Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Detection of the EBV-determined nuclear antigen (EBNA) in Burkitt's lymphoma and nasopharyngeal carcinoma biopsies by the acid fixed nuclear binding (AFNB) technique.

Epstein-Barr virus (EBV) carrying biopsies of Burkitt lymphoma (BL) and nasopharyngeal carcinoma (NPC) were used to examine the question whether the EBV-associated nuclear antigen (EBNA) can be demonstrated by the acid fixed nuclear binding (AFNB) technique, developed previously for the demonstration of EBNA in cultured cell lines [11]. Extracts of 5 BL and 5 NPC biopsies gave a brilliant, EBNA specific fluorescence after binding to acid fixed chicken red cells. Similar extracts of 3 other African tumors that are not known to carry the EBV-genome were negative, in spite of the fact that they were derived from EBV-seropositive patients with relatively high anti-EBV (VCA) antibody titers. Crude extraction and DNA-cellulose purification gave equally active extracts, provided that incubation was carried out at 4 degrees C. These results show that the acid fixed nuclear binding technique can be applied to biopsy material. This may be helpful in searching for EBNA carrying cells in heterogeneous normal and tumor tissues in vivo where the direct in situ ACIF staining for EBNA is known to meet great difficulties.     

Evaluation of epstein-barr virus-associated nuclear antigen with various human cell lines

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 temperature), provided reproducible staining results with minimum variations. The fixed smears could be stored for at least 5 weeks at ?8° 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.     

Properties of a baboon lymphotropic herpesvirus related to Epstein-Barr virus.

Three lymphoblastoid cell lines were established from splenic lymphocytes of a lymphomatous baboon (Papio hamadryas) by co-cultivation of the lymphocytes with X-irradiated cells of marmoset or baboon lymphoblastoid cell cultures; the baboon splenic lymphocytes failed to grow when cultured alone. A herpesvirus, associated with each cell line, was identified by immunofluorescence, molecular hybridization and electron microscopy. Antigenic comparison with Epstein-Barr virus (EBV) showed that the baboon herpesvirus and EBV shared cross-reacting viral capsid antigens (VCA): 20 of 20 (100%) anti-VCA (EBV)-positive human sera and 55 of 62 (89%) baboon sera reacted with the baboon lymphoblastoid cells and baboon sera stained EBV VCA in P3HR-1 and EB-3 cells. No nuclear antigen, as assayed by anti-complement immunofluorescence tests, was detected in baboon lymphoblastoid cells when human or baboon anti-VCA positive sera were used. Baboon anti-VCA-positive sera also failed to stain EBV nuclear antigens (EBNA) in Raji or P3HR-1 cells. Preliminary molecular hybridization studies showed only approximately 40% homology between viral DNA of baboon cell lines and DNA of EBV derived from P3HR-1 cells.     

High irradiation sensitivity of epstein-barr-virus (ebv) in lymphoblastoid-cells from patients with ataxia-telangiectasia and ebv genome-positive burkitts-lymphoma

Epstein-Barr virus (EBV)-immortalized lymphoblastoid cell lines derived from the peripheral blood of patients with ataxia telangiectasia (AT) and EBV genome-positive Burkitt's lymphoma (BL) were tested for expression of EBV-related lytic antigens by means of irradiation. We used 1 Gy in each experiment, according to the results of the P3HR-1 (derived from African BL) cell line. Significantly higher expression of early antigens (EA) and viral capsid antigen (VCA) was demonstrated in lymphoblastoid cell lines derived from both patients with AT and those with EBV genome-positive BL, as compared to those derived from healthy individuals. These results suggested that defective regulating mechanisms on B lymphocytes, responsible for EBV infection, may underlie for the pathogenesis of development of lymphoproliferative diseases both in patients with AT and EBV genome-positive BL.     

The detection of the Epstein-Barr virus (EBV) nuclear antigen (EBNA) by anticomplement immunofluorescence. Immunoglobulin class of antibodies and role of complement.

The Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA) was detected by anti-complement immunofluorescence (ACIF) in Raji lymphoblastoid cell line, and the mechanism of the reaction explored, using Ig fractions of anti-EBNA sera and purified components of complement. Following fractionation of serum from 13 donors, anti-EBNA antibodies were always found to be associated with IgG, but were also detectable in the IgM fraction of four sera. Sequential addition of functionally pure complement components in the ACIF reaction showed that the classical sequence of complement activation in involved. Anti-EBNA antibody reactions in Raji cell nuclei can also be detected by anti-Ig immunofluorescence with a low level of sensitivity. The same pattern of granular fluorescence was observed when C3 (beta1A/beta1C), or C4 or IgG anti-EBNA antibodies were revealed with the corresponding flurescein-conjugated reagents. Blocking of the ACIF reaction was achieved with Fab 2 anti-EBNA antibody fragments, which therefore provided an appropriate specificity control for the detection of EBNA.     

Biologic and antigenic characteristics of Epstein-Barr virus-related Herpesviruses of chimpanzees and baboons.

Leukocyte-transforming agents were isolated in baboon leukocytes inoculated with oral excretions from immunosuppressed chimpanzees. The transformed lymphoblasts had B cell surface markers and harbored herpes-type virus particles; 5-10% of the cells contained cytoplasmic antigens reactive with Epstein-Barr virus (EBV)-antibody-positive chimpanzee, human and baboon sera. These sera also neutralized the transforming activity of the chimpanzee virus. Long-term lymphoid cell lines were established from circulating lymphocytes of normal baboons: two from Papio cynocephalus and three from P. hamadryas. The cells had B cell surface markers, contained herpes-type virus particles and produced virus with leukocyte-transforming activity. No virus-associated nuclear antigen was detectable with reference baboon and chimpanzee sera; however, the cells reacted with selected human sera containing antibodies to EBV nuclear antigen (EBNA). Absorption experiments confirmed the specificity of this reaction. Baboon lymphoblasts produced baboon virus-associated soluble complement-fixing (CF/S) antigen. Baboon sera had CF antibodies to viral (CF/V) antigen derived from EBV but failed to react with EBV-associated CF/S antigen. Chimpanzee and baboon herpesviruses had similar in vitro host cell ranges but were different from those of EBV. Inoculation of baboons, rhesus monkeys and cottontop marmosets failed to produce detectable illness or palpable tumors.     

Association of Epstein-Barr virus with nasopharyngeal carcinoma in Alaskan native patients: serum antibodies and tissue EBNA and DNA

Biopsy specimens from Alaskan Native patients with nasopharyngeal carcinoma (NPC) and from other patients seen on the otolaryngology service were tested for Epstein-Barr virus-specific DNA and nuclear antigen (EBNA). Serum samples from both groups were tested for various EBV-related antibodies. EBV DNA and EBNA results were in agreement in 29 of 31 tissue specimens tested by the two methods. Ten of 11 biopsies containing NPC cells were positive for EBV DNA. Two NPC patients had biopsies that showed only atypical epithelium but were also positive for EBV DNA or EBNA. The other tissue specimens were negative except for biopsies from two patients: one with a parotid gland lymphoepithelial lesion; another with undifferentiated carcinoma of salivary gland origin.     

Demonstration of Epstein-Barr virus in malignant non-Hodgkin's lymphomas

Lymph nodes or tumor biopsies of 60 persons suspected of having a malignant lymphoma were examined for the presence of Epstein-Barr virus nuclear antigen (EBNA) by anticomplement immunofluorescence. In 8 cases the tissue specimens were also assayed for Epstein-Barr virus (EBV) DNA by nucleic acid hybridization. Serum samples of patients and controls were tested for EBV-related antibodies. The histological tests in 37 cases showed a malignant non-Hodgkin lymphoma, and in 23 cases a reactive lymphadenopathy. A Burkitt lymphoma of a European boy and a polymorphic centroblastoma contained EBNA and approximately 27 or 30 genome equivalents EBV DNA per cell, respectively. EBNA was also demonstrated in about 20% of the cells of a lymph node from a patient with recurrent reactive lymphadenopathy.     

Study of Epstein-Barr virus-determined nuclear antigen (EBNA) by chromatography on fixed cell nuclei.

Low ionic strength (50 to 100 mM NaCl) and pH 6.0 were found to be optimal conditions for in vitro conversion of Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA)-negative nuclei to EBNA-positive nuclei by addition of the complement-fixing (CF) antigen extracted from Raji cells. In vitro conversion of nuclei to EBNA-positively was sensitive to DNase but not to RNase treatment. This suggests that nuclear DNA is a specific target substance to which EBV-CF antigen binds. If nuclei were fixed with methanol/acetic acid and subsequently treated with 0.6 M NaCl, EBNA could be eluted from in vitro-converted Ramos nuclei with 0.3 and 0.4 M NaCl. The same conditions were also found to be optimal for the adsorption and elution of EBV-CF antigen in DNA-cellulose chromatography. This indicates that the DNA-binding properties of EBNA antigen can be studied by "chromatography" on fixed nuclei followed by the ACIF test. The obvious advantages of this method over chromatography on DNA-cellulose are its simplicity, the possibility of testing many samples in one experiment and, especially, the use of minimal amounts of material. Significant differences in elution patterns for EBNA were found when nuclei derived from different cell lines (Ramos, Raji, and P3HR-1) were converted in vitro to EBNA-positivity. EBNA is eluted from in vitro-converted nuclei of EBV genome-positive P3HR-1 cells at an almost 0.1 M higher concentration of NaCl than is necesssary for a similar degree of elution from nuclei of EBV genome-negative Ramos cells.     

Nasopharyngeal carcinoma. IV. Evolution of complement-fixing antibodies during the course of the disease

Sequential sera from 37 patients with nasopharyngeal carcinoma (NPC), with either controlled or uncontrolled tumours after radiotherapy, were studied for complement-fixing (CF) antibodies against a soluble antigen extracted from the QIMR-WIL lymphoblastoid line. When compared with results obtained previously with the same antigen on sequential Burkitt's lymphoma (BL) sera, the NPC sera exhibited (1) approximately ten-fold higher CF antibody titres than the BL sera; (2) an overall stability of CF titres in the uncontrolled tumour groups contrasting with the situation in BL; (3) a two- to four-fold decrease in CF titres in the controlled tumour groups; (4) similar CF titres in patients at stage I or IV of the disease. The above suggests an active and long-standing EBV infection before and during the development of the clinical NPC disease.     

Identity of the soluble EBV-associated antigens of human lymphoid cell lines

The reactions of human sera with antigens prepared from five human lymphoid cell lines were studied by immunodiffusion. Two major precipitation lines occurred with QIMR-WIL antigens; one (S line) due to a soluble antigen and the other (F line) due to a sedimentable antigen. A close association in human sera of precipitins producing the S line with antibody to Epstein-Barr virus (EBV) detectable by immunofluorescence (Henle test) and by complement fixation (CF) tests indicated a specific association of the S line with EBV, but the nature of the F line was not clarified. Soluble antigens prepared from five lymphoid cell lines (including Raji) contained at least one common identical heat-resistant component, defined as the antigen giving the S line. The ease of detection of the S antigen in cell lines by immunodiffusion appeared to be related to the titre of the soluble EBV-associated CF antigen, and the evidence suggested that the soluble CF and ID antigens were probably identical.     

EBV antigens in lymphocytes of patients with exudative tonsillitis, infectious mononucleosis and Hodgkin's disease.

A protocol utilizing isokinetic gradients to isolate human lymphocytes is combined with another that purifies the C3 receptor-bearing B lymphocyte subpopulation, thus enriching the EB virus genome-carrying population. Also, rabbit antisera were prepared to the Epstein-Bar virus nuclear antigen (EBNA) and the early antigen (EA) and utilized in an indirect immunofluorescence test (IIT) to detect these antigens in human lymphocytes isolated from various disease states. Using these methods we demonstrated excellent correlation between standard methods previously employed to detect EB virus-coded antigens and our IIT employing xenogenic antisera. Such tests were done on lymphoblastoid cell lines as well as lymphocytes isolated directly from patients with EB virus lymphoproliferative diseases. Human palatine tonsil-derived lymphocytes from children with exudative tonsillitis and peripheral blood lymphocytes of infectious mononucleosis contained only EBNA in C3 receptor-bearing B lymphocytes. However, patients with lymphoproliferative disorders, including Hodgkin's disease, harbored in their spleens and lymph nodes lymphocytes producing both EBNA and EA.     

Characterization of a second Epstein-Barr virus-determined nuclear antigen associated with the BamHI WYH region of EBV DNA

The Epstein-Barr virus-determined nuclear antigen (EBNA) is the only known virally-determined component that is regularly associated with EBV-transformed cells. A main component of EBNA, herein designated EBNA-1, has been conclusively localized to the BamHI K fragment of the viral genome. EBNA-1 is present in all EBV-carrying cell lines so far studied. Our current study deals with a second component. We have found that the EBNA reaction detected by anti-complement immunofluorescence (ACIF) in Burkitt lymphoma lines Daudi, Jijoye, and P3HR-1 could be completely removed by preabsorption of sera with any one of these 3 lines, when tested against any other of them. The same absorbed sera still gave a brilliant nuclear staining against other EBV-carrying lines, e.g. Raji or B95-8. The 3 lines in the first category carry EBV genomes that have deletions in the BamHI WYH region of the EBV genome. This region is intact in the second group of lines. This result is interpreted as showing the existence of 2 different ACIF-stainable EBV-determined nuclear antigens, one of which is associated with the BamHI WYH region. We designate this antigen as EBNA-2. We found that the two different EBNAs are different with regard to their association with metaphase chromosomes. In lines positive for both EBNA subtypes, metaphase chromosomes gave brilliant EBNA-1 staining, but could not be stained for EBNA-2, indicating differences in chromatin association of the two EBNAs. An 86 kd polypeptide was identified by immunoblotting of DNA-binding proteins from EBV-transformed lymphoid cell lines. EBV-specificity of the polypeptide was demonstrated by the presence of antibodies against this polypeptide in antisera from a population of EBV-seropositive donors, but not from seronegative donors, by the presence of the polypeptide itself in EBV-carrying but not in EBV-negative cell lines and by the appearance of antibodies against this polypeptide during the course of infectious mononucleosis (IM). The polypeptide was absent from the EBV-carrying P3HR-1, Daudi and Jijoye cell lines, which suggested that it may be encoded by the BamHI WYH region that is deleted from the viral substrains carried by these lines.     

False negative and prozone reactions in tests for antibodies to Epstein-Barr virus-associated nuclear antigen

Titration of anti-complementary human sera may yield false negative or prozone reactions in anti-complement immunofluorescence (ACIF) tests for antibodies to Epstein-Barr virus-associated nuclear antigen (EBNA) when the human complement (C') is added to the serum dilutions prior to charging of the target cell smears (two-stage ACIF test). This effect of anti-complementary sera and with it a source of error is avoided by using a three-stage ACIF technique; that is, when the target cell smears are successively overlayed with serum dilutions, C' and fluorescein isothiocyanate-conjugated antibodies to human βC/βA globulins. The two- and three-stage ACIF procedures yield comparable anti-EBNA titers with non-anti-complementary sera.     

Kaposi's sarcoma and its relationship to cytomegalovirus (CMV) III. CMV DNA and CMV early antigens in Kaposi's sarcoma

DNA-DNA reassociation kinetics, anti-complement immunofluorescence (ACIF) and ACIF-blocking tests were used to search for cytomegalovirus (CMV) gene products in Kaposi's sarcoma (KS) biopsies and early cell cultures deriving from them. Three of eight tumor biopsies were positive for CMV DNA; two of them at 0.35 genome/cell and one at one copy of 25% genome/cell. CMV-related antigens, mainly localized in the nucleus, were found in cryostat sections of seven of 31 tumor biopsies and four of 12 KS cell lines at early passage level. It appears that the antigen is present in a high number of tumor cells, like the Epstein-Barr virus nuclear antigen (EBNA) in EBV-transformed cells. Inevitably, the increasing data concerning the oncogenic potential of CMV lead on to consideration of the increasing evidence of its association with Kaposi's sarcoma, a multiple hemorrhagic sarcoma with an epidemiologic distribution in Africa similar to that of Burkitt's lymphoma.     

Detection of a nuclear, EBNA-type antigen in apparently EBNA-negative Herpesvirus papio (HVP)-transformed lymphoid lines by the acid-fixed nuclear binding technique.

In agreement with the findings of previous authors, we could not detect a virally determined nuclear antigen in Herpesvirus papio (HVP)-transformed baboon lymphoid lines by anticomplementary staining in situ, as for EBNA. However, by means of our recently developed acid-fixed nuclear binding technique an EBNA-like antigen could be readily demonstrated, after extraction from both producer and non-producer lines. We propose to designate the antigen as HUPNA. It can be detected by a human anti-EBNA antibody, suggesting cross-reactivity, if not identity, between EBNA and HUPNA. HVP-DNA carrying non-producer lines, negative for in situ ACIF stainability but capable of yielding HUPNA by the nuclear binding technique, can be superinfected with EBV, with brilliant EBNA expression as the result, suggesting that the defective in situ staining is a property associated with the baboon HVP, rather than the baboon lymphoid cell per se.     

Kaposi's sarcoma. IV. Detection of CMV DNA,CMV RNA and CMNA in tumor biopsies

In order to determine whether human cytomegalovirus- (CMV) DNA homologous sequences as well as CMV-specific RNA(s) and antigen(s) exist in tumor biopsies of Kaposi's sarcoma (KS) DNA-DNA reassociation, RNA-DNA in situ cytohybridization and anticomplement immunofluorescence test (ACIF) tests were applied. Three of 10 DNAs extracted from Kaposi sarcoma biopsies contained DNA sequences homologous to radioactively labelled human CMV DNA probe. The amount of CMV DNA in these sarcoma tissues was calculated to range from 0.7 to 1 genome equivalent per diploid cell. The presence of virus-specific RNA was also demonstrated in sections from five of 10 tumor biopsies. CMV-determined nuclear antigen(s) (CMNA) present in variable degrees were also demonstrated. In contrast, we could not detect any herpes simple virus type II (HSV-2) or Epstein-Barr virus (EBV) DNA sequences in DNA of these tumor biopsies. Furthermore, there were no detectable HSV-2 or EBV-specific RNA or virus-specific antigens in sections of these biopsies. These results provide new lines of evidence for the relationship between CMV and Kaposi's sarcoma.     

Demonstration of Epstein-Barr virus-associated nuclear antigen in nasopharyngeal carcinoma cells from fresh biopsies

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.