibodies that detect early viral antigens

A technique was applied to detect early fluorescent antigens (DEFA) of cytomegalovirus (CMV) using the E13 monoclonal antibodies in 52 immuno-compromised patients hospitalized in the Nephrology Institute of Havana. Of the 75 urine or blood (buffy coat) samples taken, 15 were found positive to CMV. Using classical diploid human fibroblast isolation technique, 12 CMV strains were isolation of previously detected positive samples by DEFA. In addition, CMV was isolated from one sample reported to be negative by DEFA. A coincidence of 80% was found between both techniques. With the ELISA test, all the sample studied have IgG antibodies to CMV.     

Effect of arginine deficiency on the reproduction of human cytomegalovirus.

In arginine-deprived human embryonic fibroblasts the reproduction cycle of human cytomegalovirus (CMV) is incomplete. Infectious virus cannot be demonstrated in cell disintergrates, and from among the CMV antigens only the diffuse cytoplasmic antigen is detectable by immunofluorescence. The antigens localized in the cell membrane and those appearing during the complete cycle as large granules or inclusion-like bodies in the nucleus do not appear in the absence of arginine. The CMV genome persists in the arginine-deprived culture; after re-feeding with arginine-containing medium, maturation of virions soon ensues. Maturation could be prevented by inhibitors of protein synthesis, but not by DNA inhibitors, added simultaneously with completion of the medium.     

Definition of human cytomegalovirus-specific target antigens recognized by cytotoxic T cells generated in vitro by using an autologous lymphocyte system

We developed an in vitro system for the generation of human cytomegalovirus (CMV)-specific cytotoxic T cells (CTL) that avoids the necessity of constituting a panel of HLA-typed fibroblasts. Autologous donor leucocytes were coated with CMV antigens and were used as both stimulator and target cells. With the use of this system, CMV-specific effector cells were efficiently generated from seropositive but not seronegative donors. These CMV-specific effectors were HLA-restricted and had characteristics of T cells. Maximum lymphoproliferation preceded the appearance of maximum CTL activity by 3 to 4 days, and a close correlation was seen between both activities. Mouse anti-CMV monoclonal antibodies were used in blocking experiments in an attempt to define target antigens recognized by CMV-specific cytotoxic lymphocytes. Monoclonal antibodies directed against an early CMV membrane antigen, against neutralization epitopes, or against nuclear inclusion body protein all specifically inhibited CMV-sensitized effector cell activity but did not affect influenza virus-specific lysis. Monoclonal antibodies directed against a normal cell determinant or against poliovirus did not affect CMV-specific CTL activity. CMV-immune cytotoxic T cells could be consistently and specifically inhibited in their lytic activity by pretreating antigen-coated target cells with monoclonal antibodies directed against CMV-related proteins.     

Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes

 Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level. Accepted: 16 February 1996     

Immunity related to cytomegalovirus after allogenic bone marrow transplantation: role of donor immunity to cytomegalovirus in the incidence of cytomegalovirus infection in recipients

Cytomegalovirus infections are severe and frequent after BMT. This study included 34 bone marrow transplant recipients (23 aplastic anaemias and 11 leukaemias), their marrow donors and 125 related or non related normal controls. Assays were performed before transplantation and every 30 days between D 0 and D 90, and then every six months. They included detection of CMV induced lymphocyte proliferation in vitro, CMV antibody determinations by complement fixation and reverse haemagglutination, viraemia and/or viruria. Similarly, cellular immunity to mitogens and to other specific antigens was evaluated. During the period of study, 22 patients developed CMV infection. The diagnostic was confirmed by virus isolation from the 12th to the 96th day after the graft. Development of positive CMV proliferation test occurred from the 9th to the 84th day after virus isolation (30 to 120th day after the graft). In one case, the CMV infection was only proved by the lymphocyte proliferation to CMV in vitro and only 60 days later by viruria and 105 days later by detection of CMV antibodies. For the other 12 patients (7 aplasies and 5 leukaemias) and 10 of their bone marrow donors, no CMV infection was proved, before or after transplant, by any of the assays performed. By selecting a donor without previous CMV infection, we hope to reduce the incidence of CM infection in recipients.     

Reliability of the RNA-DNA Filter Hybridization for the Detection of Oncornavirus-Specific DNA Sequences

Denatured DNA from leukemic myeloblasts or uninfected chicken embryos, immobilized on nitrocellulose filters, was hybridized to a vast excess of [(3)H]70S RNA from purified avian myeloblastosis virus. The viral RNA was eluted from the RNA-DNA hybrids, purified, and then rehybridized in solution to an excess of either leukemic or normal chicken embryonic DNA. This study revealed that all the slow and the fast hybridizing viral RNA sequences detectable by liquid hybridization in DNA excess had hybridized to the filter bound DNA. Both techniques also gave similar values for the number of 28S ribosomal RNA genes contained in a chicken cell genome: 210 by the liquid hybridization procedure and 218 by the filter hybridization technique. Therefore, filter hybridization can accurately detect DNA sequences present in relatively few numbers in the genome of higher organisms.     

Detection of the measles virus genome in the peripheral blood lymphocytes of patients with chronic and acute glomerulonephritis]

The presence of the measles (rubeola) virus genome was searched for in the lymphocytes from the peripheral blood of patients suffering from the acute (16 persons) or chronic (164 persons) glomerulonephritis. Dot hybridization technique with the plasmid borne probes to the measles viral genes NP, P and H have been used for the search. The measles viral genome has been detected in 58% of lymphocytes from the patients with the chronic glomerulonephritis and in 50% of lymphocytes from the patients suffering from the acute form of the disease. The genome was not found in the material from the control group including donors and traumatology ward patients. 25 samples of lymphocytes from the patients with the chronic glomerulonephritis contained the RNA that was not hybridizable with the viral genes probes by dot hybridization technique, thus containing no genes homologous to parotitis viral genes. The average titer of anti-measles antibodies in the serum from patients with chronic glomerulonephritis the lymphocytes of which contained the measles viral genome was 1:304, while it was 1:154 for patients with the negative probes. The average anti-measles antibodies titers are the same (1:166 and 1:142) for analogical groups of patients with acute form of disease.     

Enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to human cytomegalovirus

A solid-phase enzyme linked immunosorbent assay (ELISA) for determination of IgG antibodies to cytomegalovirus (CMV) is described. The assay used purified CMV and extracts of CMV infected cells as antigen. Antigens were desiccated onto the bottom surface of polystyrene microcuvettes. The antibodies bound to the antigens were assayed by anti-IgG-alkaline phosphate conjugate followed by addition of the enzyme substrate. Titration curves have been obtained from the sera of 35 blood donors and of 23 patients. Comparison of results obtained by ELISA with those obtained by complement fixation (CF) shows that there is agreement between the tests. Both purified CMV and extracts of CMV infected cells were found to be suitable antigens. Purified CMV was of value particularly in those sera which show high reactivity against control antigen. The ELISA technique described is approximately 412 to 548 times more sensitive than the CF test when purified CMV or extracts of CMV infected cells, respectively, are used as antigens. No significant heterotypic rise to CMV was observed by ELISA in three sets of sera with seroconversion to herpes simplex virus. The ELISA technique gives objective results, is easily performed, and may be adaptable as a routine test both for serological diagnosis of CMV infection and for screening of the general population.     

Monoclonal Antibodies for Detection and Serotyping of Cucumber Mosaic Virus

Two monoclonal antibodies (MAbs) to cucumber mosaic virus (CMV) were selected from a panel of MAbs for use in the direct DAS (double antibody sandwich)-ELISA. Two different test procedures were developed: an ELISA with polyclonal and monoclonal antibodies (mixed ELISA) for the routine detection of CMV and a MAb-ELISA with two MAbs directed against different epitopes for the specific detection of the N serotype which is prevalent in GDR. The conventional two-step incubation of plates precoated with IgG was compared with simultaneous incubation of test sample and labelled antibody (one-step incubation). The mixed ELISA proved to be more sensitive than the direct DAS-ELISA with polyclonal antisera in detecting CMV in crude sap of infected plants. On the other hand, the MAb-ELISA could be used for serotyping of CMV isolates which is important in epidemiological investigations and in resistance breeding. Both the two-step and the one-step procedures gave similar results with some advantages of the latter procedure. One-step incubation is not only time-saving but seems to be also more sensitive with regard to the detection limit. However, care must be taken to circumvent the hook-effect occurring at high virus concentrations.     

Long-term persistence of cytomegalovirus genome in cultured human cells of prostatic origin.

Cells from prostatic tissue obtained from a 3-year-old male donor exhibited scattered foci of cytopathology on primary culture. A virus was isolated and shown by serological analysis to be cytomegalovirus (CMV). After a number of cell culture passages, a cell line (disignated CMV-Mj-P) was obtained in which foci of infection could no longer be demonstrated, nor could virus be rescued. On continued passage the doubling time of the cells decreased markedly, and the fibroblastoid cells ceased to demonstrate contact inhibition. CMV-specific antigen(s) was detected on the surface of the cells by indirect immunofluorescence techniques after exposure of the cultures to iododeoxyuridine. Microcytotoxocity tests established that CMV-Mj-P cells, but not control human prostate cells or human embryonic lung cells, share a membrane antigen with hamster cells transformed by CMV. Nucleic acid hybridization studies revealed that virus genetic information was carried by the human prostate cells and that the cells contained an average of about 10 to 15 genome equivalents of CMV DNA. Karyotypic analysis confirmed that the CMV-Mj-P cells were of human male origin. These results indicate that the cells either have been transformed by CMV or are chronically infected with CMV and releasing virus at levels below detection.     

T and B cell responses to cytomegalovirus antigens in healthy blood donors and bone marrow transplant recipients

Abstract We measured the production of interferon-gamma (IFN-γ) from single T cells and the T cell proliferative response to different cytomegalovirus (CMV) antigens in healthy blood donors and bone marrow transplant recipients. The antigens consisted of a CMV nuclear antigen (CMV na) containing the pp65-kDa matrix protein and the immediate early antigens but lacking CMV glycoproteins, and an antigen comprising native CMV glycoproteins (CMV gp). We also measured the IgG antibodies to CMV na and CMV gp. The T cells reacted to CMV na in CMV seropositive blood donors both with the production of IFN-γ and with proliferation, while bone marrow transplant recipients had a deficient T cell response. After stimulation with CMV gp, no T cell response could be observed in CMV seropositive subjects. IgG antibodies to CMV na coexisted in plasma with similar levels of antibodies to CMV gp.     

A human monoclonal antibody to cytomegalovirus (CMV)

We report the development of a human monoclonal antibody to cytomegalovirus (CMV) produced from a human X human hybridoma. This hybrid was developed by fusion of an EBV-transformed cell line making antibody to CMV and a human lymphoblastoid cell line WI-L2. The antibody is directed to a CMV-specific antigen primarily in the nucleus of CMV-infected human fibroblasts. It cross-reacts with at least 10 different strains of CMV and may provide a method for the rapid in vitro diagnosis of CMV infections. The production of CMV-specific human man monoclonal antibodies from human-human hybridomas for future therapeutic use is now technically feasible with this specific method of production.     

Improved methods for the diagnosis of African trypanosomosis

The diagnosis of trypanosomosis in animals with low parasitaemia is hampered by low diagnostic sensitivity of traditional detection methods. An immunodiagnostic method based on a direct sandwich enzyme-linked immunosorbent assay (ELISA), using monoclonal antibodies, has been examined in a number of African laboratories for its suitability for monitoring tsetse control and eradication programmes. Generally, the direct sandwich ELISAs for the detection of trypanosomal antigens in serum samples have proved to be unsatisfactory with respect to diagnostic sensitivity when compared with traditional parasitological methods such as the dark ground/phase contrast buffy-coat technique. Consequently, antigen-detection systems exploiting various other direct, indirect and sandwich ELISA systems and sets of reagents are being developed to improve diagnosis. In addition, an existing indirect ELISA for the detection of antibodies has been improved and is being evaluated in the field in order to detect cattle that are or have been recently infected with trypanosomes. Developments and advantages of other diagnostic techniques, such as dip-stick assay and tests based on the polymerase chain reaction are also considered.     

Comparison of commercially available antibody reagents for the cytomegalovirus pp65 antigenemia assay

Background: The Argene Biosoft 1C3 and the Biotest C10,C11 monoclonal antibodies are two of the most commonly used commercially available antibody reagents for the cytomegalovirus (CMV) pp65 antigenemia assay.Objectives: The sensitivities of these two reagents were compared in peripheral blood specimens received for CMV antigenemia testing.Study design: A total of 1149 peripheral blood specimens were processed for CMV antigenemia testing. Duplicate slides were stained with the Biosoft 1C3 and Biotest C10,C11 monoclonal antibodies.Results: A total of 158 specimens gave a positive result by one or both antibodies. One hundred and forty five were positive by the Biosoft antibody and 130 were positive by the Biotest antibody. Positive cell counts were significantly higher on cell preparations stained by the Biosoft antibody (Wilcoxon signed rank, P < 0.001) and the Biosoft antibody detected twice as many low-level positive specimens as the Biotest.Conclusions: The Biosoft antibody reagent was superior to the Biotest reagent for the detection of CMV antigenemia. This is an important factor since early detection is essential for appropriate initiation of preemptive antiviral therapy, particularly in transplant recipients at high risk of CMV disease.     

Differences in the localization of Alu-repeats in various chromosomes of peripheral blood cells from normal donors and bone marrow cells from patients with acute leukemia

The dispersion of the Alu-family DNA repeats in phytohemagglutinin-stimulated lymphocytes from peripheral blood of normal donors as well as in nonstimulated bone marrow cells of four patients suffering from acute leukemia was studied by hybridization on metaphase chromosomes in situ. DNA of bacteriophage lambda CAR42 clone containing the insertion of at least 8 copies of Alu-family DNA-repeats and labelled with tritium was used as a probe in hybridization. All patients with acute leukemia had the same pattern of changes in hybridization of the bone marrow cells. It consists of silver grains clustering over 3q26, 8p12, 14q24. The pattern may reflect amplification transposition of Alu-family DNA repeats in the human genome connected with cellular differentiation or malignant transformation of blood cells.     

The development and testing of test systems for the determination of herpes simplex virus and cytomegalovirus antigens by lanthanide immunofluorescence analysis

On the basis of the lanthanide immunofluorescent assay (LIFA) test systems for the determination of herpes simplex virus (HSV-LIFA) and cytomegalovirus (CMV-LIFA) antigens have been developed. The test system HSV-LIFA includes the sandwich of monoclonal antibodies (McAb) HSV A.3.3. or B-11 to type 2 HSV, strain BH; the test system CMV-LIFA includes the sandwich of rabbit McAb to CMV, strain AD 169. The approbation of the test systems has revealed that they insure the specific detection of HSV and CMV antigens in clinical specimens (urine, blood, liquor, saliva), the LIFA results well correlating with the data on the isolation of viruses in cell cultures, with the results obtained by other diagnostic methods and with clinical manifestations of diseases. LIFA has been shown to be more sensitive than the enzyme immunoassay.     

Identification of a protein with properties of tenascin in the matrix of embryonal cartilage tissue and in a culture of human embryonal fibroblasts]

A protein with properties of tenascine has been discovered in extracellular matrix of human embryonic cartilage tissue. Its Mr, as determined by electrophoresis in PAAG with SDS, is 205 and 195 kDa. Immunoblotting with monoclonal antibodies raised against tenascine, revealed 2 precipitation bands, which permitted to identify the discovered protein as tenascine. This is the first report of detection of tenascine in rib cartilage matrix of human embryos. A protein identified as tenascine, has been also detected in a culture of human embryonic fibroblasts.     

HLA-DR-restricted cytotoxicity of cytomegalovirus-infected monocytes mediated by Leu-3-positive T cells

Mononuclear leukocytes from 14 cytomegalovirus (CMV)-seropositive and six CMV-seronegative normal healthy donors were treated with soluble CMV antigen for 5 days to generate cytotoxic T lymphocyte (CTL) activity. CMV-antigen-stimulated lymphocytes from CMV-seropositive but not CMV-seronegative donors lysed autologous peripheral blood monocyte targets infected with CMV in 13 of 14 donors (mean percentage of virus-specific lysis = 19.0 +/- 4.5%, effector to target ratio of 50:1). Freshly donated, unstimulated lymphocytes displayed little or no lysis of CMV-infected monocytes. Lysis was virus specific in that CMV-stimulated CTL did not kill herpes simplex virus-infected monocytes. The mean level of lysis of CMV-infected autologous targets was equivalent to that of HLA-DR-matched targets (20.0 +/- 8.0%), and was significantly greater than that of HLA-A/B-matched targets (6.3 +/- 2.5%, p less than 0.035) and HLA-mismatched targets (3.3 +/- 2.5%, p less than 0.01). Enrichment for T cell subsets with the use of selective depletion methods with monoclonal antibodies showed that CTL activity against autologous and HLA-DR-matched allogeneic targets was present predominantly in Leu-3-positive T lymphocytes. These results show for the first time that short term stimulation of heterogeneous lymphocytes from CMV-seropositive donors with CMV antigen can generate CMV-specific, Leu-3-positive CTL that are primarily restricted in their activity to autologous and class II, HLA-DR-matched targets. Our findings suggest a role for Leu-3-phenotypic CTL in immunity to CMV, and provide a model for analysis of this antiviral effector function during immunodeficient states.