The role of repetitive DNA sequences in the size variation of Epstein-Barr virus (EBV) nuclear antigens, and the identification of different EBV isolates using RFLP and PCR analysis.

The six Epstein-Barr virus (EBV) nuclear antigen proteins (EBNA-1-6) show characteristic size variations between different virus isolates; this is a feature that has been used to identify the source of virus isolates in epidemiological studies (Ebnotyping). We have now studied the correlation between restriction fragment length polymorphisms (RFLPs) within exons coding for the EBNAs and the molecular masses of the respective proteins. The B95-8 EBV strain was used as the prototype virus. The variation in apparent molecular mass of EBNA-1, -3 and -6 correlated positively with the size of RFLP coding for repeat sequences in these polypeptides. For EBNA-2, no correlation between apparent molecular mass and length of the repetitive sequences was found. The EBNA-4 protein showed virtually no variation in apparent molecular mass and RFLP size across the repeat sequence. Based on the strong correlation between apparent molecular mass and RFLP size for EBNA-6, we developed an EBNA-6 PCR assay that discriminated between different isolates of EBV. This assay offers the advantage of EBV characterization using uncultured material (e.g. throat washings, blood or biopsies), thus avoiding the selection against poorly transforming strains that occurs during establishment of lymphoblastoid cell lines required for Ebnotyping at the protein level.

Detection of multiple 'Ebnotypes' in individual Epstein-Barr virus carriers following lymphocyte transformation by virus derived from peripheral blood and oropharynx.

Transformation of a B lymphocyte into a lymphoblastoid cell line (LCL) by Epstein-Barr virus (EBV) results in the expression of EBV nuclear antigens (EBNAs) of which the size spectrum ('Ebnotype') is characteristic for the transforming virion. Ebnotyping has been used as an epidemiological tool for studies of EBV infection. We compared the occurrence of a single and of multiple Ebnotypes, as defined by EBNAs 1, 2 and 6, in healthy and diseased EBV carriers. Cases from which two or more LCLs could be established from peripheral blood or oropharyngeal cultures were considered informative. The frequency of multiple Ebnotypes was relatively low in healthy individuals and in patients with infectious mononucleosis or with haematological diseases who were awaiting a bone marrow transplant [blood, 11 of 74 patients (15%); oropharynx, 12 of 49 patients (24%)], whereas it was relatively high in recipients of bone marrow or cardiac allografts and one patient with AIDS [blood, 12 of 34 patients (35%); oropharynx, 11 of 16 patients (69%)]. Three patterns of the simultaneous presence of multiple Ebnotypes were distinguished. The first, most frequent, pattern observed predominantly in oropharyngeal cultures of all groups consisted of minority Ebnotypes differing from the majority type by only a single EBNA protein (usually EBNA 1). The second, less frequent, pattern observed in the healthy carriers and the (candidate) transplant recipients consisted of minority Ebnotypes differing from the majority type by two EBNA proteins (mostly EBNAs 1 and 6). The third pattern, characterized by the simultaneous presence of totally different Ebnotypes, was restricted to the (candidate) transplant recipients and the AIDS patient and was more frequently observed in the blood than in the oropharynx. We suggest that the first two patterns result from heterologous recombinations occurring during viral replication at repeat sequences within the EBNA coding regions, whereas the third pattern reflects multiple infections with exogenous viruses.

Characterization of Epstein-Barr viral strains in parotid gland saliva and peripheral blood of patients with primary Sjögren's syndrome and healthy EBV carriers.

Increased Epstein-Barr virus (EBV) replication has been reported in the salivary and lacrimal glands in Sjögren's syndrome (SS). We studied whether or not certain EBV strains would occur preferentially in the peripheral blood and parotid gland saliva of 18 EBV-seropositive patients with primary Sjögren's syndrome (pSS) and 12 EBV-seropositive control persons. Transforming EBV was detected in the blood of 11 of 18 (61%) pSS patients and 9 of 12 controls (75%). Unexpectedly, neither transforming nor Raji-superinfecting EBV strains were detected in SS parotid saliva, whereas these EBV types were detected in control saliva in 7 and 8 cases, respectively (P < 0.001). Transforming EBV strains were further characterized by 'Ebno-typing,' i.e., analysis of the size spectrum of the viral antigens EBNA 1, 2, 3, and 6 in immunoblots of lymphoblastoid cell lines (LCL). Previous work has shown that a single EBV strain (Ebnotype) dominates the blood and oropharynx of healthy carriers and that unrelated individuals carry different EBV strains, reflecting the vast polymorphism of Ebnotypes in the general population. Two unexpected observations were made. First, an identical Ebnotype was detected in 4 unrelated individuals, i.e., in the blood of 1 pSS patient and in the saliva of 3 control persons. Second, carriage of 2 to 4 different Ebnotypes by a single individual was observed in 4 cases, i.e., in the blood of 1 pSS patient, and in the blood and saliva of 3 control persons.(ABSTRACT TRUNCATED AT 250 WORDS)

Epstein-Barr virus infection in allogeneic marrow grafting: lessons for transplant physicians and virologists.

The relationship between Epstein-Barr virus (EBV) and the host is profoundly disturbed by allogeneic bone marrow transplantation (BMT) because EBV resides in the recipient's hematopoietic system, which has to be destroyed in the majority of cases, and in the donor's hematopoietic system, i.e., the marrow graft. We have shown that EBV may be eradicated from some BMT recipients and that the virus may be transferred with the marrow graft. During the immediate post-transplant period oropharyngeal EBV excretion may occur which, by infecting passing B lymphocytes, may act as co-factor for acute graft-versus-host disease and help the virus to survive, despite the temporary depletion of its reservoir. The coexistence of totally different EBV strains in BMT recipients but not in healthy, untransfused controls, suggests that superinfection may by possible in case of immunodeficiency; alternatively, transfer of the virus by the reservoir itself (the B lymphocytes) might be the only effective route for superinfection. The generation of 'variant' strains during viral replication may form the basis of the vast polymorphism between wild-type EBV isolates in the population.

Detection of multiple Epstein-Barr viral strains in allogeneic bone marrow transplant recipients.

We have previously shown in 3 allogeneic bone-marrow transplant (BMT) recipients that complete replacement of recipient marrow was associated with the elimination of the pretransplant Epstein-Barr virus (EBV) strain of the recipient. To study the kinetics of EBV elimination and reinfection in more detail, we have performed a longitudinal study of BMT recipients combining serology, virus isolation from mouthwashes and peripheral blood, and EBV strain characterization. Oropharyngeal EBV excretion was found to persist after the cytoreductive therapy prior to BMT, whereas EBV-carrying cells in the blood were detected only after 5 weeks following BMT. During the first month post-BMT, 2 different EBV strains could be isolated from sequential mouth-washes of 3 patients. The initial strains were found to persist up to 7, 21, and 29 days post-BMT, whereas the subsequent strains appeared at 21, 42, and 34 days post-BMT, respectively. Thus, the original EBV strain may persist only for a limited time after BMT, and the oropharyngeal epithelium may be reinfected by a new EBV strain from the blood within 3 weeks. With respect to the coexistence of multiple EBV strains, 2 patterns were evident. From the day 62 mouthwash of 1 patient, 1 Type A and 1 Type B strain were isolated. From the day 180 mouthwash of a second patient, a dominant Type A strain was recovered, together with 6 "variant" strains that differed from each other by only a single EBNA protein (EBNA 1). This pattern may be explained by viral recombinations during replication, which may form the basis for the vast polymorphism of EBV observed in unrelated individuals.

Expression of Epstein-Barr virus-encoded growth-transformation-associated proteins in lymphoproliferations of bone-marrow transplant recipients.

The expression of Epstein-Barr virus (EBV)-encoded, growth-transformation-associated proteins was studied in lymphoproliferations of 9 allogeneic bone-marrow transplant (BMT) recipients. Immunoblots of cell lysates were probed with polyspecific and monospecific antisera directed against EBNA 1, 2, 3 and 6, and the membrane protein LMP. All tumors expressed EBNA 1 and LMP. EBNA 2 was detected in the tumors of 8 patients, and EBNA 3 and 6 in the tumors of 5 patients. The LMP regulatory sequences, 5' of the LMP gene, were totally unmethylated in all 7 cases, while the coding sequences of LMP and EBNA 2 were more methylated in CpG dinucleotides. EBV-transformed lymphoblastoid cell lines (LCL) express EBNA 1 to 6 and LMP; in contrast, Burkitt lymphomas express only EBNA 1. In vitro experiments have shown that EBNA 2, 3 and LMP can generate targets for cytotoxic T cells (CTL). These combined observations are consistent with the hypothesis that the EBV-associated lymphoproliferative disease of the BMT recipients escapes CTL-mediated rejection due to the failure of host immunosurveillance rather than to the down-regulation of immunogenic EBV-encoded antigens.

EBNA size polymorphism can be used to trace Epstein-Barr virus spread within families.

The Epstein-Barr virus (EBV)-determined nuclear antigens EBNA 1, 2, 3, 4, and 6, regularly expressed in EBV-transformed lymphoblastoid cell lines, vary in size among viral strains. We have used this characteristic to trace the spread of the virus within seven families by using an approach called Ebnotyping. Among 33 evaluable individuals, 3 were EBV seronegative, and 17 different EBV strains could be isolated from the peripheral blood or throat washes of the remaining 30. All unrelated persons carried different strains. The EBV strain carried by 19 persons was also found in 1 or more family members. The same viral strain was carried by two members in five families, by three members in the sixth, and by five members in the seventh. The paternal strain was isolated from one child in two families, and the maternal strain was isolated from one or more children in three families. EBV was isolated from both blood and throat wash in six individuals. The Ebnotypes of both derived lymphoblastoid cell lines were identical within each individual. These results indicate that spread within families may be a relatively common route of EBV transmission. The number of horizontal transmission events required to generate diversification of the Ebnotype will require larger epidemiological studies.

Serological and molecular studies of Epstein-Barr virus infection in allogeneic marrow graft recipients.

We have shown in two allogeneic bone marrow transplant recipients that Epstein-Barr virus can be eradicated by the BMT procedure or its complications, and that these patients are susceptible to infection with a new EBV strain. This conclusion was based on a combination of EBV serology and virus strain identification ("Ebnotyping," using the size variations of 5 EBV nuclear antigens). In the present study, we conducted a serological survey of EBV infection in 153 marrow graft recipients and their donors. Ten patients who were positive for IgG antibodies against EBV viral capsid antigens prior to BMT became completely seronegative at a median of 197 days post-BMT (range 106-320 days). Four of these patients, who had received seronegative marrow, remained seronegative during prolonged periods (222 to 2105 days). Six patients had received seropositive marrow. Two of them remained seronegative during their subsequent periods of follow-up (895 and 1437 days). An additional 10 patients showed a 100-fold or greater decrease in VCA IgG antibody titers. Their titers reached a nadir of 10 (the lower limit of positive) at a median of 134 days post BMT (range 83-386 days). The serological patterns of the above 20 patients were particularly frequent among patients with chronic graft-versus-host disease; 12 of 20 patients with decreasing VCA titers (60%) developed chronic GVHD versus only 22 of 73 patients with stable or increasing VCA titers (30%). These results suggest that GVHD may contribute to the elimination of residual EBV-carrying recipient cells. Establishment of EBV-carrying lymphoblastoid cell lines (LCL) was attempted in 60 donor-recipient pairs whose cryopreserved peripheral blood mononuclear cells were available. LCL were established from 18 of 51 EBV-seropositive marrow donors and 10 of 57 seropositive recipients prior to BMT. The same EBV strain was detected in 4 of the 6 cases in which LCL could be established from both the donor and the recipient prior to BMT. The persistence of the original EBV strain was demonstrated in a recipient of a T cell-depleted graft who showed only transient hematological recovery and no GVHD, and was associated with the persistence of B cells of recipient origin.

Phenotypic study of CD4+ and CD8+ lymphocyte subsets in relation to cytomegalovirus carrier status and its correlate with pokeweed mitogen-induced B lymphocyte differentiation.

A characteristic of active cytomegalovirus (CMV) infection is its suppressive effect on in vitro assays of immune function. The expression of CD11b by the Cd4+ and Cd8+ lymphocytes allows the identification of subsets with distinct regulatory functions of pokeweed mitogen (PWM) induced B cell differentiation. In order to relate that result with our previous observation that CMV carriers have significantly increased numbers of CD4+, HNK1+ and CD8+, HNK1+ lymphocytes in their peripheral blood compared with non-carriers, we performed a three-colour flow cytometric analysis of the co-expression of Cd11b and HNK1 by CD4+ and CD8+ lymphocytes obtained from 27 CMV carriers and 42 non-carriers. The differences between CMV carriers and non-carriers were significant for the CD4+, HNK1+ lymphocytes (median [5th and 95th percentiles], 59 [18 and 123 versus 24/7 and 73 per mm3, respectively; P less than 0.001) and CD8+, HNK1+ lymphocytes (59 [18 259] versus 52 [23 and 139] per mm3; P less than 0.001), but not for the CD4+, CD11b+ lymphocytes (59 [18 and 135] versus 52 [17 and 104] per mm3) and the CD8+, CD11b+ lymphocytes (85 [34 and 293] versus 82 [21 and 248] per mm3). The CD4+, HNK1+ and CD8+, HNK1+ lymphocytes that were increased in CMV carriers compared with non-carriers included mostly CD11b-, but also CD11b+ lymphocytes. After sorting CD4+ and CD8+ lymphocytes for four CMV carriers into HNK1+ and HNK1- fractions, we analyzed their regulatory functions on PWM-driven B cell Helper function to PWM-driven B cell differentiation was exclusively associated with the CD4+, HNK1- lymphocytes; the CD4+, HNK1+ generally did not show helper or suppressor activity in this assay. Both CD8+, HNK1+ and CD8+, HNK1- lymphocytes showed suppressor activity. Thus, the NHK1 marker does not constitute a phenotypical correlate for suppressor cells of PWM-driven B-cell differentiation.

Correlation between cytomegalovirus and toxoplasma gondii serology and lymphocyte phenotypes in peripheral blood and cord blood.

Increased numbers of peripheral blood HNK1+ lymphocytes have been reported in transplant recipients, hemophilia patients treated with clotting factor concentrates, and HIV carriers. Our previous work has revealed a significant effect of cytomegalovirus (CMV) carrier status on HNK1+ lymphocytes. Since other antigenic stimuli may be also involved, we compared the effects of Toxoplasma gondii and CMV carrier status on lymphocyte subsets as defined by the CD3 and HNK1 markers in 288 healthy individuals. In contrast to CMV, T gondii carrier status had no significant effects on the CD3+, HNK1+ and CD3-, HNK1+ lymphocyte subsets. That result may be explained by the different relationships between these 2 microorganisms and their hosts. We also studied the effect of maternal CMV carrier status on the HNK1 expression by their offsprings' cord blood lymphocytes. None of the 100 newborns studied had serological evidence of congenital CMV infection. There were only few HNK1+ lymphocytes in the cord bloods, of which the majority was CD3-, and their proportions were not significantly influenced by maternal CMV carrier status. Since there has been probably no direct contact between maternal CMV and the newborns' immune systems, we suggest that the effect of CMV on the HNK1+ lymphocytes of its carriers, results from a direct interaction between virus or virus-infected cells and the immune system.

Eradication of Epstein-Barr virus by allogeneic bone marrow transplantation: implications for sites of viral latency.

Wild-type strains of Epstein-Barr virus (EBV) can be distinguished on the basis of variations in the molecular weight of virus-encoded, growth transformation-associated proteins. This approach was used to study the persistence of EBV in two seropositive recipients of allogeneic bone marrow transplants. The first patient received marrow from her EBV-seronegative brother, became EBV seronegative after grafting, and remained so for greater than 1200 days. Subsequently, she became infected with a new EBV strain that differed from her pretransplant strain but was indistinguishable from the virus isolated from her husband. The second patient received marrow from his EBV-seropositive brother. This patient showed only a transient decrease in IgG antibodies to EBV capsid antigen. His pretransplant strain differed from the virus of his donor. On days 252 and 915 after transplantation, lymphoblastoid cell lines were grown from the peripheral blood of the patient and were found to carry exclusively the virus of the donor. These results suggest that the latently EBV-infected host cells reside in a cellular compartment that can be destroyed by graft-versus-host reactivity, irradiation, or cytotoxic drugs. Hemopoietic tissue is the most likely candidate.

Effects of herpes virus carrier status on peripheral T lymphocyte subsets.

We studied the effects of herpes virus carrier status on peripheral blood T lymphocyte subsets in 334 healthy individuals. IgG-class antibodies against cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus (HSV), and varicella-zoster virus (VZV) were used as markers for the carrier status of those viruses. CMV carrier status was associated with significant increases in the numbers of some T cell subsets, whereas the carrier status of EBV, HSV, and VZV had no significant effects. The 159 CMV-seropositive individuals had higher numbers of HNK1+ T cells than did the 175 CMV-seronegative individuals [mean (SD), 292 (196)/microL v 164 (89)/microL, respectively], including the CD4+HNK1+ T cells [38 (48)/microL v 9 (13)/microL, respectively] and the CD8+HNK1+ T cells [166 (146)/microL v 73 (54)/microL, respectively]. Morphological and cytochemical studies showed that the expression of HNK1 by the CD4+ and CD8+ T cells was associated with the occurrence of azurophilic cytoplasmatic granules and a loss of nonspecific esterase activity. The numbers of CD4+HNK1+ and CD8+HNK1+ T cells increased proportionally to the levels of the IgG-class CMV antibody titers. We suggest that the increased numbers of CD4+HNK1+ and CD8+HNK1+ granular T cells in CMV carriers reflect the persistent interaction between CMV and the immune system of its hosts.

Effect of cytomegalovirus infection on T-lymphocytes after lymphocyte-depleted bone marrow transplantation.

The effect of cytomegalovirus (CMV) infection on the repopulation of the peripheral blood with T-lymphocytes was studied in recipients of lymphocyte-depleted bone marrow transplants (BMT) who had hematologic and cytogenetic evidence of engraftment. Lymphocyte depletion was performed using counterflow centrifugation and resulted in a median depletion of 98.4% (range 94.4%-99.8%) of the T cells. Between 8 and 105 days after BMT, the T-cell repopulation was characterized by a relative preponderance of T cells lacking the CD3 marker and a slow repopulation of CD3+, CD4+, and CD8+ T cells. The CD8+ T cells repopulated at a faster rate in patients with CMV infection than in those not infected with CMV. At the end of the 9- to 12-month follow-up period, patients with CMV infection had normal numbers of CD4+ and CD8+ T cells but increased numbers of HNK1+ T cells. Those without CMV infection had subnormal numbers of CD4+ T cells, normal numbers of CD8+ T cells, and numbers of HNK1+ T cells that attained the upper limit of the normal range. Most of the HNK1+ T cells in both patient groups coexpressed the CD8 marker. We conclude that the occurrence of CMV infection in recipients of lymphocyte-depleted BMT is associated with an increase in the number of T cells coexpressing CD8 and HNK1, just as in recipients of nondepleted BMT.

Reduction and repopulation of T-lymphocytes after cytoreductive therapy with or without autologous bone marrow rescue.

The reconstitution of peripheral-blood T-lymphocytes following cytoreductive therapy in standard (11 patients) or in high dosages (ten patients) was compared with that after supralethal cytoreductive therapy followed by autologous bone marrow rescue (ABMR, 20 patients). Along with the increasing cytotoxic potential of the three therapy protocols, T-cell counts fell to lower levels. Following all three forms of cytoreductive therapy, T8+ T-cell counts decreased to lower levels than T4+ T-cell counts. The greater relative reduction of T8+ T cells may indicate that T8+ T cells are more sensitive to cytoreductive therapy than T4+ T cells, and/or that T8+ T cells have shorter survival times. The contribution of residual (mainly T4+) T cells to the T-cell repopulation was significant in the patients on standard-dosage chemotherapy, less important in those on high-dosage chemotherapy, and minor in those receiving supralethal cytoreductive therapy and ABMR. The repopulation rates of T8+ T cells following ABMR exceeded those observed after chemotherapy without ABMR. The T3- (T3 negative) T-cell subset, which comprises only 5%-10% of peripheral T cells in normal individuals, decreased rapidly to low levels and remained so for the entire six-week observation period in both chemotherapy groups. Following ABMR, however, those T3- T cells rapidly increased again to normal levels. Since the T cells in bone marrow biopsies have a large T3- fraction, that rapid recovery of T3- T cells may reflect the contribution of marrow precursors in the marrow grafts to the improved T-cell regeneration following ABMR.