Journal
of General Virology (1999), 80, 441–445. Printed in Great Britain
..........................................................................................................................................................................................................
SHORT COMMUNICATION
A-type and B-type Epstein–Barr virus differ in their ability to
spontaneously enter the lytic cycle
M. Buck, S. Cross, K. Krauer, N. Kienzle and T. B. Sculley
Queensland Institute of Medical Research, Bancroft Centre, 300 Herston Road, Herston, Brisbane 4029, Australia
In this study replication of A-type and B-type
Epstein–Barr virus (EBV) strains has been assessed.
A-type and B-type lymphoblastoid cell lines (LCLs)
were established by infecting B lymphocytes, isolated from five EBV-seropositive donors, with different A-type and B-type virus isolates. The presence
of viral capsid antigens (VCA) in these LCLs was
determined by immunofluoresence assay and by
immunoblotting. All of the B-type EBV strains were
capable of spontaneously generating virus regardless of the origin of the donor cells. In contrast
the A-type strains, other than strain IARC-BL36, did
not readily produce VCA in any of the different
donor lymphocytes used. This study demonstrates
another biological difference between the two virus
types : their ability to spontaneously enter the lytic
cycle.
Epstein–Barr virus (EBV) is a herpesvirus with a DNA
genome of approximately 172 000 bp. Worldwide, the virus
infects more than 90 % of the human population, and following
primary infection with EBV all individuals retain the virus for
life. In addition, in vitro infection of B lymphocytes with EBV
leads to their immortalization, and the outgrowth of lymphoblastoid cell lines (LCL). Following in vitro infection of B
lymphocytes the EBNA gene products are expressed in a
sequential fashion (Alfieri et al., 1991). EBNA 2 and EBNA LP
appear by 12 h after infection and reach maximum levels by
32 h. Expression of EBNAs 1, 3, 4, 6 and LMP is evident by 36
h, reaching maximum levels by 46 h.
EBV strains can be categorized into one of two types (Atype or B-type) which show sequence divergence within their
BamHI WYH and HindIII E gene regions reflecting divergence
in the EBNA 2, 3, 4 and 6 gene products (Adldinger et al.,
1985 ; Dambaugh et al., 1984 ; Sculley et al., 1989). Differences
in a number of other regions of the genome have also been
identified. These include differences in EBNA LP (Sample et al.,
1986 ; Dambaugh et al., 1984) and the EBERs (Arrand et al.,
1989). Differences in the proteins expressed during EBVAuthor for correspondence : Tom Sculley.
Fax j61 7 33620106. e-mail tomS!qimr.edu.au
0001-5613 # 1999 SGM
induced transformation of B-lymphocytes are reflected in
biological differences between A-type and B-type EBV. The
two types of EBV display distinct growth phenotype characteristics with B-type transformants demonstrating lower
transformation efficiency, growth rate and saturation density
as well as greater sensitivity to seeding at limiting dilutions, as
compared to A-type transformants (Rickinson et al., 1987).
LCLs are largely nonpermissive for virus replication ;
however, in producer cell lines (usually BL and marmoset lines)
a small but variable proportion of the cells supports the viral
lytic cycle with the resulting synthesis of early antigens (EA)
and viral capsid antigen (VCA), leading to production of
progeny virus. The viral lytic cycle can be induced in latently
infected cells by superinfection with P3HR1 EBV (Henle et al.,
1970), addition of chemical inducers (Luka et al., 1979 ; zur
Hausen et al., 1978) or transfection with the transactivator
BZLF1 (Countryman et al., 1987). The VCA complex of EBV
consists of at least five components, with molecular masses of
150, 110, 40 and 21\18 kDa. Serio et al. (1996) showed that the
Table 1. Virus isolates used to establish transformed cell
lines
Virus
IARC-BL74
IARC-BL36
Ag876
B95-8
QIMR-KTu
QIMR-MBl
QIMR-JSt
QIMR-Gor
QIMR-JSm
QIMR-L19
QIMR-L4
WAN-BL
Af12
Origin of virus*
BL
BL
BL
IM patienta
IM patient
Spontaneous LCLb
Spontaneous LCLb
BL
Spontaneous LCLc
Spontaneous LCLd
Spontaneous LCLd
BL
Spontaneous LCLe
Reference
Zimber et al. (1986)
Sculley et al. (1988)
Dambaugh et al. (1984)
Miller & Lipman (1973)
Unpublished
Unpublished
Sculley et al. (1989)
Young et al. (1987)
Moss et al. (1988)
Young et al. (1987)
Young et al. (1987)
Sculley et al. (1988)
Unpublished
* a, Cell line derived by passage of EBV (from an IM patient) in a
marmoset ; b, spontaneous cell line from normal healthy donor in
Australia ; c, spontaneous cell line from rheumatoid arthritis patient ;
d, spontaneous cell line from healthy donor in Papua New Guinea ;
e, spontaneous cell line from healthy donor in Africa.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 06:59:17
EEB
M. Buck and others
Fig. 1. Immunoblot detection of the p21 component of VCA in cell lines infected with different strains of EBV. B lymphocytes
isolated from donors JBL (A), DJM (B) and ISM (C) were infected with different isolates of A-type and B-type EBV. Extracts
from each of these cell lines were subjected to immunoblotting using a human serum (MCr) which contained antibodies to the
EBNAs and p21 VCA. The EBV strains used are indicated at the top of each lane and the type of each isolate is indicated at the
bottom of each lane. VCA was also determined by IFA on cell lines established from donor JBL and the percentage of VCA
positive cells is shown at the bottom of panel (A). The positions of the EBNAs and of the p21 VCA component are indicated.
BFRF3 open reading frame encoded an 18–21 kDa protein
which was an immunodominant marker of EBV infection. EBVseropositive human sera consistently detected these proteins in
the 21 kDa range on immunoblots of B cells in which EBV had
been induced into the lytic cycle.
In this study we have assessed A-type and B-type EBV
strains for their permissiveness for virus replication. To
overcome problems associated with differences in cellular
backgrounds on the ability of each strain to undergo lytic
replication (Sculley et al., 1987), virus isolates were obtained
from parental cell lines and were used to infect and transform
B lymphocytes from different healthy individuals. To establish
the A- and B-type LCLs, B lymphocytes, isolated from five
EBV-seropositive donors (JBL, JST, DJM, ISM and MBA), were
infected with different A-type and B-type virus isolates (Table
1). After infection cells were seeded in threefold dilutions from
10& cells to 10# cells per well. After 4 weeks cell lines were
established by subculturing the cells seeded at the lowest
concentration at which transformation was visible. This
procedure was important to ensure that the established cell
lines arose by infection with exogenous virus rather than by
outgrowth of endogenous EBV-infected B lymphocytes. The
virus type in each of the cell lines was checked by Western
blotting and Southern analysis of viral DNA and in all cases the
transformed cell lines were of the same type as the exogenous
virus (results not shown). The LCLs were maintained in RPMI
1640 supplemented with 10 % foetal calf serum, benzylEEC
penicillin (0n7 mg\ml) and streptomycin (1 mg\ml) at 37 mC in
a 5 % CO atmosphere. The cell lines were not stimulated in
#
any way to induce virus production and were harvested while
the cells were in the exponential growth phase.
Initially six LCLs (three A-type and three B-type), established from donor JBL, were assayed by immunoblot for
expression of the p21 VCA component and by immunofluorescence (IFA) to determine the proportion of cells
expressing VCA. For IFA, cells were allowed to grow to
approximately 1i10' cells\ml and VCA expression in these
cells was measured according to the method of Henle & Henle
(1966), using a known VCA-positive serum (STh). For
immunoblotting cells were collected by centrifugation and the
cell pellets were dispersed by sonication in 2 % SDS–1 % 2mercaptoethanol–0n1 mM PMSF–10 mM sodium phosphate
(pH 6n8). The samples were then placed in a boiling water bath
for 2 min, allowed to cool, and centrifuged at 15 000 g for
5 min. Samples (20 µl ; 150 µg protein) were separated on 12 %
polyacrylamide gels. Electrophoresis was performed at 100 V
while the transfer of proteins from polyacrylamide gels to
nitrocellulose paper (Amersham) was performed essentially as
described by Burnette (1981). Detection of EBV antigens with
human serum (MCr) was performed using either radioiodinated
protein A (70–100 Ci\µg) (New England Nuclear) or horseradish peroxidase-conjugated sheep anti-human IgG
(Amersham) and an ECL Western blotting detection system
(Amersham).
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 06:59:17
Replicative capacity of EBV types
Table 2. Relative intensity of VCA in cell lines
transformed with A- or B-type EBV
Donor cells
QIMR-JST
QIMR-DJM
QIMR-JBL
QIMR-MBA
QIMR-ISM
Infecting virus
QIMR-JSm
Ag876
Af12
QIMR-L4
B95-8
IARC-BL36
IARC-BL74
QIMR-KTu
QIMR-L4
Ag876
QIMR-JSm
IARC-BL36
IARC-BL74
B95-8
QIMR-L4
QIMR-Gor
QIMR-JSm
IARC-BL74
B95-8
IARC-BL36
Ag876
WAN-BL
QIMR-L19
B95-8
IARC-BL74
IARC-BL36
QIMR-MBl
QIMR-L4
IARC-BL74
IARC-BL36
QIMR-JSt
p21 VCA
Virus type rel. intensity*
B
B
B
B
A
A
A
B
B
B
B
A
A
A
B
B
B
A
A
A
B
B
B
A
A
A
A
B
A
A
A
11
21n2
19n8
7
0n1
9n8
0n1
13n8
5n1
23
9n4
3n8
0n4
0n7
24n5
5n4
9n5
1n5
1n8
3n8
7n6
14
11n2
1
0n6
22
1
22n8
0n6
0n6
0n6
*Relative intensity was determined using a Molecular Dynamics
densitometer and ImageQuant software. Values can only be compared
within each donor group.
The results obtained from cell lines established from donor
JBL are presented in Fig. 1 (A). All of the B-type cell lines
expressed VCA by IFA, with 20 % of the cells transformed with
the QIMR-L4 strain expressing VCA. Of the A-type transformed cell lines only cells containing the IARC-BL36 virus
strain showed any VCA expression. Immunoblotting detected
the p21 VCA component in each of the cell lines that were
shown to be VCA positive by IFA. Quantification (using a
Molecular Dynamics densitometer and ImageQuant software)
of the p21 protein expressed in each of these cell lines (Table
2) compared favourably with the IFA results. The QIMRJBL\L4 cell line expressed the highest level of the p21 VCA
protein, followed by QIMR-JBL\JSm and QIMR-JBL\Gor ; the
A-type line QIMR-JBL\IARC-BL36 expressed a small amount
of p21. These results indicated that VCA expression as
determined by immunoblotting correlated well with the
percentage of IFA VCA-positive cells.
A number of cell lines was also assayed for the presence of
BZLF1 by IFA using an anti-BZLF1 mouse monoclonal supplied
by DAKO. All of the cell lines expressed much higher levels of
BZLF1 than VCA, with the A-type cell lines QIMR-JST\B958 and QIMR-JST\IARC-BL36 displaying 10 % positive cells
and QIMR-JST\IARC-BL74 2 % positive cells. Of the B-type
lines QIMR-JST\Ag876 had 3 % positive cells, QIMRJST\QIMR-L4 15 % positive and QIMR-JST\QIMR-JSm 50 %
positive cells. Five of these cell lines were also tested for
induction of virus replication using the phorbol ester TPA
(phorbol 12-myristate 13-acetate) (Sigma). Cells were seeded
at 5i10& cells\ml and TPA added to a concentration of
40 ng\ml. Samples were taken prior to the addition of TPA
and at 3 and 7 days after treatment. IFA VCA-positive cells
were then determined at each time-point. Each virus strain
reacted differently to addition of TPA. The A-type cell line
QIMR-JST\IARC-BL74 had 0n5 % VCA-positive cells on day
0 and 10 % VCA-positive cells by day 7 whereas TPA did not
cause an increase in virus production in the QIMR-JST\IARCBL36 cell line which had 1 % VCA-positive cells at all timepoints. Likewise, the B-type cell line QIMR-JST\QIMR-L4
displayed 5 % VCA positive cells at all time-points and was not
induced by TPA. The other two B-type cell lines tested were
induced, QIMR-JST\Ag876 going from 1 to 5 % and QIMRJST\QIMR-JSm from 5 to 70 % VCA-positive cells by day 7.
A further 25 LCLs were established by infection of B
lymphocytes from four different donors and expression of the
p21 VCA component was determined by immunoblotting.
Immunoblots of the LCLs established from donors DJM and
ISM are shown in Fig. 1 (B, C). The relative amounts of p21
VCA protein in each cell line were determined and results from
all of the cell lines analysed are shown in Table 2. In each group
of LCLs the B-type EBV-infected cell lines showed expression
of VCA, whereas of the A-type-infected cells only cell lines
containing strain IARC-BL36 demonstrated any VCA, and
then not in all donor cells (Fig. 1 C). One possibility for the
increased virus production in B-type cell lines could be related
to the rate of growth of these cell lines. To examine this three
A-type (B95-8, IARC-BL36, IARC-BL74) and four B-type
(Ag876, QIMR-JSm, QIMR-L4, Af12) LCLs, established from
donor JST, were seeded at 10& cells\ml and were cultured for
5 days in the presence of 1 or 10 % FCS with cell counts being
taken at 24 h intervals. All cell lines grew at a slower rate in 1 %
FCS ; however, there was very little difference in the growth
rate of any of the cell lines (at either 1 or 10 % FCS) except for
the A-type QIMR-JST\IARC-BL36 line, which remained in
stationary phase for the 5 day period. These results indicate
that the level of spontaneous replication observed in the Btype cell lines was not related to their growth rate.
It has been known for a long time that different cell
phenotypes can affect the permissive status of EBV strains
(Sculley et al., 1987), as is the case with the B95-8 virus which
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 06:59:17
EED
M. Buck and others
in the context of marmoset cells is permissive, but when used
to establish an LCL from human B cells is largely nonpermissive. To overcome this problem different A-type and Btype strains of EBV were used to establish LCLs from B cells
obtained from different healthy individuals. In this way it has
been possible to compare the intrinsic ability of each virus
strain to spontaneously enter the lytic cycle. The results show
that the A-type strains, other than strain IARC-BL36, did not
result in significant VCA production in any of the different
donor B lymphocytes used. In contrast, all of the cell lines
infected with B-type EBV strains were spontaneously producing VCA regardless of the origin of the donor cells.
Remarkably, 50 % of the QIMR-JSm virus-infected B cells from
donor JST were positive for BZLF-1 expression and 70 % were
of cells were VCA positive after induction by TPA. This high
degree of replication is reminiscent of P3HR-1 cells, which
were found to contain a deleted and rearranged genome
(termed het DNA) that disrupts latency and induces EBV to
replicate in vitro (Jenson & Miller, 1987). It is possible that cells
infected with B-type EBV may contain het DNA and this could
account for their ability to spontaneously enter the lytic cycle.
A condition in which EBV has been found to be actively
replicating is hairy leukoplakia, an oral lesion that occurs in
patients infected with the human immunodeficiency virus. The
presence of both A-type and B-type EBV has been demonstrated in these lesions (Walling et al., 1992), as well as the
presence of het DNA (Patton et al., 1990), raising the possibility
that the replicative nature of these lesions could be driven to
some extent by the B-type EBV (or possibly het DNA
associated with the B-type EBV).
It is widely believed that virus in circulating B-lymphocytes
and in B-cell malignancies is stringently latent. However,
Gutierrez et al. (1993) demonstrated by Southern blot analysis
that replicative forms of virion DNA could be detected in
14n5 % (8 of 55) of EBV-positive Burkitt’s lymphoma biopsies.
The percentage of BL biopsies containing replicative EBV is
similar to the percentage of individuals in Africa and Papua
New Guinea that were found to be infected with B-type EBV
(Young et al., 1987). Given the replicative nature of B-type
EBV it is possible that those biopsies identified as containing
replicative forms of EBV may also have contained B-type EBV.
Studies on immunocompromised patients have demonstrated a high incidence of B-type as well as A-type\B-type
coinfections (Sixbey et al., 1989 ; Sculley et al., 1988, 1990 ;
Kyaw et al., 1992). The high incidence of B-type EBV in
immunocompromised patients could be a consequence of the
ability of B-type EBV to replicate. Disruption to the immune
system in immunocompromised patients, restricting CTLs
directed against lytic antigens, could result in the B-typeinfected cells actively replicating virus and high levels of B
lymphocytes becoming infected with B-type EBV in these
patients.
The most significant difference between the A-type and Btype EBV strains is a reduction in the efficiency of transEEE
formation together with clearly defined phenotypic changes in
the emerging transformed cells (Rickinson et al., 1987). This
study demonstrates another biological difference between the
two virus types, namely their ability to spontaneously enter
the lytic cycle. This increased lytic ability may compensate for
the reduced transforming ability of B-type EBV and allow this
virus type to survive in vivo. A- and B-type EBV are
characterized by sequence divergence in regions of the genome
encoding EBNAs 2, 3, 4 and 6 (Sculley et al., 1989 ; Sample et
al., 1990 ; Adldinger et al., 1985 ; Dambaugh et al., 1984) ;
however, none of these gene products is involved in the lytic
cycle. The increased lytic ability of B-type EBV suggests that
other genes or their promoter regions may also be divergent
between these two EBV types.
This work was supported by grants from the National Health and
Medical Research Council of Australia, the University of Queensland and
from the Queensland Cancer Fund. N. Kienzle was a fellow of the
German Infektionsforschung\AIDS-Stipendiumsprogramm, DKFZ,
Heidelberg.
References
Adldinger, H. K., Delius, H., Freese, U. K., Clarke, J. & Bornkamm,
G. W. (1985). A putative transforming gene of Jijoye virus differs from
that of Epstein–Barr virus prototypes. Virology 141, 221–234.
Alfieri, C., Birkenbach, M. & Kieff, E. (1991). Early events in
Epstein–Barr virus infection of human B lymphocytes. Virology 181,
595–608.
Arrand, J. R., Young, L. S. & Tugwood, J. D. (1989). Two families of
sequences in the small RNA-encoding region of Epstein–Barr virus (EBV)
correlate with EBV types A and B. Journal of Virology 63, 983–986.
Burnette, W. N. (1981). ‘ Western Blottingh : electrophoretic transfer of
proteins from SDS–polyacrylamide gels to unmodified nitrocellulose and
radiographic detection with antibody and radioiodinated protein A.
Analytical Biochemistry 112, 195–203.
Countryman, J., Jenson, H., Seibl, R., Wolf, H. & Miller, G. (1987).
Polymorphic proteins encoded within BZLF1 of defective and standard
Epstein–Barr viruses disrupt latency. Journal of Virology 61, 3672–3679.
Dambaugh, T., Hennessy, K., Chamnankit, L. & Kieff, E. (1984). U2
region of Epstein–Barr virus DNA may encode Epstein–Barr nuclear
antigen 2. Proceedings of the National Academy of Sciences, USA 81,
7632–7636.
Gutierrez, M. I., Bhatia, K. & Magrath, I. (1993). Replicative viral DNA
in Epstein–Barr virus associated Burkitt’s lymphoma biopsies. Leukemia
Research 17, 285–289.
Henle, G. & Henle, W. (1966). Immunofluorescence in cells derived from
Burkitt’s lymphoma. Journal of Bacteriology 91, 1248–1256.
Henle, W., Henle, G., Zajac, B. A., Pearson, G., Waubke, R. & Scriba, M.
(1970). Differential reactivity of human serums with early antigens
induced by Epstein–Barr virus. Science 169, 188–190.
Jenson, H. B. & Miller, G. (1987). Sequences of the Epstein–Barr Virus
(EBV) large internal repeat form the center of a 16-kilobase-pair
palindrome of EBV (P3HR-1) heterogeneous DNA. Journal of Virology 61,
1495–1506.
Kyaw, M. T., Hurren, L., Evens, L., Moss, D. J., Cooper, D. A., Benson,
E. & Sculley, T. B. (1992). Expression of B-type Epstein–Barr virus in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 06:59:17
Replicative capacity of EBV types
HIV-infected patients and cardiac transplant recipients. AIDS Research 8,
1869–1874.
Luka, J., Kallin, B. & Klein, G. (1979). Induction of the Epstein–Barr
virus (EBV) cycle in latently infected cells by n-butyrate. Virology 94,
228–231.
Miller, G. & Lipman, M. (1973). Release of infectious Epstein–Barr virus
by transformed marmoset leukocytes. Proceedings of the National Academy
of Sciences, USA 70, 190–194.
Moss, D. J., Misko, I. S., Burrows, S. R., Burman, K., McCarthy, R. &
Sculley, T. B. (1988). Cytotoxic T-cell clones discriminate between A-
and B-type Epstein–Barr virus transformants. Nature 331, 719–721.
Patton, D. F., Shirley, P., Raab-Traub, N., Resnick, L. & Sixbey, J. W.
(1990). Defective viral DNA in Epstein–Barr virus-associated oral hairy
leukoplakia. Journal of Virology 64, 397–400.
Rickinson, A. B., Young, L. S. & Rowe, M. (1987). Influence of the
Epstein–Barr virus nuclear antigen EBNA 2 on the growth phenotype of
virus-transformed B cells. Journal of Virology 61, 1310–1317.
Sample, J., Hummel, M., Braun, D., Birkenbach, M. & Kieff, E. (1986).
Nucleotide sequences of mRNAs encoding Epstein–Barr virus nuclear
proteins. A probable transcriptional initiation site. Proceedings of the
National Academy of Sciences, USA 83, 5096–5100.
Sample, J., Young, L., Martin, B., Chatman, T. & Kieff, E. (1990).
Epstein–Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B and
EBNA-3C genes. Journal of Virology 64, 4084–4092.
Sculley, T. B., Moss, D. J., Hazelton, R. A. & Pope, J. H. (1987).
Detection of Epstein–Barr virus strain variants in lymphoblastoid cell
lines ‘ spontaneously ’ derived from patients with rheumatoid arthritis,
infectious mononucleosis and normal controls. Journal of General Virology
68, 2069–2078.
Sculley, T. B., Cross, S. M., Borrow, P. & Cooper, D. A. (1988).
Prevalence of antibodies to Epstein–Barr virus nuclear antigen 2B in
persons infected with the human immunodeficiency virus. Journal of
Infectious Diseases 158, 186–192.
Sculley, T. B., Apolloni, A., Stumm, R., Moss, D. J., Mueller-Lantczh, N.,
Misko, I. S. & Cooper, D. A. (1989). Expression of Epstein–Barr virus
nuclear antigens 3, 4 and 6 are altered in cell lines containing B-type virus.
Virology 171, 401–408.
Sculley, T. B., Apolloni, A., Hurren, L., Moss, D. J. & Cooper, D. A.
(1990). Coinfection with A- and B-type Epstein–Barr virus in human
immunodeficiency virus-positive subjects. Journal of Infectious Diseases
162, 643–648.
Serio, T. R., Angeloni, A., Kolman, J. L., Gradoville, L., Sun, R., Katz,
D. A., Van Grunsven, W., Middeldorp, J. & Miller, G. (1996). Two 21-
kilodalton components of the Epstein–Barr virus capsid antigen complex
and their relationship to ZEBRA-associated protein p21 (ZAP21). Journal
of Virology 70, 8047–8054.
Sixbey, J. W., Shirley, P., Chesney, P. J., Buntin, D. M. & Resnick, L.
(1989). Detection of a second widespread strain of Epstein–Barr virus.
Lancet ii, 761–765.
Walling, D. M., Edmiston, S. N., Sixbey, J. W., Abdel-Hamid, M.,
Resnick, L. & Raab-Traub, N. (1992). Coinfection with multiple strains
of the Epstein–Barr virus in human immunodeficiency virus-associated
hairy leukoplakia. Proceedings of the National Academy of Sciences, USA 89,
6560–6564.
Young, L. S., Yao, Q. Y., Rooney, C. M., Sculley, T. B., Moss, D. J.,
Rupani, H., Laux, G., Bornkamm, G. W. & Rickinson, A. B. (1987). New
type B isolates of Epstein–Barr virus from Burkitt’s lymphoma and from
normal individuals in endemic areas. Journal of General Virology 68,
2853–2862.
Zimber, U., Aldinger, H. K., Lenoir, G. M., Vuillaume, M., KnebelDoerberitz, M. G., Laux, G., Desgranges, C., Wittman, P. & Bornkamm,
G. W. (1986). Geographical prevalence of two types of Epstein–Barr
virus. Virology 154, 56–66.
zur Hausen, H., O’Neill, F. J., Freese, U. K. & Hecker, E. (1978).
Persisting oncogenic herpesvirus induced by the tumour promotor TPA.
Nature 272, 373–375.
Received 7 April 1998 ; Accepted 15 October 1998
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sat, 17 Jun 2017 06:59:17
EEF