Gary R. Armstrong

Persisting Illness and Fatigue in Adults with Evidence of Epstein-Barr Virus Infection

Clinical, serologic, virologic, and immunologic evaluations for 31 adults with chronic illness and fatigue suggested that 23 had persisting Epstein-Barr virus infection. Among these 23 patients, cellular immune mechanisms were generally normal, but 4 had mild immunoglobulin deficiencies. However, 20 patients had abnormal serologic profiles specific for Epstein-Barr virus shown by significantly elevated titers of antibodies to the viral capsid antigen or early antigen, or by a deficiency of late-appearing antibodies. In 11 of 15 patients tested, circulating immune complexes were found. Circulating interferon was not found in 18 patients tested, but the activity of 2-5 oligoadenylate synthetase, an interferon-induced enzyme, was increased in 5 patients studied. Of 19 patients, 18 had persisting suppressor T-cell activity typically found in patients recovering from acute infectious mononucleosis. We believe that the Epstein-Barr virus may be associated with chronic illness in adults.

Epstein-Barr Virus in the Bone Marrow of Patients with Aplastic Anemia

STUDY OBJECTIVE To determine whether Epstein-Barr virus is present in the bone marrow of patients with aplastic anemia. DESIGN Assay of fresh and fixed bone marrow specimens for Epstein-Barr virus using immunofluorescence for nuclear antigen, Southern analysis with an Epstein-Barr virus specific probe, and in-situ hybridization. SETTING Governmental medical referral center. PATIENTS Five patients were studied prospectively: three who previously had infectious mononucleosis, one with a recent viral pneumonitis, and one who was asymptomatic. Stored DNA samples from other patients with aplastic anemia were also screened. MEASUREMENTS AND MAIN RESULTS Epstein-Barr virus DNA and protein were detected in the bone marrow of 5 patients studied prospectively and in 1 of 40 patients studied retrospectively. As estimated by in-situ hybridization, about 3% to 5% of marrow cells were infected with virus in those patients who had not received acyclovir. In contrast, Epstein-Barr virus DNA was not detected in peripheral blood DNA of these patients, nor were Epstein-Barr virus proteins or DNA found in the bone marrow of normal donors, patients with other hematologic diseases, or in 1 patient with acute infectious mononucleosis. Analysis of DNA fragments by hybridization with Epstein-Barr virus probes showed a pattern dissimilar to the type of Epstein-Barr virus usually associated with infectious mononucleosis. CONCLUSIONS Aplastic anemia may be associated with Epstein-Barr virus more commonly than suspected by history. Localization of the virus in the bone marrow supports a causative role for Epstein-Barr virus in bone marrow failure.

Effects of 5-tungsto-2-antimoniate in oncogenic DNA and RNA virus-cell systems.

Abstract We examined the effects of the heteropolyanion 5 -tungsto- 2 -antimoniate on Herpes virus saimiri , Epstein-Barr virus, purified murine and avian viral reverse transcriptases, and on highly purified DNA polymerases α and β from lymphoblastoid cells producing Epstein-Barr virus or Herpes virus saimiri . 5 -tungsto- 2 -antimoniate enhanced the replication and cytopathic effect of Herpes virus saimiri when the virus was present prior to treatment with the compound. Moreover, a rhesus cell line chronically infected with Herpes virus saimiri showed enhancement of cytopathic effect, early and late viral antigens and virus production. In contrast, the compound had no effect on EBV replication or antigen production in lymphoblastoid cell lines, whether the EBV was transforming or nontransforming. Highly purified Rauscher leukemia virus and avian myeloblastosis virus reverse transcriptases were inhibited to different degrees with a greater effect on the mammalian enzyme. The great susceptibility of the murine reverse transcriptase may explain the inhibition of de novo infection, with murine oncornavirus, of cells in medium containing the compound. Highly purified DNA polymerases α and β from P3HR- 1 and MLC- 1 cells also showed differential inhibition with a greater effect on the β enzymes. The different effects of this compound on oncogenic DNA and RNA virus systems suggest a complex mechanism of action and require further study. In the case of HVS, TA may interfere with the synthesis or action of a cellular product inhibitory for virus replication.

Mapping of the epitopes of Epstein-Barr virus gp350 using monoclonal antibodies and recombinant proteins expressed in Escherichia coli defines three antigenic determinants.

The Epstein-Barr virus (EBV) major surface membrane antigen (MA), gp350/220, induces antibodies that neutralize virus infectivity in vitro. The MA glycoprotein is encoded by nucleotides 1784 to 4504 of the BamHI L fragment of the EBV genome. To define the antigenic epitopes on gp350, sequences encoding portions of the protein were cloned into an Escherichia coli expression system and eight recombinant clones were generated, two overlapping clones representing the C terminus and six overlapping clones representing the N terminus. The epitopes expressed by the recombinant proteins were mapped using 14 anti-MA monoclonal antibodies (MAbs) in a dot blot immunoassay. One of the MAbs reacted with clones that express the C terminus of gp350 and three others reacted with clones expressing the N terminal portion of the protein; the remaining MAbs tested were not reactive with the cloned proteins. The data identify three antigenic determinants on gp350. DNA sequences encoding these epitopes are located between nucleotides 1980 and 2307, 3186 and 3528, and 3528 and 3576 of the BamHI L fragment. In an attempt to elicit neutralizing antibodies, rabbits were immunized with gel-purified recombinant proteins from four of the clones. Neutralization assays indicate that the proteins expressed by these clones do not induce in vitro virus-neutralizing antibodies.

Characteristics ofHerpesvirus saimiri-induced lymphoma cells in tissue culture

SummaryLymphoblastoid cells were cultured from twoHerpesvirus saimiri (HVS) inoculated white-lipped marmosets and from one HVS-inoculated owl monkey. Cells from all three animals grew clumped in suspension. The cells from both species were diploid in chromo-some number and showed no unusual chromosomal abnormalities. The marmoset cell line examined was chimaeric. The marmoset cells lacked HSV-associated antigens as determined by immunofluorescence, and no evidence for the presence of virus was found by either infectivity assays or electron microscopy. Cocultivation of these cells with Vero cells resulted in cytopathology and the recovery of complete, infectious virus. The owl monkey lymphoid cells were positive to a small degree for both viral antigens and infectivity. The cells were resistant to rechallenge with HVS. Cocultivation of these cells with Vero cells led to the development of cytopathology and an increased yield of virus.

Increased Infectivity of Oncogenic Herpes Viruses of Primates with Tumor Promoter 12-O-Tetradecanoylphorbol-13-Acetate

Abstract Cell cultures of simian and human origin infected with two strains each of herpesvirus saimiri (HVS) and herpesvirus ateles (HVA) were compared for production of infectious virus and early and late antigens (EA, LA) in the presence and absence of the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). Second, Epstein-Barr virus (EBV) infected human and simian lymphoblastoid cell lines of high and low cell passages were also compared for enhanced production of early antigen and virus capsid antigen (VCA) using two concentrations of TPA, owl monkey kidney (OMK) and squirrel monkey lung. Cell cultures infected with HVS and HVA with 20 ng/ml of TPA exhibited higher percentages of early and late antigen producing cells and contained 1.0-1.6 logs more virus. Such cells also had earlier cytopathic effects, larger plaques, and a 3-fold increase in the number of plaques. The TPA also enhanced HVS-EA, and LA both in OMK and human skin fibroblast (HSF) cells. The enhancement of EA was approximately 17.0% more in OMK cells and 4.0% more in HSF. The HVS-LA was 22% higher in OMK and 6.0% higher in HSF cells. Pretreatment of OMK cells with TPA prior to HVS or HVA infection showed only a 0.5-0.7 log enhancement of virus. A dose-response of TPA in P3HRl cells showed that both 20 and 40 ng/ml doses were able to enhance EBV, EA, and VCA significantly with peak enhancement at 40 ng/ml. Higher doses of TPA (60 and 100 ng/ml) resulted in considerable cell death and reduction in antigen production. TPA (20 and 40 ng/ml) stimulated both VCA and EA antigen production in P3HRl and B95-8 cells, with lesser effects on other human and simian lymphoblastoid cells. However, the stimulation of EA and VCA with these doses of TPA varied for each cell line. Moreover, TPA-treated P3HRl, B95-8, and 407-I cells on the average also produced 1.0-1.5 logs more virus than the untreated cells. The higher percentage of EA-VCA production from P3HR1 and B95-8 cells and lower EA-VCA from other human and simian cells suggests that such virus-cell interaction may be influenced by in vitro passages.

Fatal lymphoproliferative disease in a common marmoset (Callithrix jacchus) following inoculation of Ag876 strain of Epstein-Barr virus and a tumor-promoting agent: Preliminary report

We report here the development of a fatal lymphoproliferative disease in a common marmoset (Callithrix jacchus) following inoculation of the Ag876 strain of Epstein-Barr virus (EBV) and of the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA). The marmoset showed antibodies against EBV capsid and early antigens (VCA, EA) but not against nuclear antigen (EBNA). EBNA was detected in impression smears from lymph nodes and liver, and the presence of the EBV genome was detected from the same organs by hydridization techniques. To our knowledge this is the first report of the oncogenicity of this strain of EBV. The need for further studies to assess the in vivo interaction between EBV and tumor promoting agents in the development of malignancies is stressed.

Variations in the immune response to herpesvirus saimiri in squirrel and rhesus monkeys

Humoral and cell-mediated immunity (CMI) to herpesvirus saimiri (HVS), an oncogenic lymphotropic herpesvirus, was studied in squirrel and rhesus monkeys. Natural antibody to HVS was found in five of six squirrel monkeys but there was no evidence of specific CMI directed against HVS. Rhesus monkeys did not show natural antibody or CMI against HVS antigens. Immunization with HVS, however, produced both antibody and specific CMI in the rhesus monkeys, but no CMI developed in the squirrel monkeys. These findings are important in the development of animal models for the treatment of tumors associated with lymphotropic herpesviruses.

Das nasopharyngeale Karzinom (NPC): Prognostische Aussagekraft des antikörperabhängigen zellulären Zytotoxizitätstestes (ADCC)

SummaryThe nasopharyngeal carcinoma (NPC) occupies a special position of the ENT-malignomas because the main part of these tumors shows a linkage to an Epstein-Barr-virus infection (EBV). The reactivity of the human immunological system against these tumors is well known and is the basis for using virological and immuno-serological data for diagnosis and control of therapy even as control of illness course [1–3].Specific testing of cellular immuno-reactivity against the tumor are: intracutaneous testing with isolated NPC-tumor antigens, the makrophage-inhibition-test (MIT) or measuring of the antibody-dependent-cellular-cytotoxicity (ADCC) [4] against EBV.We tested patients with NPC (n=21), patients with histomorphological identic tumors of the mesopharynx (n=15), 15 patients with inflammations of the epipharynx, even as healthy persons (n=20).ADCC-testing has been repeated before therapy and in regular intervals during therapy.ADCC-mean-titers of the different groups have been correlated with EBV-titers during therapy and tumor stage [5].

Interaction of N-methyl-N-nitro-N-nitrosoguanidine (MNNG) with owl monkey kidney cells in enhancing the yields of Herpesvirus saimiri (HVS) and its antigens

Abstract Pre- and posttreatment with N-methyl-N′-nitro-nitrosoguanidine (MNNG) of owl monkey kidney (OMK) cells infected with Herpesvirus saimiri (HVS) resulted in one to three logs higher yields of virus, depending upon the dose of MNNG. A higher percentage of cells also showed HVS early antigen (EA) and late antigen (LA) by immunofluorescence when OMK cells infected with HVS were fed with medium containing MNNG. The high yields of HVS were also observed by electron microscopy. MNNG did not induce HVS-EA in HVS nonproducer lymphoblastoid T cells, nor did it enhance TPA-induced EA to LA. The data suggest that MNNG could be useful in obtaining high yields of virus and/or antigen-producing cells for immunofluorescence or other biochemical experiments, especially from those strains of HVS which grow poorly in vitro. The interaction of MNNG and HVS could also be useful for in vitro transformation or in vivo enhancement of the malignant process.