Aspects Of The Eb Virus

ASPECTS OF THE EB VIRUS M. A . Epstein Department of Pathology. The Medical School. University

of Bristol.

Bristol. England

I . Discovery of the E B Virus . . . . . . . . . . . A . Rationale for Experiments . . . . . . . . . . B . Successful Long-Term Culture of Burkitt Lymphoblasts .’ . . C. Demonstration of Virus in Cultured Burkitt Lymphoblasts . . D . Confirmation of E B Virus in Other Cultured Cells . . . . I1. Structure and Maturation of EB Virus . . . . . . . . A . I n Thin Sections . . . . . . . . . . . . . B . I n Negative Contrast Preparations . . . . . . . . I11. Effects of E B Virus on Cells . . . . . . . . . . A. Fine Structure of Blastoid Cells of Established Strains . . . B . Fine Structural Cytopathological Changes during EB . . . . . . Virus Production . . . . . . C. Incidence of Virus-Bearing Cells . . . . . . . . . IV . Presence of E B Virus in Burkitt Lymphomas . . . . . . V . Biological Activity of E B Virus . . . . . . . . . A . Inertness in Ordinary Test Systems . . . . . . . . B . Induction of Encephalitis . . . . . . . . . . . C . Infection of Cells in Vitro and Induction of Lymphoproliferation . D . Induction of Specific Chromosomal Lesion . . . . . . E . Induction of Specific Cell Surface Neo-antigen . . . . . VI . Immunological Singularity of E B Virus . . . . . . . . . . . . . . . . . . . . . VII . Sero-epidemiology VIII . Natural Pathogenicity . . . . . . . . . . . . I X . Related Animal Tumor Viruses . . . . . . . . . . A . The Herpes Virus of Frog Kidney Carcinoma . . . . . B . The Herpes Virus of Marek’s Disease . . . . . . . C. Herpes Suimiri . . . . . . . . . . . . . x. Discussion . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . .

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At the present time. general interest in the Burkitt lymphoma (Burkitt. 1958) is widespread and this fascinating syndrome needs little introduction; excellent reviews have described both the clinical and the pathological aspects of the disease (Burkitt. 1963. 1970) . Virological interest in the tumor was aroused a t an early stage when epidemiological studies seemed to show that climatic factors governed its distribution. thus suggesting that causation might depend on some 383

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biological influence such as an arthropod vector spreading an etiological agent (Burkitt, 1962a,b). As epidemiological information accumulated, it became evident that even at the highest recorded incidence rates vector-mediated case-to-case infection could not be possible (Haddow, 1964), and that some other mechanism must therefore be involved. Despite this necessary revision of the original hypothesis of an arthropodborne disease, new epidemiological findings continue to indicate that Burkitt’s lymphoma has an infective cause. Thus, Pike, Williams, and Wright (1967) have shown that the tumor occurs in well-defined timespace clusters showing epidemic “drift” characteristic of infectious conditions. In addition, other observations make it clear that adult immigrants from tumor-free areas are unusually susceptible to the tumor on coming into areas of high incidence, suggesting that they lack an immunity present in indigenous adults (Burkitt and Wright, 1966; Wright, 1967). At the same time the climate dependence of the tumor has been confirmed in a second endemic area, New Guinea (Booth et al., 1967), and, if preliminary reports prove correct, perhaps also in tropical South America (Luisi et al., 1965; Beltrln et al., 1966). The finding that typical cases of the lymphoma can occur rarely in temperate countries outside the recognized areas of endemicity (O’Conor et al., 1965; Wright, 1966), and, indeed, almost everywhere that search has been made for it (Burkitt, 1967a), has been held by some to invalidate the idea of an infectious etiology. But such doubts are unacceptable, since it remains necessary to explain why the tumor should be exceptionally rare in most geographical regions, yet both more common than all other tumors of children added together and climate dependent in the areas where it is endemic. It seems clear that in these tropical areas of high incidence some additional factor is involved and that it is this which is climate dependent. Against the background of these changing concepts, the idea of an infectious cause has remained constant and has inspired numerous attempts to link viral agents with the tumor. It was in this context that the EB virus was first discovered. I. Discovery of the EB Virus

A. RATIONALE FOR EXPERIMENT^ Early attempts to demonstrate virus in samples of Burkitt lymphomas from Ugandan patients both by direct electron microscopy and by standard virological procedures using tissue culture systems, embryonated eggs, and animal inoculation, were uniformly negative. Consideration of certain animal tumors with a well-recognized viral

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etiology then led to the idea that if Burkitt tumor cells could be grown in vitro away from host defenses, they might yield an otherwise inapparent oncogenic virus (Epstein et al., 1364a). This notion was exactly comparable, for example, to the situation in regard to cultured cells from avian myeloblastosis where the causative agent is not readily detectable in vivo but becomes so in vitro (Bonar et al., 1960). Because of this, it was considered that in the further search for virus in the Burkitt lymphoma, high priority should be given to the establishment of strains of the tumor cells capable of growing in continuous longterm culture.

B. SUCCESSFUL LONG-TERM CULTURE OF BURKITTLYMPHOBLASTS It was known from the first histological study of Burkitt tumors that the majority were composed of poorly differentiated lymphocytic cells accompanied by randomly scattered, nonmalignant, reactive histiocytes (O'Conor, 1961). Thus, the prospects for establishing strains of the malignant cells of this tumor were unpromising since a t that time no member of the human lymphocytic series had been grown continuously in vitro despite repeated efforts ever since the first introduction of the tissue culture technique (Woodliff, 1964). Nevertheless, in the latter part of 1963 the first two strains of cultured Burkitt lymphoma cells were reported simultaneously from London (Epstein and Barr, 1964) and from Nigeria (Pulverlaft, 1964). The work in London was done with biopsy samples removed in Uganda and flown overnight to England ; material was only collected from patients with typical clinical Burkitt tumors and the diagnosis was confirmed histologically in each case. I n Nigeria, a t about the same time, Burkitt lymphoma cells were being removed from patients and placed directly in short-term cultures for the study of their behavior and cytological features. Both the details of the definitive culture method elaborated in London for the long-term propagation of the cells (Epstein and Barr, 1965) and the manner in which cells in a short-term culture proliferated continuously in' Nigeria (Pulvertaft, 1964) have been described and reviewed elsewhere (Epstein, 1970).

C. DEMONSTRATION OF VIRUSIN CULTURED BURKI~T LYMPHOBLASTS As soon as sufficient material could be spared from the first established strain of cultured Burkitt lymphoblasts (EB1) (Epstein and Barr, 1934), electron microscopy was undertaken. The cells were pelleted 75 days after the original biopsy sample was set up in vitro and were examined in thin sections by methods which have already been described in full (Epstein and Achong, 1965; Achong and Epstein, 1965).

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Within the very first grid square searched in the electron microscope a cell from this preparation was found containing numerous virus particles which were immediately recognizable as being typical morphologically of the herpes group of viruses (Epstein et al., 1964b). When first seen there was naturally no means of knowing which herpes virus was involved, but it was considered unusual that a member of the herpes family was being carried as an inapparent infection in a continuous human cell strain without causing destruction of the cultures. Preliminary biological tests for herpes viruses were applied to the virusbearing EB1 cultures using embryonated eggs, HeLa cells, and young mice inoculated by the intracerebral route, and in each case the results were negative. Exhaustive further virological investigations were therefore undertaken, and when these too gave negative results (Epstein et al., 1965a) it became clear that the agent present in the EB1 cells was unlike any known member of the herpes group of viruses since it showed a complete lack of biological activity. It is of interest to note that because of this unusual negative biological behavior the virus was inapparent and would clearly not have been readily detected other than with the electron microscope; it is fortunate that the work was undertaken in a laboratory with a tendency to resort to this type of examination where more ordinary biological tests were negative. The EB virus was thus discovered solely by electron microscopy and seems indeed to be the first agent found in this way.

D. CONFIRMATION OF EB Vmus IN OTHERCULTURED C ~ L S Following this initial work, a biologically inert and morphologicaily identical herpes-type virus was demonstrated by electron microscopy in numerous further cell strains established from Burkitt tumors occurring in patients from widely separated parts of the world (Epstein et al., 1964c, 1965b, 1967; Stewart et al., 1965a; O’Conor and Rabson, 1965; Rabson et al., 1966; Minowada et al., 1967; Hinuma et al., 1967; Osunkoya, 1967; Pope et al., 1968a). In addition, an apparently similar virus has been observed by electron microscopy in the strains of blast cells established from various types of human leukemia in North America and Australia (Iwakata and Grace, 1964; Zeve et ul., 1966; Moore et al., 1966; Pope, 1968), as well as in strains of peripheral lymphoid cells established from normal human donors (Moore et al., 1967; Gerber and Monroe, 1968) or patients with various types of cancer (Jensen et al., 1967). It may well be, as Moore has suggested (Moore et al., 1967), that these seemingly normal cells owe their unusual power of continuous proliferation in vitro to the presence of the virus. Since EB virus infection is now known to be widely prevalent in adult human

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populations, with the virus persisting in the lymphatic system (Henle and Henle, 1968a), it is not surprising that i t should sometimes be found in peripheral lymphocytes cultured from normal healthy donors or from patients with various malignant diseases. I n addition, it has been shown recently that peripheral lymphocytes from patients with infectious mononucleosis acquire the ability to proliferate in continuous culture early in the disease, and likewise carry the E B virus (Henle et al., 1968; Diehl et al., 1968; Henle and Henle, 1968a; Chessin et al., 1968; Moses et al., 1968). I I . Structure and Maturation of EB Virus

The herpes-type virus particles found in all strains of cultured hemopoietic cells examined so far are structurally indistinguishable with a morphology characteristic, both in thin sections and in negative contrast preparations, for members of the herpes family.

A. IN THINSECTIONS The virus was first studied in the E B l and EB2 in vitro strains of Burkitt lymphoblasts and its structure and mode of maturation were worked out with this material (Epstein et al., 1964b,c, 1965a) ; these findings are summarized below. Immature particles were found in both the nucleus and cytoplasm of the cells and when appropriately orientated to the plane of section showed a hexagonal profile (Fig. 1). The particles measured about 75 to 80 mp in diameter and were either empty or contained ring-shaped or dense central nucleoids (Figs. 1 and 2 ) . Maturation occurred by the immature particle budding out a t a cellular membrane and acquiring an additional coat from this membrane as i t passed through. This process of budding took place at the inner nuclear membrane, a t membrane-bounded cytoplasmic spaces, and a t the plasmalemma, so that mature particles were only observed in the perinuclear space, within cytoplasmic spaces, and close outside the plasmalemma. The mature particle with its additional enveloping membrane measured about 110 to 120 mp in diameter and contained a dense central nucleoid about 45 mp across (Fig. 3). These findings were subsequently confirmed by work on additional strains of Burkitt lymphoma cells when these became available (Stewart et al., 1965a; Epstein et al., 1965b, 1967; O’Conor and Rabson, 1965; Rabson et al., 1966; Hinuma et al., 1967; Dalton and Manaker, 1967; Pope et al., 1968a).

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B. IN NEGATIVE CONTRAST PREPARATIONS Irrespective of whether the material has consisted of partially purified virus which had been subjected to elaborate preparative procedures (Toplin and Schidlovsky, 1966) or virus examined directly within fragmented infected cells (Hummeler et al., 19S6), EB virions have shown a consistent fine structure in negative contrast preparations, characteristic of herpes-type viruses. Thus, the immature particles have measured about 100 mp in diameter, have shown hexagonal profiles, and have exhibited triangular surface facets (Fig. 4) ; empty particles without nucleoids (Fig. 4) and particles with ring-shaped nucleoids were penetrated and filled by phosphotungistic acid (PTA) . Intact particles, presumably with a fully formed dense nucleoid, have also been observed. All the immature particles were covered by hollow, tubular, surface capsomeres measuring about 12 mp in length with a central hole 4 mp in diameter (Fig. 4), and having a center-to-center spacing between individual capsomeres of 12 mp. The capsomeres had a regular arrangement, most being surrounded by six neighbors in hexagonal array but with corner capsomeres showing fivefold symmetry. Although it has not been possible accurately to determine the total number of capsomeres, all the evidence suggests that the immature particle is an icosahedron with 162 capsomeres (Hummeler et al., 1966; Toplin and Schidlovsky, 1966; Yamaguchi et al., 1967; Grace, 1967). Mature particles enveloped by their additional outer membranes have also been seen in negative contrast preparations (Hummeler et al., 1966; Toplin and Schidlovsky, 1966; Grace, 1967), but have usually been slightly damaged, allowing PTA to penetrate the membrane and reveal the immature component within. 111. Effects of EB Virus on Cells

As has already been pointed out above, EB virus has so far only been found in established long-term cultures of human blastoid cells from various sources. Before summarizing the effects of the FIG.1.’ Group of immature EB virus particles lying in the debris of an infected cell. The particles have a hexagonal profile, measure about 75-80 mp in diameter, and are either empty or have central ringshaped nucleoids. X87,500. FIG.2. Similar group of immature EB virus to that shown in Fig. 1, but with numerous particles having fully developed central dense nucleoids. X69,300.

‘All the figures except Figs. 4 and 9 are electron micrographs of thin sections of material fixed in glutaraldeh3.de followed by osmium, embedded in epoxy resin, and contrast stained in the section with uranyl acetate.

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virus on such cultured cells, their normal fine structure requires brief consideration. A. FINESTRUCTURE OF BLASTOID CELLSOF ESTABLISHED STRAINS

With few exceptions and irrespective of their origin most established strains of blastoid cells are of lymphoid type. The cells of such strains are mostly rounded and have been found to grow in suspension a s single individuals or in clumps of various sizes. Where nonspherical cells have occurred in the population, these have differed from the round forms by the possession of one or more cytoplasmic processes. Not unnaturally, it has also been found that recently fed cultures contained fewer degenerating cells than those in which the medium is spent, and that mitoses were most frequent in samples taken when cell growth was rapid. The cells tend to be relatively small, up to 15-2Op in diameter, with large rounded or indented nuclei in which dense chromatin is concentrated in a marginal zone and scattered in clumps elsewhere. Prominent nucleoli, up to 4 or 5 in number, and a well-developed nuclear envelope interrupted by the usual pores have also been observed. In many cultures, cells have been found in which the nuclear envelope was thrown up into a projection running through the cytoplasm as a characteristic flat structure enclosing cytoplasmic matrix (Epstein and Achong, 1965; Achong and Epstein, 1966). The cytoplasm appears as a narrow band, except where thrown out to form processes, and always contains very profuse free ribosomes. Mitochondria are usually few in number and tend to be grouped together a t one pole. The endoplasmic reticulum is remarkably scanty in the cells of all strains, being represented by a few vesicles and one or two scattered rough cisterni; the Golgi component tends t o be small and poorly differentiated (Fig. 5 ) . However, in rare instances FIG.3. Mature EB virus particle enveloped by its additional outer membrane. The particle measures 115 mp in diameter and lies within a cytoplasmic space bounded by a fine membrane (arrow). Xl64,OOO. FIG.4. Electron micrograph of four immature EB virus particles prepared by the negative-contrast technique. The hexagonal outline of the virus is evident, and hollow surface tubular capsomeres can be clearly seen in profile a t the edges. None of these particles contains a nucleoid and each has therefore been penetrated by the electron-opaque phosphotungstic acid used in the preparation. X330,OaO. (From Epstein, 1967.) FIG.5. Typical cultured blastoid cell from a Burkitt lymphoma. The round nucleus with prominent nucleoli is surrounded by a well-marked double membrane interrupted by pores. The cytoplasm is packed with masses of free ribosomes and contains grouped mitochondria ( m ) , poorly developed rough endoplasmic reticulum ( e r ) , and scanty Golgi elements as at g . X11,250.

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an occasional cell with more elaborate rough cisterni has been rcported (Epstein e t al., 1966a; Rabson et al., 1966) (Fig. 6 ) , but in another case stacks of annulate lamellae have been incorrectly identified as rough endoplasmic reticulum (Douglas e t al., 1967). Clear vacuoles, lipid bodies, microtubules, centrioles, and annulate lamellae have all been frequently seen. Blastoid cells have also sometimes been observed to contain an additional unusual cytoplasmic organelle (Pope e t al., 1967; Chandra, 1968) ; this consists of a regular membranous structure associated with adjacent microtubules and has been designated by Chandra (1968) as undulating tubules. The function of this organelle is unknown but it has been found in a wide range of cells of both vertebrate and invertebrate origin (Bassot, 1966; Bucciarelli e t al., 1967; Sebuwufu, 1938; Bensch and Malawista, 1988; Chandra e t al., 1958; Smith and Deinhardt, 1968), and has also, unfortunately, recently been misinterpreted as no less than two distinct kinds of viral aggregate representing the agent of such differing conditions as Degos’ disease and infectious mononucleosis (Nishida and Howard, 1968; PYIozes e t al., 1968). Although this error has already been pointed out elsewhere (Moore and Chandra, 1968; Chandra e t al., 1968; Epstein and Achong, 1970), it has nevertheless led to considerable unnecessary confusion (Anonymous, 1969). B. FINESTRUCTURAL CYTOPATHOLOGICAL CHANGE^ DURING E B VIRUS PRODUCTION Where E B virus has been observed in cells it has been present either in dead cell debris or in intact cells showing various degrees of cytopathological change. This cytopathological change consists of some or all of the following characteristic features (Epstein e t al., 1965a) : 1. Decreased electron opacity of the nucleoplasm with concentration of the nucleoli and chromatin into small marginated clumps (Figs. 7 and 8 ) . 2. Fragmentation of the nuclear envelope giving nucleocytopIasmic continuity Felween segments of nuclear membrane which often showed a multilayered reduplication (Figs. 7 and 8). 3. Alteration of mitochondria in which the cristae and matrix were replaced by a beaded or clubbed electron-opaque material (Fig. 8). 4. Sheaves of unusual electron-opaque altered spindle tubules (Figs. FIG.6 . Example of a cultured blastoid cell in which there is more rough endoplasmic reticulum ( e r ) than usual. The fine structural features are in all other respects typical, with a rounded nucleus containing nucleoli, mitochondria ( m ) grouped at one pole, and large numbers of free ribosomes. X17,ooO.

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7 and 8) whose hollow tubular construction was readily seen where they were cut transversely by the plane of section (Fig. 8 ) (Epstein et al., 1964b, 1965a). 5. In addition, intranuclear immature particles were sometimes associated with unusual ring-shaped structures of unknown nature (Fig. 7) ; such structures have long been known in relation to the immature particles of other viruses of the herpes group (Morgan et al., 1959). 6. Less frequently, virus-containing cells possessed filamentous material in the nucleoplasm (Fig. 7) similar to that which has also been reported in other herpes virus infections (Watrach, 1962; Murphy e t al., 1967). The association of the above cytopathology with EB virus production by cultured Burkitt lymphoma cells has been confirmed in numerous cell strains (Epstein e t al., 1965b, 1967; Rabson et al., 1966; Pope et al., 1968a). I n addition, identical changes have been illustrated by Toshima et al. (1967) ; unfortunately, these workers regard characteristic altered spindle tubules as “crystalline structures” and seem to imply that cells showing the overall changes associated with virus production represent a second cell type in the population. It has been repeatedly found that EB virus particles are more commonly present and in greater profusion in degenerated cells or cell debris (Figs. 1 and 2) than in intact cells (Figs. 7 and 8 ) , and it would appear therefore that the progressive cell alterations induced by virus proliferation end ultimately in cell death.

C. INCIDENCE OF VIRUS-BEARING CELLS The first strains of cultured Burkitt lymphoma cells to be examined by electron microscopy (EB1, EB2) were found during the early months in vitro to contain about 1 to 2% of cells with virus particles (Epstein et al., 1965a), and this incidence level was maintained for some two years but has tended to decrease subsequently. A much more rapid loss of virus-containing cells has been reported in the AL1 strain (Rabson et al., 1966), whereas, in contrast, the EB3 and GOR strains have shown FIG.7. Part of a virus-bearing lymphoblast showing characteristic cytopathic changes. The nucleus lies in the upper left portion of the field and shows decreased opacity of the nucleoplasm, fragmentation and reduplication of the nuclear envelope as at I , and a few scattered filaments (f).Immature EB virus particles with and without nucleoids are likewise scattered in the nucleus together with a concentration of ring-shaped structures ( r ) of unknown nature. Altered spindle tubules cut transverse-obliquely lie in the cytoplasm at s near altered mitochondria ( m ) . x37,Ooo.

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an increased incidence of such cells from 2 or 3% to 10% (Epstein et al., 1966b; Epstein and Achong, 1968a) as their time in culture increased. In the case of the Jijoye strain a clone has been established in which the incidence of virus-bearing cells has ranged in different cultures as widely as from 3 to 69% (Hinuma e t al., 1967). Although it has not proved possible to relate these spontaneous changes in virus content to recognizable changes in the environmental conditions of the cultures (Rabson et al., 1966; Epstein e t al., 1966b), it has been shown that increased virus production can be induced by growing cells in an arginine-deficient medium (Henle and Henle, 196813). The explanation for this latter phenomenon is not known, but it is clear from recent experiments on strains of Burkitt lymphoblasts derived as clones from single cells, that all the cells in a given culture carry the information enabling them to become potential producers of the virus (Maurer e t al., 1969). IV. Presence of EB Virus in Burkitt lymphomas

After careful searching of biopsy material from almost 150 Burkitt lymphomas by experienced workers in several laboratories, virus particles have not been observed in the tumor cells in situ (Epstein and Herdson, 1963; Dourmashkin, 1965; Achong and Epstein, 1966; Rabson e t al., 1966; Bernhard, 1970). Despite these substantial findings, claims have been made that both reo-virus and virus of herpes-type morphology could be observed in tumor cells in biopsy samples taken directly from the patient (Griffin e t al., 1966), but with one exception, the published pictures failed to provide supporting evidence. I n the case of this single exception herpes-like particles can indeed be seen in the nucleus of a tumor cell, but it is naturally not possible to say whether these represent the EB virus or herpes simplex virus present as a passenger in the particular jaw tumor examined, for it is well known that herpes simplex virus can be isolated from about 10% of Burkitt lymphoma samples (Woodall et al., 1965; Simons and ROSS,1965). I n every case the tumors containing this agent always communicate with the oral or nasal FIG.8. Virus-bearing lymphoblast with cell membrane (above l e f t ) and nucleus with interrupted reduplicated nuclear membrane (above T @ L ~ ) . The intervening cytoplasm contains several mature virus particles (v) within spaces enclosed by fine membranes, some immature virus particles (iv), and striking bundles of altered spindle tubules (s) cut in various planes. Rough endoplasmic reticulum ( e r ) and profuse free ribosomes are also present. X25,500. (From Epstein et al., 1964b.) FIG. 9. Light micrograph of the chromosomes of a single cultured cell from a virus-bearing strain. The preparation was stained with orcein and shows the characteristic subterminal constrictions of the long arms of the No. 10 chromosome (arrow). X2,OOO. (From Tough e t al., 1968.)

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cavities, and the isolation rate is exactly comparable to the usual oral carrier rate for herpes simplex virus (Woodall et al., 1965). V. Biological Activity of EB Virus

A. INERTNWS IN ORDINARYTEST SYSTEMS Following the negative results of preliminary attempts to isolate the virus by standard virological procedures, an extensive investigation for biological activity was undertaken using a large series of chick embryos inoculated by various routes, newborn hamsters, newborn mice of two separate strains, and nine types of test tissue culture (Epstein et al., 1965a). All these, and subsequent similar experiments (Stewart et al., 1965a; O’Conor and Rabson, 1965; Rabson et al., 1966),were invariably negative, and this negative behavior under ordinary conditions is clearly a characteristic biological attribute of the EB virus. B. INDUCTION OF ENCEPHALITIS Biological activity has only been demonstrated by certain unusual manipulations. Thus, the inoculation of virus preparations together with dimethylsulfoxide (DMSO) into thymectomized newborn hamsters produced a transmissible encephalitis (Stewart et al., 1965b), and after serial intracranial passage in these animals the virus was subsequently found to cause a fatal encephalitis on inoculation into monkeys, mice, guinea pigs, and rabbits (Stewart et al., 1968). Neuropathogenicity has been confirmed for the virus without the use of DMSO or thymectomy by intracranial inoculation in newborn kittens (Grace, 1967).

C. INFECTION OF CELLSin Vdro AND INDUCTION OF LYMPHOPROLIFERATION Despite the repeated failure of EB virus to grow in ordinary test tissue culture systems, infection of several types of hemopoietic cells has been achieved in vitro (Henle et al., 1967; Grace, 1967; Pope et al., 1968b), and some of these experiments have shown that the virus has the ability to confer the power of unlimited proliferation in culture on normal peripheral lymphocytes (Henle et al., 1967; Pope et al., 1968b). A claim that EB virus carried by Jijoye cells will replicate in dog thymus cultures with the “helper” action of Moloney sarcoma virus (Mitchell et al., 1967; Hinz et al., 1968) has not been substantiated; subsequent work has shown that the virus which replicated in the latter experiments was not EB virus, but a contaminating bovine herpes virus probably introduced with calf serum in the medium (Mitchell and Anderson, 1969).

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D. INDUCTION OF SPECIFIC CHROMOSOMAL LFSION Where cytogenetic investigations have been undertaken on cultured Burkitt lymphoma cells carrying the virus, many strains have been reported to show a characteristic chromosomal marker consisting of a constriction of the long arms of the number 10 chromosome (Kohn e t al., 1967; Miles and O’Neill, 1967; Tough et al., 1968; Kurita et al., 1968; Tomkins, 1968; Nadkarni et al., 1969) (Fig. 9). I n this connection it is of great interest that normal hemopoietic cells which proliferated in vitro following successful infection with E B virus acquired the same chromosomal abnormality (Henle et al., 1967; Pope et al., 1968b), and it would seem that infection by the virus induces this particular cytogenetic lesion. E. INDUCTION OF SPECIFICCELL SURFACE NEO-ANTIGEN

It has been known for some time that many experimental tumors carry transplantation antigens on the surface of their cells which can be demonstrated by the membrane fluorescence technique applied to live tumor cells (Moller, 1961; Klein and Klein, 1964; Irfin, 1967; Lherbson et al., 1967; Tevethia et al., 1968; Morton et al., 1968). Murine lymphoid tumors of viral etiology have the additional property of carrying specific virus-determined neo-antigens on the surface of their cells (Klein and Klein, 1964; Pasternak, 1965). It was therefore of considerable interest when Klein and his collaborators first reported that Burkitt lymphoma cells appeared to carry a specific surface neo-antigen detectable by the membrane immunofluorescence test using live target cells (Klein et al., 1966, 1967). Further work has indicated that this neo-antigen is E B virus-determined (Klein et al., 1968a,b, 1969), and the significance of this in relation to a possible etiological role for the E B virus in the Burkitt lymphoma has recently been discussed extensively by Klein elsewhere (Klein, 1970). The occurrence of natural antibodies to this surface neo-antigen in many patients with Burkitt’s lymphoma might well be related to the well-known tendency of this tumor to show bemporary remissions following the administration of convalescent sera (Ngu, 1967; Burkitt, 196713; Clifford et al., 1967) and even well-documented instances of spontaneous regression (Burkitt and Kyalwasi, 1967). VI. Immunoloqical Singularity of EB Virus

As soon as it became apparent th at’ the E B virus did not behave biologically like any known member of the herpes group of agents

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(Epstein et al., 1965a; Stewart et al., 1965a; O’Conor and Rabson, 1965; Rsbson et al., 1966), attempts were made to investigate its immunological nature. Early experiments of this kind soon established that antisera t o known herpes viruses would not react in indirect immunofluorescence tests with cultured Burkitt lymphoma cells carrying EB virus (Henle and Henle, 1966a,b). At the same time it was found by Henle and Henle (1966a) that sera both from patients with Burkitt’s lymphoma and from normal individuals were capable, when appropriately conjugated, of giving a positive fluorescence response with a percentage of the cells from virus-bearing strains of cultured Burkitt lymphoblasts but not with cells infected with known herpes viruses. I n repeated experiments on different Burkitt cell strains, the number of cells reacting with the fluorescent sera corresponded roughly with the number known from electron microscopy to contain virus particles (Henle and Henle, 1966a,b), and it was then actually shown that if individual fluorescing cells were removed and examined in the electron microscope they were virus-bearing, whereas nonfluorescing cells were not (Zur Hausen et al., 1967). These findings were rapidly confirmed (Grace, 1967; Hinuma et al., 1967). Further confirmation of the distinctness of the E B virus has been obtained by tests for antibody coating and agglutination of the virus particles (Henle e t al., 1966; Moore et al., 1966; Mayyasi et al., 1967). While this work was going forward, other immunological studies were being undertaken in this laboratory; antisera were produced in rabbits by injections of purified E B virus and were used in a direct immunofluorescence test whose specificity was authenticated by combining phase-contrast, fluorescence, and electron microscopy (Epstein and Achong, 1968b). Using this test it has been shown that the viruses in five different Burkitt lymphoma cell strains as well as in cultured cells from an American case of myeloid leukemia were immunologically identical and quite distinct from known human herpes viruses with which the antiserum would not react (Epstein and Achong, 1968a). I n addition to demonstrating the distinctness of the E B virus from other herpes viruses, immunological experiments have also established that all the examples of this virus in the many different cell strains examined are antigenically identical. For, specific antisera have reacted in various types of test with a range of different E B virus-bearing cell strains indicating that a single virus is involved or a least members of a group so closely related as to be a t present indistinguishable (Henle and Henle, 1966a,b, 1967; Mayyasi et al., 1967; Klein, 1968; Epstein and Achong, 1968a; Gerber and Monroe, 1968).

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VII. Sero-epidemiology

Following the demonstration that certain normal human sera contained high-titer specific antibodies to E B virus (Henle and Henle, 1966a,b), serological surveys for these antibodies were undertaken in various populations using an indirect “sandwich-type” immunofluorescence test. I n this way it has been shown that 100% of sera from patients with Burkitt’s lymphoma had antibodies to E B virus in remarkably high titer (Levy and Henle, 1966; Moore et al., 1966), while control sera from African children sampled both in areas of high tumor incidence and in areas where the tumor is rare or absent, showed only relatively low-titer antibodies in some 50% of individuals (Levy and Henle, 1966). These antibodies in normal individuals have been found in populations lhroughout the world (Henle and Henle, 1966b, 1967; Moore et al., 1966; Goldman et al., 1968; Svedmyr and Demissie, 1968). Complement fixing antibodies to the virus have also been found in worldwide normal human populations as well as in several species of nonhuman primate, but not in lower mammals (Gerber and Birch, 1967; Gerber and Rosenblum, 1968). The antibodies detected by both the indirect immunofluoresence test and by complement fixation have shown an identical age-distribution curve. Thus, they are present in a high proportion of very young infants, where they are presumably of maternal origin since by four to 24 months the incidence level drops to about 10%. Thereafter, the proportion of individuals showing the antibodies increases with age to about 8 0 4 5 % in late adolescence and persists a t this level in all subsequent age groups (Henle and Henle, 1966b, 1967; Moore et al., 1966; Gerber and Birch, 1967; Svedmyr and Dimissie, 1968). I n contrast to this, i t has recently been found that 100% of patients with nasopharyngeal carcinoma have high titer antibodies to E B virus (Henle, personal communication) ; the significance of this curious relationship is not yet known. VIII. Natural Pathogenicity

For a long time EB virus was without known effects in man or animals other than the eliciting of specific antibodies. Recently, however, striking evidence has been obtained that E B virus is the etiological agent of human infectious mononucleosis (Henle et al., 1968; Niederman et al., 1968). It appears that infection by the virus in the early years of life passes unnoticed but is accompanied by permanent sero-conversion ; in contrast, exposure of previously uninfected subjects as adolescents or young adults frequently leads to the development of classical

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Paul-Bunnell positive infectious mononucleosis (Henle and Henle, 1968a). It might be that the route of infection or size of viral dose also play a part in determining development of the disease at this age; the relationship of outbreaks of infectious mononucleosis to kissing among youthful groups has long been recognized, and this association could well depend on the transfer by the oral route of virus-containing salivary secretions. Every patient with infectious mononucleosis subjected to appropriate investigation was found to be free of antibodies to EB virus before the disease and to have developed these in persisting high titer as the syndrome progressed. Indeed, it has even been demonstrated that such high titer antibodies continued to be present in one of Paul and Bunnell’s original patients when his serum was examined for antibodies to EB virus 37 years after his original disease episode. In other cases, besides this serological conversion, it has been possible to demonstrate that patients’ peripheral lymphocytes acquired the unusual ability to proliferate in continuous culture early in an attack of infectious mononucleosis, and that these replicating tissue culture cells not only carried EB virus, but also showed the subterminal constrictions of their number 10 chromosomes characteristic of infections with this agent (Fig. 9) (Henle et al., 1968; Niederman et al., 1968; Diehl et al., 1968; Henle and Henle, 1968a; Kohn et al., 1968; Evans et al., 1968). Confirmation of each of these findings has quickly been forthcoming (Chessin et al., 1968; Moses et al., 1968), and a recent attempt to cast doubt on the significance of this work (Glade et al., 1968) has already been shown to be erroneous (McCollum et aZ., 1969). Furthermore, identical observations have been made using a specific complement fixation test for EB virus; 21 patients were found to be sero-negative before the disease, and each developed persistent complement-fixing antibodies during the early phases of the illness (Gerber et al., 1968). Finally, the etiological relationship of EB virus to infectious mononucleosis has been dramatically established by a successful transmission experiment using a human volunteer (Grace et al., 1969) and by study of the disease as a post-perfusion syndrome (Gerber et al., 1969). IX. Related Animal Tumor Viruses

A. THEHEaPEs VIRUS O F FROG KIDNEYCaRCINOMA It has been known for some time that the renal carcinoma of leopard frogs was associated with a virus infection which was probably responsible for the disease (Luckk, 1938). Later, the virus was shown to be a typical member of the herpes group (Fawcett, 1956), but there has

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been great difficulty in propagating the virus since, like E B virus, it has proved to be avidly cell associated. Very recently, the virus has indeed been successfully isolated and has been shown both to cause kidney carcinomata on injection into developing tadpoles (Mizell e t al., 1969) and to share a common antigen with E B virus (Fink et al., 1968). B. THEHERPESVIRUSOF MAREK’SDISEASE For more than 60 years Marek’s disease has been a well-recognized lymphomatous syndrome of chickens (Marek, 1907) and although its infectious nature has long been obvious, it has not proved possible over the years to identify the causative agent. It was of considerable importance, therefore, when a virus was isolated recently, in tissue culture, from Marek’s disease both in Britain (Churchill and Biggs, 1967) and in the United States (Solomon et al., 1968), when this agent was found to be morphologically a typical member of the herpes group (Churchill and Biggs, 1967; Epstein et al., 1968; Nazerian et al., 1968; Ahmed and Schidlovsky, lW), and when solid evidence was obtained that it caused the condition (Biggs et al., 1968; Churchill et al., 1969). This was the first time that a herpes virus had been shown to cause an animal malignancy and it is particularly relevant in relation to the Burkitt lymphoma since the animal tumor in question is likewise of lymphomatous nature. Furthermore, the herpes-type virus of Marek’s disease shows the same extreme cell avidity as E B virus, a characteristic which has been responsible in both cases for the great difficulty experienced in isolating these agents in test systems. I n addition, the two agents cause similar fine-structural cytopathic changes, in particular the production of unusual altered spindle tubules (Epstein et al., 1964a, 1965a, 1968) (Figs. 7 and 8). Incidentally, such altered spindle tubules are otherwise known only in cells of the renal carcinoma of leopard frogs, which is likewise caused by a herpes virus. C. HERPES Saimiri I n recent months a further example of a seemingly oncogenic herpes virus has been reported. Herpes Saimiri was isolated in tissue culture from normal kidney cells of the squirrel monkey (Saimiri sciureus) (Melhndez et at., 1969a); although apparently having no pathogenic effect on its natural squirrel monkey host, this herpes virus regularly induces a fatal lymphoreticular tumor on inoculation into both owl monkeys (Aotus species) and marmosets (Xanguinus species) (Mel6ndez e t al., 1969b). Although these findings are, of course, preliminary, and much further investigation and confirmation is required, the fact that a

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herpes virus seems to have proved oncogenic in primates is of the greatest importance. X. Discussion

The finding of the highly unusual EB virus in relation to such an unusual human tumor as Burkitt’s lymphoma has been of considerable interest. As a result of this, much new information has been accumulated on the EB virus and has raised important questions as to the relationship of this agent to both benign and malignant human lymphoproliferative diseases. It has already been pointed out that the original hypothesis that Burkitt’s lymphoma might be caused by a climate-dependent arthopodborne oncogenic virus (Burkitt, 1962a,b) has required revision in the light of later findings (Haddow, 1964). But in addition to this it has also been stressed that the climate dependence of the tumor remains unquestioned in Africa and has been extended to a second endemic area (Booth et at., 1967), and that in such endemic areas evidence continues to accumulate strongly suggesting the infectious nature of the condition (Burkitt and Wright, 1966; Pike et al., 1967). From what we now know, if a virus is involved in causing Burkitt’s lymphoma it would have to be widely present in human populations, inducing a tumor in temperate countries as a very unusual effect involving a rare individual out of the large numbers infected, in the same way as widespread murine and avian tumor viruses may initiate neoplasia only in a minority of infected animals. In the tropics, in contrast, tumor production would be a much more common result of this widespread infection, the increase in oncogenicity being connected with climatic factors. Thus, the virus might act in this way through being introduced by an abnormal route such as directly into the blood stream if carried sometimes by a hemophagous climatedependent vector, or through having its properties slightly altered in the direction of oncogenicity by replication in such a vector or in an animal reservoir from which a vector might carry it to man. Alternatively, the high incidence and climate dependence in the tropics might be determined by some independent co-factor whose presence might make oncogenic activity on the part of a widespread virus much more frequent than in a temperate areas where the co-factor is lacking. This latter hypothesis has recently been excitingly elaborated in connection with hyperendemic malaria (Burkitt, 1969) ; it has been persuasively suggested that constant assaults on the reticulo-endothelial systems of the population in areas where malaria is ever present might well provide an appropriate altered cellular soil which a normally quiescent widespread virus could more readily transform to malignancy.

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Within this currently postulated pattern, EB virus fits the role of oncogenic agent increasingly well. It is a virus which has indeed been shown to be widely present in human populations throughout the world (Henle and Henle, 1966a,b; Levy and Henle, 1966; Moore et al., 1966; Gerber and Birch, 1967; Gerber and Rosenblum, 1968; Goldman et al., 1968; Svedmyr and Demissie, 1968). But beyond this, the crucial significance of the EB virus lies in the fact that it is a stimulator of lymphoproliferation, not only capable of conferring the power of unlimited growth on normal human lymphocytes in vitro (Henle et al., 1967; Pope et al., 1968b), but also responsible for the in vivo lymphoproliferation of infectious mononucleosis (Henle et al., 1968; Niederman et al., 1968). Furthermore, EB virus is known to induce a specific chromosomal aberration in human cells (Kohn et al., 1967, 1968; Miles and O’Neill, 1967; Tough et al., 1968; Chessin et al., 1968), and causes the formation of a surface neo-antigen on the cells of the Burkitt lymphoma (Klein et al., 1966, 1967) in the same way as those caused in murine lymphoid neoplasias by well-recognized mouse leukemogenic tumor viruses (Klein and Klein, 1964; Old and Boyse, 1964; Kaplan, 1967). The EB virus would thus seem to fulfill in its effects on human lymphoid tissue many of the criteria established for malignant transformation of normal animal cells by known oncogenic viruses (Henle, 1968). Equally striking is the fact that high titer antibodies to EB virus are present both in 100% of patients with Burkitt’s lymphoma (Levy and Henle, 1966; Moore et al., 1966), as well as with infectious mononucleosis (Henle et al., 1968; Niederman et al., 1968; Chessin et al., 1968; Gerber et al., 1968; Evans et al., 1968). Apart from this body of indirect circumstantial evidence, EB virus has assumed a new importance following the demonstration that herpes viruses play an etiological role in frog kidney carcinoma (Mizell et al., 1969), in a malignant lymphoreticular proliferation of primates (Melhndez et al., 1969b), and in Marek’s disease of chickens (Biggs et al., 1968; Churchill et al., 1969). I n the case of the herpes virus of Marek’s disease it appears that the agent is widespread in chicken populations and exerts its oncogenic potential only under certain individual, environmental, and genetic circumstances (Biggs et al., 19&3), the exact mechanisms being little understood at the present time. In a similar way, EB virus is widespread in human popuIations. Infection in youth commonly results only in sero-conversion without recognizable illness, exactly as occurs with poliomyelitis where only a tiny minority of people infected develop the paralytic disease. The antibodies acquired are associated with subsequent immunity to infectious mononucleosis, but for those who remain uninfected and seronegative as young adults, 15% develop the disease (Evans et al., 1968)

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with its characteristic lymphoproliferative lesions. Furthermore, if EB virus does in fact play a role in human neoplasia, it is not difficult to imagine, in view of its transforming capabilities, that it might induce actual malignant lymphoproliferation in a very rare, unusual individual anywhere in the world where sporadic Burkitt lymphomas occur. Many currently believe that E B virus could well cause such a wide spectrum of human lymphoproliferative conditions (Sinkovics et aZ., 1968 ; Rosenkranz, 1968; Allison, 1968) and it is of interest in this connection that the Friend and Rauscher murine leukemia viruses can induce a mononucleosis-like syndrome in mice which are insensitive to the leukemogenic effect of these agents (Schneider et al., 1967). Where Burkitt’s lymphoma is very common in Africa and New Guinea, some additional climatedependent co-factor must clearly be superimposed on this pattern to account for the local areas of high incidence, and the possible role of malaria in this context has been discussed above. Such a factor might act to cause frequent lymphoid tumors in the same way as the environmental factors which influence the oncogenic behavior of the Marek virus. It has been repeatedly recognized from the start that E B virus may well prove t o be no more than an opportunistic passenger living as a commensal in lymphoid cells (Epstein et aE., 1964a; Epstein, 1967), particularly where these are hyperactive in proIiferative disease states. However, although it is quite possible that E B virus may turn out to be a wild goose, it is clearly one which it is urgently necessary to chase, if only for the purpose of exclusion. Over the last five years evidence pointing to a carcinogenic role for EB virus has steadily continued to accumulate. Whether or not this agent ultimately proves to be a human tumor virus, enough is known a t the present time to make it a prime suspect. The considerable body of evidence associating the EB virus with a t least one type of human malignant lymphoid tumor has been reviewed, but from the point of view of future policy the human situation presents a considerable dilemma. Once a virus has been found which, from indirect and circumstantial evidence, is suspected of oncogenicity in man, direct proof of its role in the causation of malignancy may be impossible to obtain. The question then remains as to the value of accumulating yet more and more evidence for the association of this agent with a given human malignancy, for, information of this type can never give a final definitive answer. At some stage a more dynamic approach must be decided upon, and it might well be that this should be undertaken sooner rather than later. It would appear that a t the present time the only way to resolve this difficulty might, be the long-term development of an experimental vaccine and the undertaking of a trial pilot vaccination program in an

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area of high endemicity of the tumor, followed by a prospective surveillance to detect any consequential decrease in the expected number of cases. If it could be shown in this way that elimination of the virus materially affected the incidence of the disease, not only would a causal relationship have been demonstrated, but also the usefulness of prophylactic vaccination. Decisions on these difficult questions of policy in the coming years will be needed not only for EB virus but equally for any other agents which may be suspected of carcinogenicity in man. The resolution of this problem is likely to prove even more difficult than the laboratory investigation of the complex and obscure situation surrounding the question of human viral neoplasia.

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