Epstein-Barr virus in a malignant lymphoproliferative disorder of B-cells occurring after thymic epithelial transplantation for combined immunodeficiency.

A fatal disseminated polyclonal malignant lymphoproliferative disorder of B-cells (immunoblastic sarcoma) developed shortly after a second thymic epithelial peritoneal implant in a 5-yr-old girl with combined immunodeficiency. The immunodeficiency was characterized by low T-cell numbers and function, very low levels of thymic hormone, dysgammaglobulinemia, and an inability to mount a primary antibody or cell-mediated response to new antigens. At necropsy, the thymus fulfilled morphological criteria for thymic dysplasia. Epstein-Barr virus (EBV) antigen and DNA were identified in neoplastic infiltrates in the lymph nodes and thymus by immunofluorescence for the EBV nuclear antigen and by EBV-specific complementary RNA/DNA hybridization. No antibodies to nuclear antigen, early antigen, or viral capsid antigen of EBV were identified in the serum. The concurrence of these events suggests that the thymic epithelial implant itself may have been instrumental in the pathogenesis of this neoplasm. It is proposed that the thymus may have provided factors which indirectly potentiated the proliferation of EBV-infected B-cells, possibly by induction of nonspecific T-helper cells and perhaps through other thymic humoral factors. It is suggested that some forms of immunoblastic sarcoma, even when polyclonal, and especially those which arise in immunocompromised hosts, may, in some instances, represent an opportunistic form of EBV-induced B-cell neoplasia.

Epstein-Barr virus polymorphic B-cell lymphoma associated with leukemia and with congenital immunodeficiencies.

Polymorphic B-cell lymphoma seen in four patients with congenital immunodeficiencies and in two patients with leukemia receiving chemotherapy was associated with the Epstein-Barr virus (EBV). The tumors had characteristic histologic features: they were polymorphic consisting of a mixture of lymphoblasts and differentiated cells including plasma cells, and areas of hemorrhagic necrosis were prominent. The tumors were either polyclonal, monoclonal, or multiclonal. Patients with congenital immunodeficiencies who developed these tumors died despite radiotherapy, corticosteroids plus acyclovir, or a combination of intravenous (IV) immunoglobulins and alpha 2 interferon. Patients with leukemia recovered when immunosuppressive drugs were discontinued and leukemia has not recurred over a period of 2 and 4 years, respectively, in the two patients.

Comparison of two methods in the determination of the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to acyclovir and alpha-interferon.

Two methods, the colorimetric method (neutral red dye uptake), and DNA hybridization using a HSV thymidine kinase gene probe (TK) have been used to examine the sensitivity of 84 herpes simplex virus (HSV) type 1 and 2 clinical isolates to two antiviral drugs, acyclovir (ACV) and alpha-interferon (alpha-IFN). Using the colorimetric method, HSV isolates had ED50s ranging from 0.03 +/- 0.02 micrograms/ml to 0.164 +/- 0.03 micrograms/ml for ACV and 6.3 +/- 5.2 IU/ml to 55.0 +/- 11.4 IU/ml for alpha-IFN. With the DNA hybridization method, ED50s ranged from 0.033 +/- 0.012 micrograms/ml to 0.190 +/- 0.031 micrograms/ml for ACV and 8.5 +/- 5.0 IU/ml to 43.5 +/- 6.0 IU/ml for alpha-IFN. Two strains of HSV-1 were found to be resistant to very high concentrations of ACV (greater than 50.0 micrograms/ml). The values obtained by the two methods showed good correlation (r = 0.724, P = 0.002). Furthermore, our results demonstrate that the two methods are reproducible, reliable and the dye uptake assay is suitable for use in a diagnostic virology laboratory.

Herpes encephalitis. Rapid diagnosis and treatment with antiviral drugs.

The use of specific IgM antibodies and direct electron-microscopic examination of brain biopsies or vesicle fluid was tested as means of raped diagnosis in 6 cases of herpes simplex encephalitis seen consecutively in Montreal. In 2 of 3 biopsies herpes viruses were seen by negative staining of a cell extract within 1 hr. In the negative case, the biopsy was done almost 1 month after onset. In 2 additional cases herpes virus particles were found directly in the fluid of isolated vesicles. In the last 2 cases, who survived, the diagnosis of herpes encephalitis rested upon the demonstration of a greater than 4-fold rise in complement fixing herpes simplex virus antibodies in convalescent sera and upon the appearance late in the course of the encephalitis of specific antibodies in the cerebrospinal fluid. The early appearance of specific IgM antibodies contributed to the diagnosis in 4 of the 6 cases. Antiviral therapy was attempted in alternate cases (3 cases) but was not successful. Brain biopsy is rarely performed for diagnostic purposes but when prompt antiviral therapy is contemplated, the examination of the biopsy material for herpes virus particles by electron microscopy in negative staining and thin sections can rapidly and reliably confirm the diagnosis.

Standardization and kinetics of in vitro bovine blood lymphocyte stimulation with bovine rotavirus.

Two groups of 3-month old calves were immunized intramuscularly with attenuated bovine rotavirus and boosted 21 and 42 days later. The first group of three calves were vaccinated with live virus emulsified with incomplete Freund's adjuvant (IFA) and the second group was immunized with live virus suspended in phosphate buffered saline (PBS). Three other calves, serving as controls, were inoculated with PBS emulsified with IFA. The specific cell-mediated and antibody responses of the animals were studied. Preliminary analysis of in vitro peripheral blood lymphocyte transformation to bovine rotavirus determined optimal conditions as: 96 h culture period, 5 X 10(5) cells per culture in RPMI 1640 medium containing 10% heat-inactivated bovine fetal serum and the use of inactivated virus in the cell culture at a concentration of 5 X 10(6) median tissue culture infective dose before inactivation. Specific blastic stimulation was observed on calves immunized with the rotavirus emulsified with IFA after the second and third vaccine inoculation with stimulation index values varying from 2.00 to 5.73. Serum neutralizing antibody titers of 1/25,600 were also induced in the same calves. Calves immunized with rotavirus-PBS suspension developed a mean antibody titer of 1/1,600, but showed no specific lymphocyte stimulation. No increase in specific immune responses was detected in the control animals.

Giant cell pneumonia due to respiratory syncytial virus. Occurrence in severe combined immunodeficiency syndrome.

A 6-month-old male infant with a severe combined immunodeficiency syndrome was hospitalized for progressive respiratory distress. Examination during hospitalization disclosed widespread pulmonary infiltrates that did not respond to intensive therapy. The patient died eight days after admission. Autopsy disclosed Pneumocystis carinii pneumonia and widespread giant cell pneumonia. Respiratory syncytial virus (RSV) was grown from a lung specimen obtained at autopsy. Specific immunofluorescent staining of the cytoplasm of alveolar lining cells with RSV antiserum was demonstrated. The electron microscopic appearance of giant cells was compatible with RSV infection. The RSV should be added to the list of viruses causing giant cell pneumonia.

Immune response of pregnant heifers and cows to bovine rotavirus inoculation and passive protection to rotavirus infection in newborn calves fed colostral antibodies or colostral lymphocytes.

The efficacy of an adjuvanted bovine rotavirus vaccine in pregnant cattle (15 heifers and 2 cows) was studied. Each of 4 animals was inoculated IM at 8, 5, and 2 weeks before parturition with a water-in-oil emulsion containing live purified bovine rotavirus, mineral oil, and a mannide oleate compound. Four other animals were treated identically, except that muramyl dipeptide was added to the virus preparation. Five additional animals were inoculated orally at the same time intervals with adjuvant-free viral suspension, and 4 other pregnant animals inoculated only with buffer served as uninoculated controls. Kinetic studies of the specific immune responses were determined by quantification of the rotavirus-neutralizing antibodies and by a rotavirus lymphocyte stimulation test in vitro. Results showed that only the emulsions induced marked enhancement of rotavirus antibody titers in the serum, colostrum, and milk of inoculated cows. Colostral and milk lymphocytes isolated from these cows had a positive in vitro proliferative response to rotavirus stimulation, which lasted at least 21 days after parturition. The values of the stimulation index obtained with the colostral/milk lymphocytes were higher than those of the blood lymphocytes, reflecting increased lymphocyte activity in the colostrum/milk. However, addition of muramyl dipeptide to the emulsion preparation did not exert any potentiating effect on the immune response to rotavirus. Calves fed for the first 5 days after birth with a rotavirus-immune cell-free colostrum supplement were protected from a rotavirus challenge exposure on the third day after birth. Virus was not detectable in their feces.(ABSTRACT TRUNCATED AT 250 WORDS)

The diagnosis of Epstein-Barr virus-associated polymorphic B cell lymphoma in immunocompromised patients: Review of methods.

Polymorphic B cell lymphoma and diffuse B cell lymphoproliferation associated with Epstein-Barr virus infection is increasingly reported in immunodeficient patients. Accurate diagnosis of these pathologies is essential because the appropriate treatment regimens for the patients in question differ from those for patients with other lymphoproliferative diseases. Two complementary techniques are currently used in the diagnosis and characterization of Epstein-Barr virus-associated B cell lymphomas and diffuse B cell lymphoproliferation. Immunofluorescence allows specific detection of Epstein-Barr nuclear antigens in lymphomatous tissue. Molecular hybridization with the Bam H1-W and/or Bam H1-NJ probes confirms the presence of the Epstein-Barr virus genome in tumour cells. The Bam H1-NJ probe is also useful in determining the clonality of the tumour and the replication mode, episomal or linear, of the viral genome. The polymerase chain reaction method allows detection of the Epstein-Barr virus genome within 24 h in these tumours and is more sensitive.

Viral attributes and host factors in carcinogenesis: herpesviruses.

This paper describes two different experiments of nature: 1) the persistence of unusual virus strains of Epstein-Barr virus (EBV) (which proved oncogenic in vitro) and cytomegalovirus (CMV) in lymphoid cells following congenital or early acquired infection; 2) the occurence of multiple cases of Burkitt's lymphoma and nasopharyngeal carcinoma in one family. All the members of this family were EBV viral capsid antigen (VCA) and nuclear antigen (EBNA) antibody positive. The two patients with nasopharyngeal carcinoma had high titers of EBV-VCA, EA, and EBNA antibodies. The only member of this family having EBV early antigen (EA antibodies in addition to the patients with tumors was the mother. Borderline IgA deficiency was documented in 3 members of this family. These findings illustrate the importance of host factors (intracellular resistance to transformation and secondarily, immunological surveillance) in the outcome of the host-virus challenge whether cancer or infectious disease is the outcome. Extensive studies of these cases may provide the best insight into the mysteries of viral oncogenesis.

Biomolecular analysis of a defective nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection.

A virus recovered from the saliva of a child with chronic active Epstein-Barr virus (EBV) infection for 8 years was shown to induce EBV early antigen (EBV-EA) in Raji cells and to be expressed into EBV-EA in fresh EBV-negative peripheral blood leukocytes. However, it did not replicate its DNA. Oropharyngeal epithelial cells scraped from recurrent mouth lesions were similarly positive for EBV-EA. DNA extracted from these cells and digested with BamHI contained a 6-kilobase-pair fragment homologous to BamHI fragment V and B1 EBV DNA probes. Furthermore, Southern blots of the BamHI and EcoRI digests of the DNA extracted from the cell lines of the patient (transformed with EBV strain B95-8) and of her mother (spontaneous) revealed, in addition to the expected BamHI G, H, H2, and B1 fragments used as probes, additional shorter ones of a presumably endogenous defective virus.

Radioimmunoprecipitation in the diagnosis of chronic active Epstein-Barr virus infection.

Chronic active Epstein-Barr virus (EBV) infection occurs sporadically in a small fraction of individuals infected with EBV. A clear definition of the disease and an unambiguous diagnostic test are still lacking. In an attempt to identify a serologic marker to facilitate the diagnosis, immunoblot and radioimmunoprecipitation assay (RIPA) were compared with standard immunofluorescence on 39 available sera. Results by RIPA revealed that antibodies to a 120 kDa viral protein correlated with the presence of chronic active EBV infection; these antibodies were not detected in sera from other EBV-seropositive individuals, with the exception of one of two patients with ataxia telangiectasia. Also, RIPA was the most sensitive technique for detecting EBV antibodies in sera weakly or doubtfully positive for antibody to EB viral capsid antigen by indirect immunofluorescence. All these sera had antibodies to the 150 kDa protein, also known as p160, the major viral capsid antigen.

DNA polymorphism among isolates from multiple sites of a patient with chronic herpes simplex virus, type 1 infection.

DNA polymorphisms among independent isolates of herpes simplex virus (HSV) type 1 were studied from a 7-year-old male patient with recurrent infections of the skin and internal organs. In the patient's serum, HSV antibodies could not be detected by complement fixation, enzyme-linked immunosorbent assay (ELISA), or neutralization tests. ELISA tests for the presence of antibodies to human immunodeficiency virus were also negative. One HSV isolate was obtained from mesenteric nodes biopsied in 1983; one from skin in 1984; and three (postmortem) from brain, lungs, and liver in 1985. Restriction enzymes Eco RI, Bgl II, Hind III, Kpn I, and Bam H1 digestion patterns of the five isolates were similar. However, Sal I digests of isolates from skin, mesenteric nodes, lungs, and liver showed variations that were distinct from that of the brain isolate. Although Sal I digests of skin, mesenteric nodes, lungs, and liver isolates share a common variation in lacking F and G, the liver isolate can be further differentiated because of the gain of a restriction site on the H fragment. Thus, the three distinct variants observed were the isolates from brain (variant 1); from skin, mesenteric nodes, and lungs (variant 2); and from liver (variant 3). The fragments involved in variations among these isolates (presence or absence of Sal, G and H) are from the unique short and long regions (invariable regions) of the genome and therefore do not show heterogeneity in size. The extent of variation among these isolates is less than that seen among epidemiologically unrelated strains, suggesting that they originated from a single infecting strain, probably the brain isolate.

[Fatal infectious mononucleosis in a leukemic child in remission]. / Mononucléose infectieuse fatale chez une enfant leucémique en rémission.

The authors report the case of a 5 year-old girl who died from primary Epstein-Barr virus infection while in remission from acute lymphoblastic leukemia. There was a diffuse, polymorphic and polyclonal proliferation of B lymphocytes, associated with a strong histiocytic reaction with hemophagocytosis. The role of Epstein-Barr virus was ascertained by the appearance of specific antibodies and the finding of EBNA antigen on liver smear.

Lytic, nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection.

A new wild-type isolate of Epstein-Barr virus (EBV) was identified in follow-up studies of a case of chronic active EBV infection in an 8-year-old girl who had high titres of antibody to viral capsid antigen and early antigen (EA) (greater than 20 480 and 2560 respectively), persistent splenomegaly and abnormal immunologic features. More than 10 throat washings from this patient failed to transform cord blood lymphocytes (CBL), but at least 7 were able to induce EA in Raji cells. Supernatants from cultures of the lymphoblastoid cell line obtained by in-vitro infection of this patient's leukocytes with the B95-8 strain of EBV revealed a herpesvirus particle when examined by electron microscopy. The same supernatants were unable to transform CBL but could induce EA in Raji cells upon superinfection. In 30 or more trials the patient's lymphocytes never transformed spontaneously but did become positive for EBV nuclear antigen and EA in the first week of culture at least twice. Parallel studies performed on the father of the patient yielded similar results. This, then, is the first report documenting lytic activity associated with a wild-type EBV isolate.

Rash associated with Coxsackie A9 infection.

Coxsackie A9 virus was identified by the authors during the fall of 1965 in Montreal in six children with fever and exanthem. Three of the six children were siblings. The exanthem was centrally distributed as described by Lerner et al. and consisted of discrete maculopapules 3 to 4 mm. in diameter. The viral agent was recovered and identified in tissue culture in five cases, while in the sixth Coxsackie type-A lesions were produced in suckling mice. Serological confirmation was obtained in two patients from whom sera were available. In contrast, no exanthem was observed in three older patients with a diagnosis of aseptic meningitis associated with Coxsackie A9 virus. In only one of 16 patients with Coxsackie B virus infection was an exanthem observed during the same period.The true incidence of Coxsackie A9-associated exanthems is difficult to determine because of the benign nature of the disease.

Persistence of both human cytomegalovirus and Epstein-Barr virus genomes in two human lymphoblastoid cell lines.

By DNA-DNA reassociation kinetic analysis, less than one genome equivalent per cell of human CMV-DNA was found in two lymphoblastoid cell lines, one derived from the peripheral blood of a congenitally infected male infant at the age of 21 months (D4 cell line), the other obtained by co-cultivation of lethally X-irradiated cells from the 9-month lymphoblastoid cell line previously described by Joncas et al. (1975) with cord blood leukocytes of a female newborn (M1 cell line). Human CMV antigens could not be detected and virus could not be rescued from these cells by co-cultivation with fully permissive human fibroblasts. It may be that the CMV-DNA is defective. Epstein-Barr virus DNA as well as EBNA and EBV-EA antigens were present in these cell lines. Both lines express surface markers characteristic of thymus-independent, B lymphocytes. The CMV-DNA of the CMV-DU strain, isolated from this infant's urine five times successively from the age of 1 day to 30 months, appears to be closely related to the DNA of the AD-169 strain by reciprocal hybridization and by electrophoretic pattern analysis of the restriction enzyme cleavage products. Experimental attempts to transform cord blood leukocytes with this urine strain of CMV before or after u.v. irradiation have so far failed.

Improved Epstein-Barr virus immunoglobulin M antibody test.

The successful demonstration of Epstein-Barr virus immunoglobulin M (EBV IgM) antibody in human sera has been accomplished to date by at least four groups of workers. Many, however, including ourselves, have had difficulty in getting reproducible results with the techniques described. The three-coat technique described by H. Schmitz and M. Scherer (1972) on both fractionated and unfractioned sera was adopted with minor modifications. The Hyland antihuman IgM antiserum used in the second coat was made specific by absorption on Cohn fraction II. This step in the procedure was found to be the single most important factor in arriving at reproducible results in the IgM test. The EBV IgM antibodies from our results to date with this method in 14 cases of heterophil-positive cases of mononucleosis appear short lived, lasting 2 months or less. These antibodies were found in only 2 of 18 selected non-mononucleosis cases, in both associated with EBV-viral capsid antigen antibody rise or seroconversion. The successful elimination of nonspecific fluorescence by a simple, inexpensive procedure and the possibility of testing unabsorbed, unfractionated sera directly will facilitate the use oe the EBV IgM antibody test in the future.

Limitations of immunofluorescence tests in the diagnosis of infectious mononucleosis.

The relative value of heterophil agglutinins (HA) and of specific EBV antibodies in the diagnosis of infectious mononucleosis (IM) was assessed in 108 cases of the disease and in 280 controls. Among the 108 cases 93 were HA-positive by sheep cells in at least one of their sera, while 15 were HA-negative by the same test. Among the 280 controls false-positive HA tests were not encountered except in eight cases with the horse cell microtitre tests. With one of the two slide tests at least two false-positive tests and 12 false-negative tests were also found but these sera had low titres in microtitre tests. The HA life-span was found to be unexpectedly long in a few cases, sheep cell HA lasting up to 8 to 10 months and horse cell HA up to 21 to 23 months.Many false-positive tests may therefore not be true false-positives and may result from the persistence of HA following unrecognized mononucleosis months before. Virtually all cases of IM had (or developed) antibodies to Epstein-Barr virus, viral capsid antigen (EBV-VCA), whereas only half of the controls were EBV-VCA-positive. The comparative analysis of nonspecific and specific test results in mononucleosis allows the following conclusions: (1) horse cell microtitre tests and the monospot test are more sensitive than sheep cell microtitre tests and the monotest; (2) false-negative results are occasionally seen with the latter tests but not with the former; (3) more false-positive results, however, are probably seen with the former tests; and (4) specific EBV-IgM and EBV-EA antibody tests are useful in the diagnosis of selected borderline cases of mononucleosis.