Detection of human serum antibodies to the BFRF3 Epstein-Barr virus capsid component by means of a DNA-binding assay.

A novel assay for antibodies to an immunodominant component of the Epstein-Barr virus (EBV) capsid antigen (VCA) complex was developed by creation of a chimeric protein containing the DNA-binding domain of the yeast GAL4 protein fused to the capsid antigen encoded by the BFRF3 gene of EBV. GAL4-BFRF3 antigen fusion protein bound specifically to a duplex DNA oligonucleotide containing GAL4-binding sites. Antibodies to the antigen were revealed by retardation of the electrophoretic mobility of the DNA-protein complex. Antibodies to the BFRF3 component of VCA became detectable approximately 2 months after onset of infectious mononucleosis. Kinetics of the antibody response to BFRF3 were identical using supershift or immunoblotting assays. Concordance between the DNA-binding assay and the classical indirect immunofluorescence assay for antibody to VCA was 97%. The GAL4 epitope assay is applicable for detection of antibodies to many cloned gene products.

Geographical and temporal distribution of babesial infection in Connecticut.

Human babesiosis was first recognized in Connecticut in 1989, nearly 15 years after Lyme disease, a similarly transmitted spirochetosis, was detected in the state. To determine the seroprevalence for the babesial pathogen and whether it was recently introduced, we used an indirect immunofluorescence assay to test for Babesia microti antibody in 1,285 Connecticut residents. Four groups were studied: I, people seropositive for Lyme disease, tested from 1986 to 1989; II, randomly selected outpatients tested in 1989; III, college students residing in Connecticut, tested from 1959 to 1989; and IV, healthy people without tick exposure or Lyme disease, tested in 1989. Babesia seropositivity was significantly higher in group I (9.5%; n = 735) than in groups II (2.6%; n = 304, P less than 0.0001) and III (1.0%; n = 206, P less than 0.0001) but not group IV (2.5%, n = 40). Babesia seropositivity for group I ranged from 9.2 to 10.2% between 1986 and 1989, and Babesia seropositivity for group III ranged from 0% between 1959 and 1985 to 2.9% between 1986 and 1989. There is a considerable risk of babesial infection among residents of the Connecticut mainland who are seropositive for Lyme disease, a risk that appears to have remained constant over the past 5 years.

Clinical and serological features of human herpesvirus-6 infection in three adults.

3 adult patients with serological evidence of human herpesvirus-6 (HHV-6) infection had mild, afebrile illnesses with nonspecific symptoms. In each case, the characteristic clinical feature was the presence of enlarged, bilateral, non-tender, anterior and posterior cervical nodes early in the illness which persisted for up to 3 months. HHV-6 IgG antibody reciprocal titres of 160 to 2560 were found during acute infection, and decreased to a titre of 10 in 1 patient 3 years later. IgM responses were detected at low reciprocal titres (10) in 2 patients and disappeared after several months.

Fragment length polymorphisms among independent isolates of Epstein-Barr virus from immunocompromised and normal hosts.

DNA restriction fragment length polymorphisms of Epstein-Barr virus (EBV) DNA were used as a molecular epidemiological tool to study multiple isolates of virus from the same and different individuals. We studied 35 EBV isolates: 19 from seven immunocompromised children and 16 from seven college students with mononucleosis. Analysis of the fragment length polymorphisms in this collection of isolates permitted several conclusions. Sites of polymorphism were most often encountered in regions with repetitive DNA. Epidemiologically unrelated patients harbored viruses that could be readily distinguished; by contrast, two infants and their mothers harbored similar viruses. Isolates from different sites in the same patient were similar. Variations between different clinical isolates of EBV mimic those found between different laboratory strains of the virus. Fragment length polymorphisms thus provide a useful marker for studying transmission and pathogenesis of EBV infections.

Some recent developments in the molecular epidemiology of Epstein-Barr virus infections.

We have applied two different recombinant DNA techniques to the study of the epidemiology of Epstein-Barr virus infections. In the first application, cloned subfragments of viral DNA were used as probes to detect EBV DNA in a variety of lymphoproliferative disorders and in lymphoid cell lines. Patients who are epidemiologically unrelated harbor EBV genotypes which can readily be distinguished from each other. Patients who are epidemiologically related (such as mothers and infants) have similar EBV genotypes. Some patients, especially those who are immunocompromised, are infected with two distinct genotypes. In the second application, we have examined the immune response to specific EBV antigens expressed from small cloned viral DNA subfragments. We have identified a group of patients with presumed chronic EBV infection who selectively fail to recognize one subcomponent of the EB nuclear antigen complex.

Selective lack of antibody to a component of EB nuclear antigen in patients with chronic active Epstein-Barr virus infection.

The sera of 12 patients with presumed chronic active Epstein-Barr virus (EBV) infection lacked antibody to a component of the Epstein-Barr nuclear antigen (EBNA) complex encoded by the BamHI K fragment of viral DNA. This anomaly, detected in approximately 18% of sera obtained from patients with a diagnosis of "chronic mononucleosis," was more often found in patients with severe disease (approximately 32%) who had objective clinical findings and markedly elevated antibody titers to EBV replicative antigens than in those patients with the "fatigue syndrome" (10%). The lack of antibody to the K nuclear antigen is specific because most of those who did not have antibody to the K antigen made antibody to other latent nuclear (EBNA 2) antigens or nuclear early antigens. Such patients are thus able to lyse immortalized cells, release nuclear products, and present them to the immune system. Three hypotheses are suggested to explain the lack of antibody to the K antigen: a viral mutation, a failure of immune recognition, or lack of in vivo expression of the antigen due to extensive viral replication. Lack of antibody to one component of EBNA may serve as an objective serological marker for certain patients with chronic EBV infection.

Enzyme-linked immunosorbent assay of antibodies to Epstein-Barr virus nuclear and early antigens in patients with infectious mononucleosis and nasopharyngeal carcinoma.

A sensitive enzyme-linked immunosorbent assay was used to measure titers of IgG antibodies against bacterially synthesized Epstein-Barr virus nuclear antigen and early antigen in sera from 100 healthy North Americans, 40 North American patients with infectious mononucleosis, and 48 Asian patients with nasopharyngeal carcinoma. All healthy persons previously infected with Epstein-Barr virus had antibodies to nuclear antigen, and 70% had very low but detectable antibody titers to early antigen. In contrast, patients with mononucleosis had nondetectable or very low levels of antibodies to nuclear antigen and high antibody levels to early antigen. High levels of antibody to early antigen also were seen in patients with nasopharyngeal carcinoma, and a decrease in this response during the first 12 months after diagnosis and treatment was a significant prognostic indicator of survival. The probability of survival was 75% for patients whose antibody concentration to early antigen remained constant or decreased, and near 0% for patients with increasing levels of antibody.

Antibody responses to two Epstein-Barr virus nuclear antigens defined by gene transfer.

By transfecting small fragments of Epstein-Barr virus (EBV) DNA into cells, we defined two nuclear antigens, termed M and K, and examined serum from 258 subjects for antibodies against these antigens. We hoped to learn whether such single-antigen systems would clarify the association of EBV with various diseases. Although reactivity to M antigen was found in only 14 per cent of healthy EBV-seropositive subjects, 90 per cent of Chinese and North African patients with nasopharyngeal carcinoma had antibody to M. Nearly all persons (96 per cent) who were EBV seropositive, as judged by their serologic reaction to a nuclear antigen encoded by the complete virus (EBNA), had a reaction to K antigen. However, serum samples from three patients with chronic active EBV infection did not react to K, even though the serum contained anti-M titers above 1:1000. Lymphoid cells from one such patient carried a normal gene for K and made K protein of correct size. Therefore, in this patient the absence of antibody to K had not resulted from a viral mutation that destroyed the K protein. These serologic studies show that some patients with chronic active EBV infection have an abnormal immune response to a specific viral gene product.

Infectious mononucleosis: a polyclonal B cell transformation in vivo.

We obtained spontaneous formation of Epstein-Barr virus-transformed B lymphocyte colonies from the blood of four patients with mononucleosis with the use of soft-agar medium. The colonies were propagated into separate cell lines and analyzed for their immunoglobulin secretion. Of 52 such lines, 44 produced immunoglobulin composed of a single class of heavy chain and single type of light chain. Among these clonal transformants, 27 (62%) of 44 produced mu-chain, 13 (29%) of 44 produced gamma-chain, and 4 (9%) of 44 produced alpha-chain. Analysis of light-chain secretion revealed that of the 44 cell clones secreting complete, monoclonal immunoglobulin products, 31 produced kappa-chain and 13 produced gamma-chain. One clone secreted mu-heavy chain and no light chain, an observation suggesting the clone is a pre-B cell phenotype. Seven lines derived from clones had aberrant patterns of immunoglobulin secretion that produced either two heavy chains or two light chains. B lymphocytes in different states of immunoglobulin gene expression are, therefore, transformed in vivo by Epstein-Barr virus.

Failure to demonstrate concomitant antibody changes to viral antigens other than Epstein-Barr virus (EBV) during or after infectious mononucleosis.

A search for antibody rises to viral antigens other than to Epstein-Barr virus, the causative agent, has been carried out in serial serum samples from 82 patients with infectious mononucleosis (IM). Fourfold or greater rises in titer rarely occurred and did did not cluster in time. No rises occurred to cytomegalovirus, only 1.2 percent to herpes simplex virus, and 8.5 percent to varicella zoster virus. Rises to measles antibody were found in 7.5 percent of patients and to rubella in 10.4 percent; these may represent natural infections or immunizations. A few patients also showed rises to respiratory viruses but there was no apparent connection to IM.

An enzyme-linked immunosorbent assay (ELISA) for measurement of heterophile antibody.

An enzyme-linked immunosorbent assay (ELISA) test has been developed for measurement of heterophile antibody. The microtiter test utilizes a bovine erythrocyte monolayer as antigen and anti-human IgM antiserum conjugated with horseradish peroxidase to measure the degree of binding of the heterophile antibody in the test serum with the erythrocytes. A single serum dilution yields quantitative results when read in a spectrophotometer. The ELISA test showed a sensitivity comparable with the immune adherence hemagglutination assay (IAHA) and other heterophile tests, good reproducibility, and high specificity.

Infectious mononucleosis: observations on transmission.

Epsten-Barr virus oropharyngeal shedding has been demonstrated in infectious mononucleosis patients many months after acute illness and long after the disease hallmarks, atypical lymphocytes and heterophile antibody, have disappeared. Extracellular virus is present more frequently in saliva than in other oropharyngeal samples. Prolonged excretion of EBV in asymptomatic carriers explains the difficulty in tracing case-to-case spread and increased transmissibility in age groups in which salivary exchange is high.

EBV-IgA and new heterophile antibody tests in diagnosis of infectious mononucleosis.

An evaluation has been made of the EBV-IgA tests and the immune adherence hemagglutination assay (IAHA) as compared with standard diagnostic procedures in 119 serial sera from 22 clinical and 113 sera from 42 subclinical cases of EBV infectious mononucleosis. EBV-IgA antibody was demonstrable in 86.4 per cent of patients using EB3 cells as antigen and in 68.2 per cent with P3/HRIK cells. For heterophile antibody the IAHA test was more sensitive, gave higher titers, and was positive longer than the standard absorbed horse or sheep RBC tests in both clinical and subclinical EBV infections.

Expression of Epstein-Barr viral early antigen in monolayer tissue cultures after transfection with viral DNA and DNA fragments.

Antigens associated with the Epstein-Barr virus (EBV) replicative cycle were found in the nucleus and cytoplasm of human placental, Vero, BSC-1, and owl monkey kidney cells transfected with EBV DNA prepared from several different strains of virus. The number of antigen-positive nuclei increased when transfection was followed by cell fusion induced by inactivated Sendai virus. About 1,200 antigen-positive foci were induced per micrograms of EBV DNA. On the basis of their reactivity with various well-characterized human sera, it appears that the antigens are part of the early antigen complex. None of the four restriction endonucleases, EcoRI, HindIII, SalI, and BamHI, destroyed the ability of EBV DNA to induce early antigen. However, only SalI seemed to leave intact the full spectrum of antigen expression by the HR-1 and FF41 strains of EBV DNA. By means of transfection with recombinant DNA plasmids containing different EBV (FF41) DNA fragments regenerated by EcoRI, we showed that the coding region for early antigen was at least partially contained on the 17.2-megadalton EcoRI B fragment.

Plasmacytic differentiation of circulating Epstein-Barr virus-infected B lymphocytes during acute infectious mononucleosis.

During the acute phase (1 wk of symptoms or less) of infectious mononucleosis (IM), 70--80% of circulating Epstein-Barr virus nuclear antigen (EBNA)-positive cells have differentiated toward plasma cells. Thus the characteristics of the infected cells in the majority of IM patients during early disease are indistinguishable from EBNA-positive tumor cells of a previously reported child who developed lymphoma during IM. IgA and IgG were the most frequent and IgM the least frequent immunoglobulin isotypes detected in EBNA-positive cells. In acute disease EBNA was present in 5.5--20% of T cell-depleted blood lymphocytes but in the 2nd or 3rd wk of illness the number of EBNA-positive cells sharply decreased to 0.4--1.4%. At the same time the fraction of antigen-positive cells containing cytoplasmic immunoglobulins also diminished, suggesting either that differentiation of infected cells was altered during the disease or that nondifferentiated antigen-positive cells had a survival advantage. Both the high proportion of plasmacytic EBNA-positive cells seen during acute disease and the apparent loss of differentiation by these cells later in disease may be regulated by host immunologic factors. Immunoglobulin-producing EBNA-positive cells may be the source of heterophile antibodies and other seemingly inappropriate antibodies usually found in serum during IM; however, increased numbers of noninfected plasma cells were present in some patients and may also be a potential source of these unusual antibodies.

Mitotic EBNA-positive lymphocytes in peripheral blood during infectious mononucleosis.

Infectious mononucleosis (IM) is usually a benign lymphoproliferative disease caused by Epstein-Barr virus (EBV). Although EBV induces a state of continuous proliferation in infected B lymphocytes in vitro, the most prominent lymphoproliferation during IM is of activated, or atypical, T lymphocytes presumably responding to the virus or virus-infected cells. However, EBV genome-carrying cells are known to be circulating during IM, as cultured peripheral blood leukocytes from patients with the disease give rise to continuous lymphoblastoid cell lines, each cell of which contains the EBV genome and expresses the EBV determined nuclear antigen (EBNA). The proposal that EBV-infected cells in IM blood are not endowed with enhanced growth potential but are merely latently infected is supported by demonstrations that cells infected in vivo enter a viral replicative cycle when placed in vitro and that most cell lines derived from cultured lymphocytes of IM patients are infected by virus released in vitro. However the cells could also be capable of proliferation in vivo, since virus production and transformation are not mutually exclusive properties of EBV-transformed cells. Recently, EBNA has been detected in a very small fraction of peripheral blood lymphocytes of IM patients after T cells were first removed and this has been interpreted to indicate that cell transformation occurs in vivo during IM. The isolation of colonies of EBNA-positive cells from IM blood leukocytes cultures in soft agar suggests that at least some infected cells are capable of direct outgrowth into transformed cells. We report here direct evidence that circulating EBV-infected cells exhibit increased growth properties during IM.