Benign and Malignant Disease Caused by E B V

(1)

0022-202X/84/830lS-0088s$02.00/0

Vol. 83, No. 1 Supple;^{; l 1} Printed in U, ,\

Benign and Malignant Disease Caused by E B V

H A N S W O L F , M . D . , P H . D . , A N D R U D O L F S E I B L

Max von Pettenkofer-Institute for Hygiene and Medical Microbiology, University of Munich, Munich, Western Germany

Epstein-Barr virus (EBV) causes infectious mononu- cleosis as a primary disease. The virus infects more than 90% of the average population and persists lifelong in peripheral B-lymphocytes. The virus is produced in the parotid gland and spread via the oral route. Serology suggests that the Epstein-Barr virus might be involved in the causation of two neoplastic diseases of humans:

African Burkitt's lymphoma and nasopharyngeal car- cinoma. Whereas the development of the lymphoma has an even better linkage with chromosomal rearrange- ments, nasopharyngeal carcinoma shows a unique asso- ciation with Epstein-Barr virus. Environmental factors, including traditional Chinese medicine, may be respon- sible for the enhanced risk of nasopharyngeal carcinoma in certain, predominantly Chinese, populations of south- ern Asia. Possible mechanisms leading to the establish- ment of the neoplastic manifestations will be discussed.

E p s t e i n - B a r r virus ( E B V ) , p a p i l l o m a virus, a n d hepatitis B virus have a few remarkable properties i n common. T h e i r mobile nucleic acid is D N A that is circular or integrated during latency, they persist lifelong i n carriers, and they resisted all attempts to grow them i n vitro i n a simple a n d fully lytic system. T h e y are, however, efficiently t r a n s m i t t e d i n nature, where they may cause a more or less severe p r i m a r y infection followed by a lifelong carrier status, i n d i c a t i n g the delicate interaction of these viruses w i t h their hosts. E B V * was first isolated a n d characterized as a herpes group virus from l y m - phoblastoid cell lines of the B lineage established from A f r i c a n B u r k i t t ' s lymphomas ( B L ) . V i r a l particles or structural a n t i - gens have never been detected i n fresh biopsies. T h e possibility to grow these cell lines from the neoplastic tissue was not only technically very helpful, because these cells could be used as an antigen source for serologic studies, but it was also supposed to be a significant observation as far as the mechanisms of onco- genicity are concerned. T h e role of E B V i n this process was stressed by the fact that B cells of n o r m a l u m b i l i c a l cord blood are efficiently i m m o r t a l i z e d by preparations of E B V . W h e n some years after these i n i t i a l findings marmosets died of i y m - phoproliferative disease after i n o c u l a t i o n w i t h E B V , it ap- peared as i f the H e n l e - K o c h postulates for the causation of a neoplastic disease of humans by a virus h a d been fulfilled for the first time.

However, if one looks to the H e n l e - K o c h postulates i n their original version w i t h a l l their l i m i t a t i o n s , especially for assess- ing secondary or chronic disease, one can see that the situation

R e p r i n t requests to: D r . H a n s W o l f , M a x von Pettenkofer-Institut, Pettenkoferstrasse 9a, 8000 M u n i c h 2, West G e r m a n y .

A b b r e v i a t i o n s :

B L : B u r k i t t ' s l y m p h o m a E A : early antigens

E B N A : E p s t e i n - B a r r nuclear antigen E B V : E p s t e i n - B a r r virus

N P C : nasopharyngeal c a r c i n o m a P B L : peripheral b l o o d lymphocyte T C : T o n s i l l a r c a r c i n o m a

V C A : V i r a l capsid antigens

* M o s t of the data presented i n the first part of this paper are extensively referenced i n available review literature [1-3].

should not be seen as s i m p l i s t i c . T h e H e n l e - K o c h postulates (in a translation of R i v e r s [4]) are as follows:

1. T h e parasite occurs i n every case of the disease i n question and under circumstances that can account for the patho- logic changes and c l i n i c a l course of the disease.

2. It occurs i n no other disease as a fortuitous a n d nonpath- ogenic parasite.

3. After being fully isolated from the body a n d repeatedly grown i n pure culture, it can induce the disease anew.

For A f r i c a n B L , the first postulate is not fulfilled i n only 3%

of cases. However, c l i n i c a l l y very s i m i l a r l y m p h o m a s outside Africa—where they are m u c h r a r e r — c a n be associated with E B V i n only 2 5 % of cases. T h i s w i l l be c o m m e n t e d on later.

T h e second postulate is not exactly fulfilled because E B V is regularly associated w i t h infectious mononucleosis, clinically inapparent seroconversion a n d nasopharyngeal carcinoma. The t h i r d postulate is also not fulfilled i n its strict sense because the experimentally infected marmosets present w i t h a polyclonal, although fatal, lymphoproliferative disease. T h i s observation w i l l also be commented on later.

S E R O L O G Y O F E B V - R E L A T E D D I S E A S E S T h e B - c e l l lines that can be established from B L or, as was shown later, from anybody's peripheral blood leukocytes after primary E B V infection v a r y i n their a b i l i t y to produce virus, although every single cell contains E B V genomes a n d expresses a nuclear antigen, E B N A . Some cell lines, e.g., one named Raji, can be treated w i t h a variety of chemicals, such as I U D R , butyric acid, phorbol esters, or cycloleucine. A l l these com- pounds are inhibitors of D N A - m e t h y l a t i o n and induce synthesis of additional virus-related antigens t h a t have been named early antigens ( E A ) . O n l y few cell lines stably produce virus and viral capsid antigens ( V C A ) by spontaneous activation of the latent genome i n a characteristic fraction of the cells. The various cell lines proved of tremendous value for molecular biology a n d seroepidemiology. U s i n g these antigens, the Henles and a number of other investigators found that B L patients had invariably h i g h antibody levels to E B V - r e l a t e d antigens.

T h e seroconversion to E B V positivity of one laboratory tech- nician following severe mononucleosis was the i n i t i a t o r of a study w i t h college students at Y a l e t h a t clearly showed that E B V is the cause of infectious mononucleosis a n d that this frequent disease of higher socioeconomic groups is the primary disease caused by E B V .

Sera from nasopharyngeal c a r c i n o m a revealed s i m i l a r E B V specific antibody patterns to those observed for B L . Sera from a variety of other diseases ( H o d g k i n ' s disease, chronic lym- phatic leukemia, chronic m y e l o n l e u k e m i a , a n d sarcoidosis, just to name some) also showed significantly elevated antibod}

titers. However, i n a l l these cases, a significant fraction of sera was negative and antibody levels to other latent viruses (vari cella virus, cytomegalovirus) were also elevated.

I N F E C T I O U S M O N O N U C L E O S I S A S T H E P R I M A R Y D I S E A S E C A U S E D B Y E B V

Infectious mononucleosis seems to be u n k n o w n i n such areas as the P h i l i p p i n e s [5] or M a l a y s i a [6], where infection by E B V occurs very early i n life a n d almost everybody has antibodies 88s

(2)

(3)

by he age of 10 years. C l i n i c a l symptoms seem to be a conse- qu nee of juvenile or a d u l t infection, and one might speculate wh >ther a vaccine-primed organism will be infected without

Se\ are clinical s y m p t o m s .

' 'he clinical diagnosis of infectious mononucleosis is usually dei ived from a c o m b i n a t i o n of four signs:

. H i g h leukocyte count ranging from 10,000 to 20,000 a n d reaching up to 50,000

::. 1 0 % atypical cells

;;. L y m p h a d e n i t i s 4. Fever

The most frequent s y m p t o m s are listed i n Table I.

T A B L E I. Clinical symptoms of infectious mononucleosis

Symptom Incidence

Enlarged l y m p h nodes 8 0 - 1 0 0 %

Fever 8 5 - 9 8 %

P h a r y n g i t i s 8 0 - 9 5 %

T o n s i l l i t i s 9 0 - 9 8 %

Splenomegaly 4 0 - 9 0 %

Hepatomegaly 3 0 - 4 0 %

P e r i o r b i t a l edema 1 0 - 3 0 %

E n a n t h e m a 3 0 - 5 0 %

E x a n t h e m a 3-15%

Jaundice 5-10%

Source: M o d i f i e d from [7].

Several authors described a m u c h higher prevalence of rash.

T h i s is, however, i n most cases due to a typical concomitant hypersensitivity to a m p i c i l l i n . A b o u t 1 percent of the infectious mononucleosis cases show one or more of the complications listed i n Table II, either already at the onset of the disease or as a late consequence. M o s t complications are due to a u t o i m - mune mechanisms a n d are i n some cases indiscernible from graft versus host disease, a m e c h a n i s m by w h i c h the body could clear itself from the excess of E B V - c o n v e r t e d proliferating B cells.

U n d e r rare circumstances, the T - c e l l response might be i n - sufficient because of temporary influences such as treatment w i t h high doses of cyclosporin A i n c o m b i n a t i o n w i t h cortico- steroids or because of A I D S or a certain genetic predisposition as described by P u r t i l o et a l (Duncan's syndrome, X L P , X - chromosome-linked lymphoproliferative disease) [9]. I n these cases, occasional B cells have a chance to escape host control and grow without l i m i t a t i o n , as they do when they are kept i n vitro. T h e consequences have been described as B L - l i k e disease in 3 A I D S patients [10] or as polyclonal lymphoproliferative disease for X L P patients [9] or kidney transplant recipients [in.

T h e first standard test for the diagnosis of infectious m o n - onucleosis is still the test for heterophile antibodies w h e n specific tests are not available. F o r young c h i l d r e n , these tests are false negative i n up to 30% of cases. E B V - s p e c i f i c antibodies

T A B L E II. Complications of EBV infection

1. Hematologic: A g r a n u l o c y t o s i s , granulocytopenia, a u t o i m m u n o h e - molytic anemia, t h r o m b o c y t o p e n i a , panmyelopathy

2. H e p a t i c : Hepatitis, massive necroses

3. Neurologic: C e r e b e l l i t i s , encephalitis, G u i l l a i n - B a r r e syndrome, meningoencephalitis, transverse myelitis, S S P E

4. R e n a l : G l o m e r u l o n e p h r i t i s , nephrotic syndrome 5. Cardiovascular: M y o c a r d i t i s , pericarditis

6. Respiratory: A c u t e l a r y n g i t i s , interstitial pneumonia, bronchopneu- m o n i a

7. Others: A r t h r i t i s , d e r m a t i t i s , splenic rupture, immunodeficiency, myoglobinuria, subacute t h y r o i d i t i s , transitory anergia

8. Neoplastic: C o n v e r s i o n o f p o l y c l o n a l B - c e l l proliferation to mono-

clonal B - l y m p h o b l a s t m a l i g n a n c y ⁰ X L P as a n example o f i m m u n o l o g i c a l l y deprived hosts.

b D e t e r m i n e d by i m m u n o p r e c i p i t a t i o n of G P 240/200.

T A B L E III. Serologic parameter associated with various disease conditions

V C A

Disease - - E A E B N A M A "

IgG IgM IgA

N o r m a l adults + —

- -

₊ ₊

A c u t e adults (early) + + +

-

⁺

- -

C h r o n i c infection + +

-

± ± ±

Reactivation + +

-

⁺ ⁺ ⁺

X L P⁷ +

- -

⁺ ⁽⁺⁾ ⁹

N P C + +

-

⁺ ^+(D) ⁺ ^{+ +}

B L + +

- -

+(R) ⁺ ^4-

F I G 1. I n situ E B V D N A h y b r i d i z a t i o n )f cryosections of n o r m a l p a r o t i d g l a n d a, b), n o r m a l tonsil (c), and n o n k e r a t i n - zing t o n s i l l a r c a r c i n o m a (d). C l o n e d v W D N A that was labeled i n v i t r o w i t h

!' l ] t h y m i d i n e triphosphate by nick n n s l a t i o n was used as a probe. N o t e I at the producing cells (arrow) i n the ); rotid glands s u r r o u n d or are present r the ducts (a, b), t h a t the n o r m a l tonsil s E B V - g e n o m e - f r e e (c), a n d that the ( asillar carcinoma c o n t a i n s E B V - c a r -

"Ung cells (arrow) (d). (From Wolf et al

a » !

a i l

•

* # '

**-

(4)

vary i n their value for diagnosing disease. Table III gives the appearance and persistence of the antibodies with their respec- tive reactivities.

There is no cure for infectious mononucleosis other than time, although the clinical symptoms and the antibody titers reportedly reach normal values much faster, especially in severe cases, when the tonsils are removed [12]. T h e tonsils may be the primary site of immunologic defense and the reservoir or

"recruitment center" of B-lymphocytes able to proliferate. F o l - lowing convalescence, about 0.01% of B cells supposedly contain E B V genomes or E B N A and are able to grow in vitro. The chances of growing EBV-immortalized B cells are higher if leukocyte preparations are infected with certain strains of E B V (e.g., from the B-95-8 marmoset cell line). When this i n vitro immortalization is done with leukocyte preparations from E B V - i m m u n e individuals, the T cells have to be removed. A t least 60% of patients with infectious mononucleosis shed E B V in their saliva. Virus shedding does not require special preven- tion against spreading the disease because epidemics are rare and infection of persons in close contact (roommates) is scarce [13]. Virus shedding does not stop with recovery from disease, and at least 30-40% of the adult population sheds E B V .

N A S O P H A R Y N G E A L C A R C I N O M A A S A P O S S I B L E

" S E C O N D A R Y D I S E A S E " R E L A T E D T O E B V The other disease i n which E B V so far shows a 100 percent association is nasopharyngeal carcinoma ( N P C ) [3]. N P C most frequently starts at the fossa rosenmuelleri at the postnasal space, although patients frequently report only after the first typical metastases in the cervical lymph nodes have developed.

The disease has an incidence of up to 40 per 100,000 per year, the most frequent neoplasia of humans occurring in some areas of southern China and among Chinese i n Singapore and M a - laysia. The incidence is also elevated i n other parts of the world, such as Borneo or Tunisia. In most other areas, the incidence is around 0.2 per 100,000 per year. The age distribu- tion shows a clear single peak around the ages of 40 to 50 i n almost all high-risk areas. There is, however, a second peak at an early age, ranging from 5 to 15 years i n Borneo and to some extent i n Tunisia [3]. Possible exogenous risk factors will be discussed later. A genetic disposition for the disease is difficult to test, although the decline of tumor incidence i n second- generation Chinese emigrants to the United States, expecially if they live in rural areas, does not support a genetic involvement [1-3]. The relatively simple H L A patterns of the Chinese allowed, however, a significant association of relative risk with certain H L A types. A2 and B W 4 6 have elevated relative risks of 1.5 and 1.9, respectively, and of 2.5 i f expressed i n combi- nation, and B 1 7 / B W 5 8 is associated with a relative risk of 2.2.

A l l , however, is associated with a relative risk of 0.5. Thus a relative risk factor of 5.0 exists between the best and the worst constellation of H L A types [14]. The neoplasia leads invariably to death of the patient, frequently with extensive cranial inva- sion unless the tumor is recognized early and treated. The most successful therapeutic approach is early detection combined with careful radiation [15], and survival rates exceeding 90%

have been reported ( Y i Zeng, personal communication). The use of inexpensive screening methods becomes evident in face of the correlation between a rapid decline i n survival rates and the elapsed time between the putative earliest detection with careful screening and the actual detection of disease and first intervention. The determination of IgA a n t i - V C A antibodies has been used with good success to define a group at high risk, since 3% of persons positive in this test had N P C upon clinical examination. Between 35 and 70% of tumor bearers have IgA antibodies to E A ; therefore, this test can be used with good success as a second screening step. About 30 percent of patients positive in the IgA a n t i - E A test have detectable N P C [43]. New and improved methods for the detection of viral nucleic acid and the development of sampling devices that aspirate cells

without taking biopsy [16] may further improve the diagn< tic possibilities. The introduction of single-stranded hybridiza on probes and the availability of label other than V2P with its inhibitory short half-life have helped to bring nucleic acid hybridization over the threshold of a routinely usable method.

Considering cost-effectiveness, necessary equipment, and sen- sitivity, we have found that chemical iodination of a collection of viral fragments cloned i n the single-stranded D N A phage m l 3 gives excellent hybridization probes usable for in situ hybridization and hybridization on Southern blots of D N A or spotted D N A or cell lysates [44].

B U R K I T T ' S L Y M P H O M A A N D I T S A S S O C I A T I O N W I T H E B V

Burkitt's lymphoma has been discussed earlier, and addi- tional remarks will be found i n the Discussion section.

SERUM ND

^{6 5 6 6} 7070 8 4 6 3

EBNA -

¹⁶ ³² ³²

EA - ^-

⁴⁰⁹⁶

^-

VCA -

2042 16384 1024

F I G 2. A n analysis of EBV-specified polypeptides by immunopr cipitation using sera with different reactivities to EBV-specified an gens. N D is a serum free of E B V antibodies; 6966 and 8463 have hn titers against V C A but not E A ; serum 7070 has high titers against bo V C A and E A . Proteins 138, 88, 45, 40, 38, and 37 are only precipitate by the EA-positive sera. These results are representative for a mu<

larger panel of sera which we have tested. (From Bayliss and Wc [30].)

(5)

F I G 3. a, R N A from induced P 3 H R 1 was hybridized to the cloned E B V Bam fragments and two clones ( M 3-1-1 and M 3-5-6) that subdivide the Bamk fragment (see map). T h e bound m R N A was eluted and translated i n vitro using the rabbit reticulocyte system. T h e translation products were immunoprecipitated and analyzed on SDS-polyacrylamide gels.

S O M E O P E N P R O B L E M S A N D A P P R O A C H E S T O S O L V I N G T H E M

The presented observations on the involvement of E B V with ] leoplastic and nonneoplastic disease raised a number of ques- 1 ions that will be discussed in this section:

1. What is the source of virus in the throat washings of apparently healthy persons, and why do apparently normal individuals who shed virus have antibodies only to V C A and E B N A and not to E A , which are produced in large amounts during viral replication?

2. How does the virus infect epithelial cells that apparently lack receptors for the virus?

3. How does the virus persist i n cells lifelong, and what are the molecular mechanisms underlying the regulated expression of E B V ?

1. One might postulate that E B V - c a r r y i n g lymphocytes in the oropharynx that may have escaped host control mechanisms could be the source of the virus that can be obtained from healthy individuals. Other studies [17] indicated that E B V could be isolated in relatively high titers from saliva collected

(6)

P R O T E I N S IN I N D U C E D R A J I C E L L S

110

P R O T E I N S IN I N D U C E D P 3 H R 1 C E L L S 37,5 34

31

3 0 4 9

4 1 2 6 . 5 2 5 21 73 21

9 6

6 9 4 3

4 1 . 5 140 140 4 0 . 5 6 4 6 4 4 0 . 5 4 6 4 6

3 5 3 5 150

140 135 1 3 0 1 2 3 1 1 5

78 4 4 3 5 102 3 5 3 5 138

4 0 2 9 4 0 ^ 0 39 31

W W W W W W W W W W W W Y H Q U P O a M S L E • Z R K O c b T X V d

M 3 - 5 - 6

C H 4 A 2 6 - 3 6 C H 4 A 3 8 - 4 7 C H 4 A 5 3 - 6 1 C H 4 A 6 9 - 7 9 C H 4 A 8 3 - 9 3

C H 4 A 0 - 8 C H 4 A 3 2 - 4 1 C H 4 A 4 5 - 5 4 C H 4 A 6 1 - 7 2 C H 4 A 75 - 8 4 C H 4 A 9 0 - 9 9

B R a p E c o C

R a p E c o 0 cue

F I G 3. b, M a p of the in vitro translated proteins with hybrid-selected R N A from induced P 3 H R 1 cells and induced Raji cells. The Bam fragments were cloned from B 95-8 derived E B V D N A . B y hybridizing to sheared fragments of E B V D N A cloned in Charon 4A, a finer map of the coding regions of some proteins could be achieved. The narrow coding regions for proteins 18, 90, 47, 73, and 69 in the induced Raji cells are based on the assumption that these proteins correspond to proteins with the same molecular weights in P 3 H R 1 cells.

from Stenson's duct. Studies on parotid salivary gland tissue [18,19] have suggested that the cells which surround or fill the lumen of the ducts of this gland are capable of supporting a productive cycle of E B V replication (Fig 1, a, b). Production of E B V in salivary duct cells could also explain the absence of certain EBV-specific serum antibodies in normal adults (see also Discussion). Searches with other tissues of the oropharynx (e.g., tonsils from healthy individuals; Fig 1, c) proved negative, since no productively infected cell could be found using in situ hybridization techniques. This situation resembles somewhat that seen with Marek's disease virus, where the transformed cells are T cells, but the lytic expression of the virus occurs in the germinative epithelium of the feather follicles [20]. In situ hybridization studies using tonsillar carcinoma (TC) tissue, however, showed that 25% of the specimens tested so far carried E B V D N A in the epithelial cells (Fig 1, d).

2. To date, receptors for E B V have been demonstrated only on B-lymphocytes [21] and no other normal nonmalignant cell type [3]. How then does the virus enter such cells and persist within them? Initially, microinjection of E B V D N A into a wide variety of receptor-negative cells [22] and, later, transfection with E B V D N A using the calcium phosphate coprecipitation technique [23,24] and the implantation of receptors into the membranes of receptor-negative cells [25] have been used to demonstrate that once E B V overcomes the barrier of penetra- tion, normal expression of the virus can occur, although synthesis of E B N A was not observed when only lytic expression was induced. It was proposed some time ago that syncitium- inducing viruses, such as paramyxovirus, might induce fusion between lymphocytes and epithelial cells, thus allowing the virus to gain access to such cells. Since then, we have demonstrated that E B V itself can induce fusion. When densely packed monolayers of lymphoblastoid cells [26] were infected with E B V derived from the EBV-producing cell line P 3 H R 1 , the formation of polykaryocytes could be observed. Further studies

with mixed monolayers containing both receptor-positive (Raji) cells and receptor-negative cells (human fibroblasts, epithelial cells, or T-lymphoblastoid cells) showed that an infected E B V - antigen-expressing B-lymphoblastoid cell was capable of fusing with a noninfected receptor-negative cell [27]. A close cell-to- cell contact, such as occurs in monolayers, is necessary for the development of polykaryocytes. The viral nature of the fusion event and the mechanisms involved have been studied in detail using chemical activation of latent genomes and various met- abolic inhibitors. It was shown that an early viral protein was responsible for the fusion event [28]. More recent experiments [29] have further substantiated the viral origin of the fusion- inducing protein(s). Purified E B V D N A was transfected into unrelated cells ( N I H 3T3 cells). In addition to the synthesis of E B V E A , fusion of the transfected cells was also observe 1.

Further studies with this technique using cloned fragments f the E B V genome should permit the mapping of gene(s) encod- ing the fusion on the E B V genome [29].

3. Very little is known at the molecular level of the mecb nisms that regulate the response of the cell to infection wi- E B V . As far as we know, all E B V - c a r r y i n g cell lines expres;

single viral a n t i g e n — E B N A . T h i s protein could be the repr<

sor protein that prevents lytic expression. If E B V - c a r r y i n g c lines are infected with a sufficiently high multiplicity, a ly cycle will occur [30], during which at least 30 virus-induced specific proteins will be synthesized. These proteins can divided into three groups according to their kinetics of syntl sis, response to inhibition of D N A synthesis, and requireme for virus-specified factors.

Do these observations have any relevance to regulat expression of E B V during the stepwise induction of a lytic eye by the virus in the absence of inhibitors? If Raji cells t infected with decreasing amounts of virus, then the intern diate and late proteins are no longer synthesized at a cert;

cutoff value [29,31].

(7)

However, a certain subset of the E A complex is made, and it is the same subset as that obtained by chemically induced Raji ctlls. Similar observations have been made using an E B V grnome-negative cell line (BJA). Immunoprecipitation of E B V p oteins allows further characterization and a linkage to sero- lcgic data. These data (Fig 2) show also that proteins of the EA complex are synthesized in considerable amounts during the replication of E B V ; yet normal adults do not have antibodies against these proteins even if they shed virus.

In an attempt to substantiate further the viral nature of the polypeptides and to map the genes on the viral genome, both of which are essential for detailed studies of the molecular basis for regulation of viral gene expression, we selected m R N A from a producer cell line (P3HR1) by hybridization to cloned E B V D N A fragments. This m R N A was then translated in vitro using a rabbit reticulocyte translation system. W i t h this technique we were able to identify proteins as EBV-specific and have mapped their positions on the E B V genome (Fig 3, a, b) (Rudolf Seibl and Hans Wolf, in preparation).

One interesting observation that awaits explanation is that if m R N A from EBV-negative cells ( B J A - B ) is selected using E B V D N A , an m R N A is obtained that hybridizes to the BamHl K fragment of the E B V genome. This R N A can be translated in vitro to yield polypeptides of 84 kd and 92 kd molecular weight (Fig 4). This region of the E B V genome is known to hybridize to cellular D N A under stringent conditions and may

Bam R K B

FlG 4. R N A from B J A - B cells was hybridized to cloned Bam frag- ments of E B V as given on top of the gel slots. The bound m R N A was eluted and translated in vitro. Translation products were analyzed on SDS-polyacrylamide gels.

represent cellular sequences that have been incorporated into the viral genome during the course of evolution.

D I S C U S S I O N

A model has been developed that attempts to include the available data and should be useful to predict certain events or measurable parameters as well as to design further experiments, thus allowing the hypothesis to be further tested (Fig 5).

1. Normal Situation

a. Primary infection: Development of antibodies to V C A , E A , and E B N A

As suggested, E B V infects B-lymphocytes during acute or primary infection, and because of the lack of immune response, a number of cells enter into the lytic cycle and produce a full set of viral antigens ( E A and V C A ) that are shed into the bloodstream. It seems probable that not all B-lymphocytes are capable of supporting a fully lytic infection because of a cellular factor that prevents expression of E B V (Fig 5, block 2). These cells will be selected for and grow on to become the cells that carry E B V latently for the rest of the host's life.

Because of the release of all the EBV-specified antigens, antibodies will be developed against E A , V C A , and E B N A (Table III).

b. Convalescence: Disappearance of antibodies to E A and maintenance of antibodies to V C A and E B N A

As the immune defense mechanisms of the body remove the lytically infected cells from the circulation (Fig 5, block 2), the antibody levels will start to fall during the convalescent phase.

After a certain period, antibodies to the E A disappear. However, as mentioned above, E B V is produced in the parotid gland. The viral particles and intracellular virus-associated antigens i n - cluding E A will be shed into the saliva and reach the oropharynx. Here the viral particles (but not E A ) could bind to the B - lymphocytes and be presented to the body as antigens, thus maintaining the antibody titers to V C A . E A cannot bind to the lymphocytes and will be degraded by proteases and not be available to the body as antigens. Circulating lymphocytes that carry E B V latently contain E B N A and as far as we know no other EBV-specified protein. These lymphocytes will be subject to the normal turnover processes, and as they die, they will release E B N A into the bloodstream. Therefore, antibodies to this antigen will persist. As a consequence, normal convalescent sera will have low-level IgG a n t i - V C A and a n t i - E B N A antibodies (Table III).

2. Special Conditions

a. Nonspecific secondary antibody titer increase to V C A and E A

If immunosuppression occurs, either because of other disease (e.g., Hodgkin's disease) or because of immunosuppressive therapy, some of the circulating peripheral blood lymphocytes (PBLs) will escape the normally tight control mechanism and enter into a cycle of virus replication, causing a secondary increase in the titers to E A , V C A , and E B N A .

b. Development of Burkitt's lymphoma (monoclonal disease) Under rare circumstances, a cell clone that differs in its antigenic makeup may arise and be selected. T h i s clone may then lead to the development of B L . Environmental mutagens [32] and unspecific "mitogens" (malaria) [33] that facilitate clonal selection through proliferation may favor this event.

Specific karotypes in the selected clones may correlate to the altered antigenic makeup of these cells.

Following original observations by Manolova et al [34], several authors have indicated that chromosomal rearrangements occur in all B L cases independent of their association with E B V [35]. The most important step seems to be a translocation

(8)

0 0 0

cell lysis V C A

EA •

immunol e l i m i n a t i o n

cytogenetic error clonal s e l e t i o n

Lymphoepithelial Carcinoma of Waldeyer s ring

( m a i n t a i n a n c e of p o p u l a t i o n ) E B N A

c y t o g e n e t i c error u n s p e c i f i c stimulation clonal selection

FlG 5. T h e scheme summarizes the suggested biological effects of E B V described i n the text. Blocks 1 and 2 inhibit the lytic expression of E B V , and block 3 suppresses the proliferation of E B V genome containing lymphoid cells in the periphery. Block 1, Block at cellular level (endogenous block). Evidence: Only a certain percentage of cells from cloned cell lines produce virus. T h i s block is responsible for poor production of virus.

Block 2, Block from the outside (exoge- nous block, immunologic control?). Evi- dence: In peripheral blood from patients with infectious mononucleosis, i n fresh B L or N P C biopsies no viral particles can be found. After explantation into tissue culture, a few cells start to produce virus. O : E B V , O: activated E B V genes,

v: E B V r e c e p t o r s , ^ : virus-specific changes. (From Wolf et al [29].)

Lymphoma

of part of the distal arm of chromosome 8 carrying the cellular oncogene myc to alternate acceptor chromosomes 2, 14, or 22, respectively. T h e oncogene is i n all cases translocated to a transcriptionally active site close to immunoglobulin genes.

Whether the transcription of the myc gene after translocation is enhanced and what impact this may have are not clear yet [36,37]. In any case, the involvement of myc alone cannot explain the patterns or mechanisms of appearance of B L .

It should be noted that all E B V genomes containing B - lymphoblastoid cells have unlimited growth potential i n vitro and after intracerebral injection into nude mice. Only the lymphoma-derived cells, however, can grow in the nude mouse after subcutaneous application [3]. This behavior seems rather suggestive of a loss of control elements on the surfaces of the selected clones. One might speculate that H L A or similar determinants are altered or lost during the process of clonal selection.

Transfection experiments have shown that two oncogenes frequently may be involved in oncogenesis [38]. Such a model could include an E B V gene as a possible member of a comple- mentation group of oncogenes and myc as a member of another group. Thus E B V might still play a major role i n the causation of African B L .

c. Development of polyclonal proliferative disease

Specific genetic constellations ( X L P ) or acquired conditions (immunosuppressed transplant recipients, A I D S patients; see above) determine a reduced efficiency of block 3 (cellular response to proliferating cells). Therefore, the selective pressure on peripheral B-lymphocytes (which already have a potential for unlimited growth by virtue of the resident E B V genomes) may be weaker. Under less stringent selective pressure, more cells may proliferate in vivo and lead to a polyclonal lymphoma [9]. Nonspecific growth stimuli such as the graft may favor initial proliferation and facilitate the selection of cell clones. A specific cytogenetic error [39] may occur under therapy and may induce a change from polyclonality to monoclonality.

d. Relation to T-cell leukemia

As a side effect of primary E B V infection, the initial proliferation of B-lymphocytes due to the early absence of block 2 apparently leads to a proliferation of T-lymphocytes [8]. This

may well be caused by growth factors. This polyclonal activation of T-lymphocytes may occasionally activate T cells with malignant growth potential and lead to a T-cell leukemia following infectious mononucleosis.

e. Development of nasopharyngeal carcinoma

On the specific conditions of the site of initiation of lymphoepithelial tumors. A close contact between epithelial cells and B-lymphocytes that may carry E B V genomes has been described for the lymphoepithelial ring of the throat (Waldey- er's ring). T h i s unique tissue may provide the necessary conditions for EBV-induced fusion between the two cell types, thus enabling the E B V genome to enter the epithelial cells. If this hypothesis is true, then E B V should be associated with other tumors that arise within this tissue. Evidence for this association has been independently derived by our group [28] and by Brichacek et al [40]. The major difference between T C and N P C is that only 25% of the limited number of T C s so far tested were serologically related to E B V , whereas all N P C s are EBV-genome-positive. A t the moment, we cannot explain these findings. T h i s proposal would be i n good agreement with the proffered model.

On the role of environmental factors on the relative risk ol N P C . It has been observed that certain plant extracts car induce latent E B V and somehow increase the risk for NP(

[41]. Croton o i l was one of the first suspected medications However, it is highly irritating and is used only under clos*

medical supervision and is therefore unlikely to be an N P C related risk factor. More recently, Zeng et al [42] have teste- over 500 different extracts from 100 plant families and foum that 20 of them were capable of activating latent E B V . Som of the extracts were active as aqueous extracts and thus oper the possibility that traditional Chinese herbal medicine, ofte applied as teas, may contain E B V - i n d u c i n g principles. On difference in the E B V serology of the populations with low an«

high risk for N P C development is that i n high-risk areas t h level of antibody to E B V antigens remains high throughout lit when compared with low-risk populations ( Y i Zeng and Sh Y a n G u , personal communication). Thus EBV-activating sub stances could continuously stimulate the latently infected I cells to enter into a lytic cycle, explaining the differences i

(9)

a itibody titers. If follow-up studies could demonstrate a match b .tween the areas with high risk for N P C and the use of the d scussed plant extracts, a multifactorial causation of N P C w ould be substantiated.

R E F E R E N C E S

1. The Epstein-Barr Virus. Edited by M A Epstein, B G Achong.

Berlin, Springer Verlag, 1979

2. Viral Oncology. Edited by G K l e i n . New York, Raven Press, 1980, pp 1-842

3. The Biology of Nasopharyngeal Carcinoma. U I C C Technical Re- port Series, V o l . 71. Edited by M J Simons, K Shanmugaratnam.

Geneva, International U n i o n Against Cancer, 1982, pp 1-87 4. Rivers T M : Viruses and Koch's postulates. J Bacteriol 33:1-10,

1937

5. Evans A S , Niederman J C : The Epstein-Barr virus. (Submitted for publication)

6. T a n D S K : "Absence" of infectious mononucleosis among Asians in Malaysia. M e d J Malaysia 21:358, 1967

7. Wahlen YV, Ortgiese H J , Kirsch W : Infektiose Monokleose und Epstein-Barr Virus-Infektion im Kindesalter. Z Allg M e d 55:1911,1979

8. Purtilo D T : Immunopathology of infectious mononucleosis and other complications of Epstein-Barr virus infections, Pathology Annual, part 1, vol. 15. Edited bv S C Sommers, P P Rosen. New York, Appleton-Century-Crofts, 1980, pp 253-299

9. Purtilo D T , Sakamoto K , Barnabei V , Seeley J , Bechtold T , Rogers G, Yetz J , Harada S, and the X L P Collaborators: Epstein-Barr virus-induced diseases in boys with the X - l i n k e d lymphoproliferative syndrome ( X L P ) . Update on studies of the registry.

A m J M e d 73:49-56, 1982

10. Ziegler J L , M i n e r R C , Rosenbaum E , Lennette E T , Shillitoe E , Casavant C, Drew W L , M i n t z L , Gershow J , Greenspan J , Beckstead J , Yamamoto K : Outbreak of Burkitt's-like lymphoma in homosexual men. Lancet 2:631-633, 1982

11. Hanto D W , Frizzera G, Purtilo D T , Sakamoto K , Sullivan J L , Saemundsen A K , Klein G , Simmons R L , Najarian J S : Clinical spectrum of lymphoproliferative disorders in renal transplant recipients and evidence for the role of Epstein-Barr virus. Cancer Res 41:4253-4261, 1981

12. Sauerbrei A , Sprossig M , Wutzler P, Farber I, Schweitzer H , Swoboda R, W i l k e J : Antikorpertiterverlauf gegen Epstein-Barr- Virus-spezifische Antigene bei infektioser Mononukleose mit Tonsillektomie in der akuten Krankheitsphase. Laryngol Rhinol Otol (Stuttg) 62, 1983

13. Evans A S : The transmission of E B viral infections, Viral Infections in Oral Medicine. Edited by J Hooks, G Jordan. Amsterdam, Elsevier/North-Holand 1982, p 211

14. Chan S H , Day N E , Kunaratnam N , Chia K B , Simons M J : H L A and nasopharyngeal carcinoma in Chinese: A further study. Int J Cancer 32:1711, 1983

15. H o J : Treatment of Cancer. Edited by K G Hainan. London, Chap- man and H a l l , 1982, pp 249-267

16. Richter W , G u S Y , Seibl R, W o l f H : A new method for examination of carcinomas of the nasopharynx, Nasopharyngeal Carcinoma:

Current Concepts. Edited by U Prasad, D V Ablashi, P Levine, G Pearson. In press

17. Morgan D G , M i l l e r G , Niedermann J C , Smith H W , Dowalby J M : Site of Epstein-Barr virus replication in the oropharynx. Lancet 1:1154-1155, 1979

18. W o l f H , Wilmes E , Bayliss G J : Epstein-Barr virus: Its site of persistence and its role in the development of carcinomas. Hae- matol Blood Transfusion 26:191-196, 1981

19. W o l f H , Bayliss G J , Wilmes E : Biological properties of Epstein- Barr virus, Cancer Campaign, vol 5: Nasopharyngeal Carcinoma.

Edited by E Grundmann. Stuttgart, Gustav Fischer Verlag, 1981, pp 101-109

20. Calnek B W , Hitchner G B : Localization of viral antigen in chickens infected with Marek's disease herpesvirus. J N a t l Cancer Inst 43:935-949, 1969

21. Jondal M , K l e i n G: Surface markers on human B and T lymphocytes: II. Presence of Epstein-Barr virus receptors on B lymphocytes. J E x p M e d 138:1365-1378, 1973

22. Graessmann A , W o l f H , B o r n k a m m G W : Expression of Epstein- Barr virus genes in different cell types after microinjection of viral D N A . Proc N a t l Acad Sci U S A 77:433-436, 1980

23. Stoerker J , Parris D , Yajima Y , Glaser R: Pleiotropic expression of Epstein-Barr virus in mouse lymphocytes. Proc N a t l Acad Sci U S A 78:5852-5855, 1981

24. Miller G, Grogran E , Heston L , Robinson I, Smith D: Epstein- Barr viral D N A : Infectivity for human placental cells. Science 212:452-455, 1981

25. Volsky K J , K l e i n G , Volsky B , Shapiro I M : Production of infectious Epstein-Barr virus in mouse lymphocytes. Nature 293:299-401, 1981

26. Bayliss G J , W o l f H : The spontaneous and induced synthesis of Epstein-Barr virus antigens in Raji cells immobilized on surfaces coated with antilymphocyte globulin. J Gen Virol 54:397-401, 1981

27. Bayliss G J , W o l f H : Epstein-Barr virus induced cell fusion. Nature 287:164-165, 1980

28. Bayliss G J , W o l f H : A n Epstein-Barr virus early protein induces cell fusion. Proc N a t l Acad Sci U S A 78:7162-7165, 1981 29. W o l f H , Bayliss G J , Seibl R: New results on the biology of Epstein-

Barr virus. Current Topics in Veterinary Medicine and A n i m a l Sciences. Edited by G W i l l m a n n , A Rziha, R Geskell, M Nijhoff.

In press

30. Bayliss G J , W o l f H : The regulated expression of Epstein-Barr virus: III. Proteins specified by E B V during the lytic cycle. J Gen V i r o l 56:105-118, 1981

31. K a l l i n B , K l e i n G : Epstein-Barr virus carried by Raji cells: A mutant in early functions? Intervirology 19:47-51, 1983 32. Birnboim H C : D N A strand breakage in human leukocytes exposed

to a tumor promotor phorbol myristate acetate. Science 215:1247-1249, 1982

33. Burkitt D P : Etiology of Burkitt's lymphoma: A n alternative hypothesis to a vectored virus. J N a t l Cancer Inst 42:19-28, 1969 34. Manolova Y , Manolov G, Keller J , Levan A , K l e i n G : Genesis of

the 14g+ marker in Burkitt's lymphoma. Hereditas 90:5-10,1979 35. K l e i n G : Specific chromosomal translocations and the genesis of B-cell derived tumors in mice and man. Cell 32:311-315, 1983 36. Erikson J , A r - R u s h d i A , Drwinga H L , Nowell P C , Croce C M :

Transcriptional activation of the translocated c-myc oncogene in Burkitt's lymphoma. Proc N a t l Acad Sci U S A 80:820-824, 1983

37. Maguire R T , Robins T S , Thorgeirsson SS, Heilman C A : Expres- sion of cellular myc and mos genes in undifferentiated B cell lymphomas of Burkitt and non-Burkitt types. Proc N a t l Acad Sci U S A 80:1947-1950, 1983

38. Land H , Parada L F , Weinberg R A : Tumorigenic conversion of primary embryo fibroblasts requires at least two cooperating oncogenes. Nature 304:596-602, 1983

39. Hanto D W , Frizzera G, Gaji-Peczalska K J , Sakamoto K , Purtilo D T , Balfour H H J r , Simmons R L , Najarian J S : Epstein-Barr virus-induced B-cell lymphoma after renal transplantation. N Engl J M e d 306:913-918, 1980

40. Brichacek B , Suchankova A , Hirsch I, Sibl O, Rezacova D , Zava- dova H , Vonka V : Presence of Epstein-Barr virus D N A in tonsillar tissues. A c t a V i r o l 25:361-370, 1981

41. Ito Y , Kishishita M , Yanase S, Harayama T : Epstein-Barr virus- activating principles in medicinal plants, Herpesvirus: Clinical, Pharmacological and Basic Aspects. Edited by H Shiota, Y C Cheng, W H Prusoff. Amsterdam, Excerpta Medica, 1981 42. Zeng Y , Zhong J M , M o Y K , M i a o X C : Epstein-Barr virus early

antigen induction in Raji cells by Chinese medicinal herbs.

Intervirology 19:201-204, 1983

43. Zeng Y , Zhang L G , L i H Y , J a n M C , Zhang Q, W u Y C , W a n g Y S , Su G R : Serological mass survey for early detection of nasopharyngeal carcinoma in Wuzhow city, C h i n a . Int J Cancer 29:139-141, 1982

44. G u S Y , W o l f H : Study on recombinant D N A of E B virus and phage m l 3 . I. Establishment and indentification of recombinant nucleic acid (in Chinese). Cancer 2:129-135, 1983

Benign and Malignant Disease Caused by E B V