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Antibody reactivity to Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was investigated by means of the anticomplement immunofluorescence technique on sera from patients with Hodgkin's disease (HD) and from appropriate controls. Antibody levels to other EBV-determined antigens, i.e. viral capsid (VCA) and early antigens (EA), and to measles and rubella viruses, to cytomegalovirus (CMV), and to toxoplasma gondii were also measured. The results of anti-EBV antibody titrations demonstrated that anti-VCA, anti-EA and anti-EBNA reactivity was significantly higher in HD patients than in healthy subjects. There was no significant difference between the distribution of high rubella and measles antibody titers in HD and control sera. The GMT and the incidence of high titer anti-CMV and toxoplasma antibodies were greater in HD patients than in controls. The analysis of the data, according to histological subtypes, showed that the condition of lymphocyte depletion was associated in HD patients with the highest anti-EBNA antibody levels and the lymphocyte predominance with the lowest. This pattern seemed to be peculiar for anti-EBV reactivity, since anti-CMV and anti-toxoplasma antibody levels in the lymphocyte-depleted group of patients did not significantly differ from those of controls. No correlation was found between anti-VCA and anti-EBNA in individual sera of HD patients. This observation suggests that different mechanisms are probably responsible in HD for the release of EBV-related antigen from infected cells.

Transformation of human leukocytes by the QIMR- WIL strain of EB virus in vitro involves co-operation between adherent cells (probably macrophages) and non-adherent cells (predominantly lymphocytes). It was shown that the proliferating cells were derived from the non-adherent population. Unlike macrophages, autochthonous fibroblasts did not enhance proliferation when cultured with non-adherent cells, but apparently were inhibitory. A strain of allogeneic fibroblasts was not inhibitory. Inoculation of nonadherent cells with EB virus was essential for transformation, while inoculation of macrophages only was insufficient. It appears from these results that infection of non-adherent cells with QIMR- WIL EB virus induces some cells to proliferate, and that the enhancing role of the macrophage does not involve infection with virus.

Two lymphoblastoid lines, CCRF-CEM and HSB-2, with properties of malignant cells, derived from children with leukemia secondary to lymphosarcoma, have T-cell properties and lack Epstein-Barr virus antigens.

Fresh nasopharyngeal carcinoma (NPC) biopsies were treated in several ways to yield satisfactory cell preparations for detection of Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) by anti-complement immunofluorescence tests. If the cells were well dispersed (trypsinization or extensive mechanical dispersal) few or none of them were EBNA-positive. In contrast, if the biopsy preparations contained small to moderately sized tissue fragments (touch preparations or limited mechanical dispersal), nuclear staining was detected in nearly every cell at the margin of the fragments or in cell sheets protruding from them. The stained cells corresponded to the carcinoma cells when compared to histologically stained replicate cell preparations. Nuclear staining was obtaining with anti-EBNA positive sera [healthy donors, NPC patients, convalescents from infectious mononucleosis (IM)], shown to be free of antibodies to other nuclear antigens, but not with anti-EBNA negative sera (healthy donors or patients in the early acute phase of IM). These results confirm and extend previous reports that the carcinoma cells harbor EBV genomes. The implications of these findings are discussed.

A micro-51chromium-release assay was used to examine the possible relationship between antibodies cytotoxic to lymphoid cells and EBV infections of the donors. No correlation was found between sera capable of killing lymphoid cells pre-labelled with51 chromium and the presence or absence of antibodies to the viral capsid antigen (anti-VCA). Sera obtained from patients with infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma and Hodgkin's disease known to have elevated titers to anti-VCA showed cytotoxicity similar to control values. There appeared to be both heat-stable and heat-labile complement-dependent cytotoxic antibodies.

Previous reports have emphasized that the transitory antibody responses to Epstein-Barr virus (EBV)-induced early antigens (EA) in the course of infectious mononucleosis are usually directed against the D (diffuse) component of the EA complex. In this report clinical and serologic data have been presented on 14 patients with infectious mononucleosis who responded either solely with antibodies to the R (restricted) component of the EA complex or revealed anti-R after the initial anti-D responses had subsided weeks or months after onset of the disease. Anti-R persisted usually for many months , as many as 39, but in some patients it was no longer detectable in late follow-up serum spectimens. Although many patients had an unremarkable course of illness, others, mostly those with late anti-R responses, showed unusual or protracted clinical manifestations and several complained over periods of 4 to 28 months of recurrent symptoms resembling those experienced during the acute stage of disease. The presence of anti-R and the relatively high continuous titers of antibodies to EB viral capsid antigens (VCA) may reflect a more than usual, persistent EBV activity which, in turn, may account for the recurrent symptoms.

• A group of patients with a history of recurrent tonsillitis were observed during an acute episode to determine the cause of the infection. The microbial pathogen that was consistently implicated was the Epstein-Barr virus. Seventeen (65%) of 26 patients exhibited a substantial seroconversion to the early antigen of Epstein-Barr-virus-infected lymphoblastoid cells (P3HR-1). We conclude that there is a high incidence of tonsillitis associated with the Epstein-Barr virus. The propensity of the virus for the palatine tonsils, a rich source of B cells, is suggested. Furthermore, the value of monitoring early antigen titers to confirm the nature of the infection is apparent, bearing relevance to future studies of this virus. ( Arch Otolaryngol 101:552-556, 1975)

Biologic activities of extracellular Epstein-Barr virus (EB virus) from two laboratory strains, namely, P 3 J-HR-1 (P-H) from Burkitt lymphoma and B95-8 (B95) from infectious mononucleosis, were compared. Virus stocks from both sources contained approximately the same number of virions. Virus from the P-H line induced “early antigen” in six nonproducer EB virus genome carrier cell lines; virus from B95 did not induce “early antigen.” Extracellular virus from B95 regularly caused lymphocytes from human umbilical cords to form continuous lines (immortalization); P-H virus did not cause primary cultures of human lymphocytes to grow continuously. B95 virus stimulated DNA synthesis as determined by rate of incorporation of [ 3 H]thymidine into acid-insoluble material; P-H virus did not stimulate DNA synthesis. Pretreatment of lymphocytes with undiluted P-H virus inhibited immortalization and stimulation of DNA synthesis by B95 virus. The inhibitory properties of the P-H virus were sedimented at 100,000 × g and inactivated by heat and UV irradiation; interference by the P-H virus was neutralized by human serum with antibody to EB virus and not by antibody-negative human serum. The hypothesis most consistent with these results is that the P-H virus is defective in gene(s) needed for initiation of immortalization. We speculate that the absence of this gene allows early antigen to be expressed upon super-infection of nonproducer cell lines. The availability of two laboratory strains of EB virus which differ in biologic behavior provides starting material for analysis of the mechanism of lymphocyte immortalization by EB virus and of virus structural differences which affect immortalization.

The growth inhibition of human lymphoblastoid cells by human interferon preparations (HIF) and a mock-interferon preparation (Mock-IF) was studied. HIF was induced in suspensions of primary human leukocytes by Newcastle Disease Virus. Mock-IF was prepared from uninduced cell suspension of a comparable batch. The highest degree of inhibition of cell propagation was ob­ tained on the EBV producer line P3HR1, the inhibition of the cell lines Raji and SKL 1 was significantly lower, no effect was observed on the cell line RPMI 1788 originating from a healthy donor. Mock-IF also caused a growth inhibition but to a lower degree as HIF. HIF and Mock-IF are similar in the following properties: 1. Stability at pH 2 at 4 °C for four days, 2. sensitivity to tryptic digestion, and 3. molecular weights in the range between 5,000 and 40,000 daltons

Programming of the Epstein-Barr (EB) virus productive cycle was studied in synchronized nonproducer Raji cells and producer EB-3 cells after activation by 5-iododeoxy uridine (IdU) added for 60 min during the S-1 period of the cell cycle. The activated Raji cells synthesized only the virus-associated early antigen (EA) complex, while some activated EB-3 cells also synthesized virus structural antigens (VCA). Synthesis of EA occurred at a time which corresponded to the early G-2 period of the cell cycle. DNA synthesis after removal of the IdU was not required for subsequent synthesis of EA. The time of EA synthesis, including associated RNA and protein requirements, was the same in cells allowed to complete the S phase as in cells where DNA synthesis was inhibited by 1-B- d -arabino furanosylcytosine (ara-C) or hydroxyurea (HU) added at the time of removal of the IdU. The findings indicated that synthesis of EA was independent of the cells' S phase and suggested a possible association between EA synthesis and synthesis of cell G-2 proteins. Synthesis of EA in abortively infected Raji cells was followed by low levels of deoxycytidine (dCyt) incorporation which persisted for several hours. At least some EA-positive cells were also able to reach mitosis, indicating that synthesis of EA did not result in immediate cell death. Synthesis of VCA occurred 3–4 hr after synthesis of EA in approximately 50% of the activated EB-3 cells. Synthesis of VCA required a short period of DNA synthesis (approximately 60 min) after EA synthesis. The inability of approximately 50% of the activated EB-3 cells to synthesize VCA was ascribed to toxic effects of the incorporated IdU which precluded the cells from carrying out events required for initiation of the short period of DNA synthesis subsequent to completion of the S phase and EA synthesis. Those EB-3 cells which did progress to VCA synthesis showed productive replication of virus DNA which continued for several hours after the initial appearance of VCA.

Summary The effect of interferon on expression of Epstein-Barr virus (EBV) early gene functions was investigated. The ‘early antigen’ synthesis which follows either EBV superinfection of established lymphoid cell lines or 5′-iododeoxyuridine activation of the intrinsic EBV genomes harboured by these cells could be suppressed with interferon. In contrast, the spontaneous early antigen expression that occurs in a few per cent of the cells in the producer cell lines could not be blocked with interferon. The lymphoid cell lines tested differed in their ability to acquire an antiviral state after exposure to interferon. Several cell lines were also growth inhibited by the interferon preparations. The antiviral and growth inhibitory activities of different interferon preparations could not be separated by a number of criteria.

We have compared the properties of the DNA of Epstein-Barr virus (EBV) purified from HR-1 (EBV HR-1 DNA) and B95-8 (EBV B95-8 DNA) continuous lymphoblast cultures. Our data indicate that (i) the S suc of native EBV DNA relative to T4D DNA is 55S. Using the modified Burgi-Hershey relationship (5), we estimate the molecular weight of native EBV DNA is 101 (plus or minus the molecular weight of native FBV DNA by measurement of the length of 3) times 106. Estimation of the molecule relative to form II PM2 DNA yields a value of 105 (plus or minus 3) times 106. (ii) After alkali denaturation, less than 50% of EBV DNA sediments as a single band in alkaline sucrose gradients in the region expected for DNA of 50 times 406 daltons. (iii) Intact EBV HR-1 and EBV B 95-8 DNAs band at 1.718 g/cm3 and a smaller band (approximately 25% of the DNA) AT 1.720 G/CM3. (IV) EBV HR-1 DNA possesses greater than 97% of the sequences of EBV B95-8 DNA. Hybrid DNA molecules formed between (3H)EBV HR-1 DNA and EBV HR-1 DNA or EBV B95-8 DNA had identical thermal stability. EBV B95-8 DNA lacks approximately 15% of the DNA sequences of EBV HR-1 DNA. We interpret these data to mean that EBV B95-8 is derived from a parental EBV through loss of genetic complexity. This defect may be linked to the ability of EBV B95-8 to "transform" lymphocytes invitro.

Mononuclear peripheral blood leukocytes from 21 patients with infectious mononucleosis and 16 healthy controls were tested in a 51Cr-release assay for cytotoxicity against two human lymphoblastoid cell lines derived from the same donor. One line contained the Epstein-Barr virus (EBV); the other did not. Acute-phase leukocytes were significantly more cytotoxic against the EBV-infected cell line than were control leukocytes. Mean (+/- S.E.) lysis at a leukocyte-target-cell ratio of 100:1 was 10.6 +/- 1.6 per cent for patients and 3.4 +/- 0.6 per cent for controls (P less than 0.0005). Cytotoxicity correlated with the percentage of atypical lymphocytes. Cells of three patients with acute mononucleosis-like illnesses failed to show killing activity above those of normal controls. Cytotoxicity against the EBV-negative line was not significantly different for each group. The finding of cytotoxic cells in infectious-mononucleosis patients with atypical lymphocytes suggests that these cells operate in vivo to limit the proliferation of altered EBV-transformed B lymphoblasts.

Redistribution and consequent cap formation of Epstein-Barr virus adsorbed to human lymphoblastoid cells were studied by indirect membrane immunofluorescence carried out at 0 degrees C. When EBV was adsorbed on cells at 0 degrees C, the cell surface fluorescence had a mostly r-ng-like pattern. However, the ring cells could be transformed into cap cells when warmed at 37 degrees C. This cap formation could be induced by EBV alone without participation of antibodies involved in the immunofluorescence procedure. The cap formation was temperature- and pH-dependent, and was reversibly inhibited by sodium azide or some sugars. Thus the EBV-induced cap formation was analogous to that induced by antibodies or ligands on other lymphoid cells.

The amount of HL-A antigens present on the peripheral blood lymphocytes of a single human donor was increased about 11-fold after stimulation with phytohemagglutinin and 36-fold after transformation with Epstein-Barr virus. This increase applied to all four HL-A specificities of these cells. The response to phytohemagglutinin was dependent on dose and was first observed at 12 hr of incubation. Measurements of the amount of surface membranes by geometry, by radioiodinatable surface proteins, and by 5'-nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) assay all indicated that the enhanced representation of HL-A antigens after stimulation by phytohemagglutinin or transformation by Epstein-Barr virus must be due to an increase in the density of these antigens on the cell surface.

An in vitro line was derived from an American Burkitt lymphoma, designated Ra No. 1, which produced malignant tumors when inoculated into thymus-deficient nude mice. The cells have B-lymphocyte characteristics, with surface-associated mu and kappa chains and Epstein-Barr virus (EBV) receptors, and can be readily infected with EBV in vitro. B95-8 virus induced EBV-determined nuclear antigen (EBNA) but not early antigen (EA) in Ra No. 1 cells, whereas P3HR-1 virus induced both EBNA and EA, but the EA level was much lower than in the prototype Raji strain, EBNA and EA levels were comparable in Ra No. 1 and in the previously established, EBV-infection-sensitive BJA-B lymphoma. The two lines differed, however, because BJA-B could be converted into a permanent EBV-carrying line by the B95-8 but not by the P3HR-1 virus strain, whereas Ra No. 1 could be converted by the P3HR-1 virus. This demonstrates that different B-lymphocyte-derived lymphoma lines can vary with regard to the restrictive control they can exert on the same virus strain. Furthermore, virus strains vary in their sensitivity to the restrictions of the same host cell. Permanent EBV conversion of the Ra No. 1 cell did not appear to change the cellular controls, as judged by the unchanged sensitivity of the cell to viral antigen (EA, viral capsid antigen) induction by P3HR-1 virus superinfection.

Human lymphoid cells isolated from the peripheral blood of adults, from cord blood, and from fetal liver, spleen, bone marrow, and thymus were cultivated with or without a cell-free preparation of Epstein-Barr virus (EBV) with demonstrated transforming activity. The cultures were examined for the EBV-associated nuclear antigen (EBNA) and for transfromation into permanent lymphoblastoid cell lines (LCL). EBNA, seen only in cultures that had received exogenous EBV, was detected between days 1 and 6 after addition of EBV, most frequently on day 3. EBNA-positive cells had a lymphoblastoid appearance. Transformation into established LCL became apparent between days 12 and 19. The addition of pokeweed mitogen to cultures containing EBV enhanced the development of EBNA, whereas phytohemagglutinin or concanavalin A had no such effect. Neither EBNA nor transfomration was observed in lymphoid cells from fetal thymus. In fetal spleen, bone marrow, and liver cells, EBV regularly induced EBNA and LCL transformation.

The antibody against viral capsid antigen (VCA) of Epstein-Bair (EB) virus was studied in 40 patients with malignant tumors of the nasopharynx. Out of 40 cases, there were 32 carcinomas (squamous cell carcinoma 18, undifferentiated carcinoma 7, lymphoepithelioma 3, transitional cell carcinoma 2 and adenoid cystic carcinoma 2) and 8 sarcomas. Patients were treated by radiotherapy alone, radiotherapy accompanied by chemotherapy or radiotherapy followed by radical neck dissection. Titration of anti-VCA antibody was done by means of fluorescent antibody indirect method (Kawamura's method). The results obtained were as follows: 10 patients before tretment had a geometric mean titer (GMT) of 1 : 421 and a positive rate of 60%. In 13 patients during treatment, there was an increase in the anti-VCA antibodies (GMT 1 : 880) and the positive rate was up to 77%. In patients at the end of treatment, GMT was 1 : 1381 and the positive rate 89%. From the above results, it is concluded that in the present study the anti-VCA antibodies increased during and at the end of tretment. In 16 patients with recurrence or metastasis a mean titer was 1 : 1809 and a positive rate was 100%. On the contrary, 6 patients in remission of more than 3 years had a titer of 1 : 319 and a positive rate of 50% . Therefore, it is concluded that in cases of recurrence or metastasis, there was a significant increase in the anti-VCA antibodies and in cases in remission of more than 3 years, there was a decrease in the antibodies.We conclude that the anti-VCA titer can be applied clinically as an useful parameter for the prognosis of the nasopharyngeal carcinoma.

Epstein-Barr virus DNA in virus non-productive cells was separated from high-molecular-weight cell DNA by sedimentation through a neutral glycerol gradient after gentle lysis of cells by Pronase and Sarkosyl. The isolated Epstein-Barr virus DNA had a density of 1.716-1.717 g/cm 3 in CsCl equilibrium centrifugation, which is very close to the virus DNA density of 1.718 g/cm 3 . The results indicated that the majority of Epstein-Barr virus DNA in virus nonproductive cells is not covalently integrated into high-molecular-weight cell DNA.

Antigens determined by Epstein Barr virus (EBV) have been detected and quantitated in human lymphoblastoid cell culture lines by specific radioiodine-labeled antibody elution (RIE). The peak of specific elutable antibody has been shown to quantitatively increase with increasing doses of EBV 2–3 days after superinfection of susceptible lines. Increasing antibody concentrations on constant cell numbers produced a linear increase in elutable antibody binding specifically to the producer cells. Increasing cell numbers in the presence of a constant antibody concentration, likewise, produced a graded increase in the specific antibody eluted from the producer cells. Antigen suspensions prepared from frozen and thawed cells were used to complete with the antigens on the target cells for the antigen-combining sites of the labeled antibodies. Experiments were performed using suspensions from cells containing all the EBV-determined antigens for competition as well as limiting the competition to early antigen (EA).

Critical evaluation of the anti-complement immunofluorescence (ACIF) test for Epstein-Barr virus-associated nuclear antigen (EBNA) in known EBV-associated cell lines was carried out. The ACIF procedure, in which the cell smears were thoroughly air-dried and fixed with carbon tetrachloride (for 15 min at room termperature), provided reproducible staining results with minimum variations. The fixed smears coulb be stored for at least 5 weeks at minus 80 degrees C without loss of reactivity. On the basis of the above evaluation, a total of 59 human cell lines, which had unknown or uncertain association with EBV, were examined for the presence or absence of EBNA by the ACIF procedure. All of the cell lines tested could be judged positive or negative for EBNA. All of 21 lines growing as monolayer or in a mixed state of monolayer and suspension, were negative. Of 38 lines grown in suspension, 36 lymphoblastoid lines were positive but the remaining 2 lines (seemingly not lymphoblastoid) were negative.

Three exceptional cell lines have been tested for the presence of the Epstein-Barr virus genome by nucleic acid hybridization (complementary RNA·DNA) and Epstein-Barr virus-determined nuclear antigen tests. Two lines were derived from Swedish lymphoma cases and one from an African Burkitt-like lymphoma biopsy that was negative for Epstein-Barr virus DNA and the virus-determined nuclear antigen. All three lines apparently lacked the viral genome. Two of the three lines clearly had characteristics of B-cells (bone-marrow-derived).

Cells from Burkitt lymphoma (BL) biopsies were examined for Epstein-Barr virus (EBV)-associated antigens by complement fixation (CF) tests and by the anti-complement immunofluorescence (ACIF) test. In CF tests, anticomplementary factors made it difficult to test all the biopsies available. However, biopsies from 19 patients were effectively tested and 12 of these (including two from one patient) contained antigen reacting with a battery of human sera with antibody to EBV but not with sera lacking such antibody. Technical difficulties prevented further characterization of the EBV-related antigens in the biopsies. Application of the ACIF test to BL revealed the presence of EBV-related nuclear antigen in biopsies from 11 of 13 patients. Absorption studies indicated that the nuclear antigens of the biopsies were closely related antigenically to the EBV-determined nuclear antigen (EBNA) previously described in lymphoblastoid cell lines. It is concluded that cells of BL biopsies may contain EBNA in addition to the EBV-related membrane antigen previously described. The results provide further evidence that BL cells from African patients resemble non-producer lymphoblastoid cell lines in containing EBNA and therefore appear to be transformed by EBV.

Sera from 67 Hodgkin's disease patients, 71 leukemia patients, and 186 healthy subjects were tested for antibodies to Epstein-Barr (EB) viral antigens by immunofluorescence methods. In both disease categories, in particular Hodgkin's disease patients, levels of antibodies to the viral capsid antigen (EBV-VCA) and MGT were higher than in the healthy controls. Significantly higher titers were found in Jewish patients of Asian-African origin, as compared to Jews of European origin, with Arab patients as intermediates. The effect of ethnic origin was independant of age and histopathologic type. Sex had no effect on titer. Inconsistent differences in titer were found between age groups in the various ethnic-histopathologic type groups. Some of the leukemia patients had no detectable antibodies to EBV, while all Hodgkin's disease patients showed previous contact with EB virus. Antibodies to the early antigen (EBV-EA) were found in 27% of Hodgkin's and 37% of leukemia patients, and in none of the healthy controls tested.

Binding of Epstein-Barr virus (EBV) to lymphocytes has been demonstrated by addition of extracts of virus-producing lymphoid cell lines (LCL) to suspension of human lymphocytes followed by fluoresceinated antibody to EBV. Combined tests for EBV binding and cell surface markers for T and B lymphocytes demonstrated that virtually all B lymphocytes have binding sites for EBV, whereas cells of the T developmental axis (thymus cells, T lymphocytes, T lymphoblasts) have no demonstrable receptors. This result was confirmed by analysis of purified T and B cell suspensions and by results with lymphocytes from children with selective immunodeficiencies. The relationship between lymphocytes bearing EBV binding sites, cell in LCL which carry the EBV genome, and atypical mononuclear cells in infectious mononucleosis (IM) have been investigated. Fifteen out of 16 LCL analyzed by a panel of seven cell surface markers were B cell-like and only one had the T cell surface phenotype. Atypical cells from IM were predominantly T lymphoblasts although some B lymphoblasts also appeared to be present.

A rapid microradioimmunoassay (RIA) technique was adapted for quantitatively measuring antibody titers to antigens occurring in Epstein-Barr virus (EBV)-infected lymphoid cells. In these experiments two EBV-infected cell lines, HR1K and EB-3, were used as antigen-positive cells and Molt-4 was used as the negative control cells. The antibody titers of sera from suspected infectious mononucleosis patients were compared by RIA and indirect fluorescent antibody (IFA) methods. As determined by each of the methods, 14 of 19 sera had positive antibody titers and the remainder of the sera had negative antibody titers. Thus, the two methods agreed completely in differentiating sera with antibodies to EBV antigens. To further evaluate the antibody specificity of the RIA, the antibody titers of paired sera, pre- or early infection and postinfection, from five confirmed infectious mononucleosis patients were determined by RIA and IFA. Seroconversion was demonstrated by both RIA and IFA for each of the patients. Thus, the sensitivity and specificity of the two procedures are about the same.

Investigations with the electron microscope on peripheral blood of patients with Hodgkin’s disease revealed parallel tubular structures in the cytoplasm of the lymphocytes, similar to those described in the literature. In normal subjects 2–9% of the lymphocytes were found to contain these structures. The number of such lymphocytes is increased in most patients as compared to normals. A correlation between an increase of inclusions containing lymphocytes and the histological or clinical classifications could not be established. An elevation of lymphocytes with tubular structures could be found in both favourable and unfavourable histological and clinical subgroups of Hodgkin patients, but especially in all groups with an unfavourable prognosis. The origin and function of the parallel tubular structures is still unknown, but their increase in lymphocytes of patients with Hodgkin’s disease is of some interest in view of the involvement of these cells.

Sera from healthy donors and individuals with primary Epstein-Barr virus (EBV) infections were titrated for EBV-neutralizing antibodies, and the results were compared with those obtained in indirect immunofluorescence assays for antibodies to EB-viral capsid antigens (anti-VCA) and to EBV-induced early antigens (antiEA). Sera from more than 100 healthy donors without anti-VCA failed to show neutralizing activity, whereas, with one exception, anti-VCA-positive sera from nearly 150 donors neutralized the virus. The ratios between the neutralizing and anti-VCA titers varied considerably, however, indicating that different antibodies were measured. The general correspondence with neutralizing activity explains why anti-VCA serves as a dependable indicator of immunity to infectious mononucleosis (IM). Accordingly, sera taken before infection from individuals who subsequently developed IM or seroconverted with respect to anti-VCA antibodies without evident or significant signs of disease were devoid of neutralizing activity. Titers of anti-VCA antibody usually attained peaks early in the course of the disease (within two weeks) and subsequently declined to lower, persistent levels, whereas maximal neutralizing titers were reached in six to seven weeks and showed no significant declines thereafter. Because of their slow development