Demonstration of a Herpes Group Virus in Cultures of Peripheral Leukocytes from Patients with Infectious Mononucleosis

Abstract

Cultures were initiated with peripheral leukocytes from individuals: (i) in the acute stage of infectious mononucleosis (IM); (ii) with past histories of IM; and (iii) without histories of IM and without antibodies to EB virus (EBV). In confirmation of other reports, the first group of cultures developed readily and rapidly into lines of blastoid cells. Cellular replication commenced in 24 of 25 attempts within 17 to 28 days, regardless of the technique employed; i.e., initial seeding of the leukocytes with or without addition of phytohemagglutinin onto monolayers of human diploid cells (WI-38) or direct establishment of suspension cultures. EBV was detected in all cultures by immunofluorescence and also by electron microscopy when tested. Furthermore, the nine cultures which were examined cytogenetically revealed the C-group (#10) chromosomal marker previously found in cultured Burkitt tumor cells. These findings supported the earlier conclusion that EBV is related to, if not identical with, the causative agent of IM. Cultures of the second group commenced growth only within 30 to 60 days in five of seven attempts, depending apparently upon the early presence of WI-38 cells. These cultures also revealed the presence of EBV and, in the three examined, the C-group chromosomal marker. Leukocytes of the third group, seeded onto WI-38 monolayers, failed to become established in four attempts. The possible implications of these findings are discussed.

Establishment of continuous lymphoblast cultures from leukocytes of gibbons (Hylobates lar)

“…In the present study, no continuous cultures of lymphoblasts were obtained directly from gibbon leukocytes. This was not unexpected with lymphocytes from antibody-negative animals since no lines can be derived from leukocytes of antibody-negative human donors (Diehl et al, 1968(Diehl et al, , 1969Gerber and Monroe, 1968;Nilsson rt a/., 1971. That no lines were obtained from anti-VCA positive animals, which is in contrast to the not infrequent success obtained with human leukocytes of anti-VCA positive donors, might be explainable on a quantitative basis.…”

“…The Kaplan line derived from a patient with infectious mononucleosis (Diehl et were maintained in medium RPMI-1640, supplemented with 10% fetal calf serum, 1% glutamine, and antibiotics as described (Diehl et a/., 1968(Diehl et a/., , 1969. Both lines are overt carriers of EBV, with virus antigen-producing cells ranging from 1-10% of the population.…”

“…In the acute phase of IM, EBV-stimulated lymphoid cells circulate regularly in the blood. In culture they grow readily (in nearly 100% of attempts) and rapidly (within 2 to 4 weeks) into permanent lines of lymphoblasts, all harboring EBV in a small proportion of the cells (Diehl et al, 1968(Diehl et al, , 1969; Moses et al, 1968). As a rule, primary EBV infections lead to a persistent, latent carrier state in the lymphoreticular system so that EBVstimulated cells are present in lymph nodes (Nilsson et a/., 1971) and a few of them occasionally enter the circulation.…”

“…As a rule, primary EBV infections lead to a persistent, latent carrier state in the lymphoreticular system so that EBVstimulated cells are present in lymph nodes (Nilsson et a/., 1971) and a few of them occasionally enter the circulation. Thus, depending upon the source of the cells, the techniques employed, the total number of leukocytes cultured, and the proportion of stimulated cells, continuous EBV-positive lines of lymphoblasts may be established with variable success ranging from about 10 to nearly 100o/o of attempts (Diehl et a/., 1968(Diehl et a/., , 1969Gerber and Monroe, 1968;Glade et a/., 1968;Jensen et al, 1967; Moore et a/., 1967; Nilsson et a/., 1971), but growth generally becomes evident only after S weeks to -3 months. It has not been possible to derive continuous lymphoblast lines from leukocytes of donors who had no antibodies to EBV; that is, donors who had not been previously exposed to EBV and thus were not carrying the virus (Diehl et al, 1968(Diehl et al, , 1969Gerber and Monroe, 1968), nor from cord blood leukocytes or fetal lymphoid tissues (Pope ef a/., 1969(Pope ef a/., , 1971 ; Nilsson ef a/., 1971).…”

Karyotypes in Infectious Mononucleosis

“…At present, the general consensus is that Epstein-Barr virus (EBV) is the common etiologic agent to Burkitt's lymphoma (BL) and IM (Henle & Henle 1974, Pattengale et a1 1974, Carter 1975. Several reports concerning the chromosome complement of BL-tumour cells (Jacobs et a1 1963, Stewart et a1 1965, and EBV-containing-cell lines derived from BL (Kohn et a1 1967, Huang et a1 1970, Zajac & Kohn 1970, Macek & Benyesh-Melnick 1972, IM lymphocytes (Diehl et al 1968, Huang et a1 1970, Steel et a1 1971, Macek & Benyesh-Melnick 1972, and from in vitro EBV-infected cells (Gerber et a1 1969, Whang-Peng et a1 1970, Wahren et a1 1972, Amano et a1 1973 are available. The so called 'C marker' which is a C group chromosome, presumably a no.…”