PERSPECTIVES

agent responsible for causing tumours

TIMELINE
in chickens, so was extremely receptive
to the somewhat unfashionable notion
Epstein–Barr virus: more than that viruses might play a role in the
development of human cancer. He had
50 years old and still providing the further advantage of having worked
on the application of electron microscopy
surprises to the study of RSV, having learned the
technology in the Rockefeller Institute
laboratory of George Pallade, who
Lawrence S. Young, Lee Fah Yap and Paul G. Murray subsequently won the Nobel Prize for his
development of electron microscopy to
Abstract | It is more than 50 years since the Epstein–Barr virus (EBV), the first human probe cellular ultrastructure. So it was that
tumour virus, was discovered. EBV has subsequently been found to be associated Epstein, by then working at the Middlesex
with a diverse range of tumours of both lymphoid and epithelial origin. Progress in Hospital, attended a lecture entitled ‘The
the molecular analysis of EBV has revealed fundamental mechanisms of more commonest children’s cancer in tropical
Africa: a hitherto unrecognized syndrome’,
general relevance to the oncogenic process. This Timeline article highlights key given by Burkitt on 22 March 1961. In
milestones in the 50‑year history of EBV and discusses how this virus provides a Epstein’s own words — “I was struck from
paradigm for exploiting insights at the molecular level in the diagnosis, treatment the outset by the highly unusual nature
and prevention of cancer. of the new tumour and by the exciting and
unprecedented findings on epidemiology
Approximately 2 million cases of cancer infection can result in EBV-associated B cell which seemed to show that its distribution
each year are caused by infectious agents1. tumours. The role of EBV in the pathogenesis depended on climatic factors indicating that
Studying the role of infectious agents in of epithelial tumours — namely, undifferen- some biological agent might be involved
cancer pathogenesis, in both humans and tiated nasopharyngeal carcinoma (NPC) and in the aetiology” (REF. 6). As Burkitt was
animals, has provided important insights into EBV-associated gastric carcinoma (EBV‑GC) talking, Epstein formulated the notion that a
the mechanisms that drive the oncogenic — remains unclear but is thought to result climate-dependent arthropod vector might
process. This understanding has also from the aberrant establishment of latent be involved in the spread of an oncogenic
identified opportunities for therapeutic viral infection in epithelial cells with existing virus. Although it later became clear that this
and prophylactic intervention. premalignant genetic changes4. Although the was not precisely the case, this idea marked
Epstein–Barr virus (EBV) was the first precise contribution of EBV to the oncogenic a crossroads in Epstein’s career and heralded
human tumour virus to be discovered. It is process is uncertain, the presence of the virus the advent of human tumour virology.
estimated that EBV accounts for more than in all tumour cells provides opportunities Epstein met Burkitt immediately after
200,000 cases of cancer each year and that for the development of novel therapeutic the lecture, and in discussions a few days
1.8% of all cancer deaths are due to EBV- interventions and diagnostic approaches. later Burkitt agreed to send fresh tumour
attributable malignancies1,2. The higher In this Timeline article, we provide an biopsies from Kampala for analysis
prevalence of EBV-associated tumours in the overview of the major milestones in EBV in Epstein’s laboratory using electron
developing world compared with developed research and highlight the insights that microscopy, along with more traditional
regions and the contribution of EBV to this virus continues to provide into cancer virus culture approaches, in an attempt
cancer development in immuno­suppressed pathogenesis and treatment (FIG. 1). to identify and rescue the putative virus
patients1 highlight important facets of the resident within Burkitt lymphoma (BL)
oncogenic process, including the role of The discovery of EBV cells3. Over almost 3 years none of these
multiple risk factors and the contribution In the context of the discovery of EBV it is attempts was successful and so Epstein, by
of loss of immune control. eminently appropriate that Louis Pasteur, then working with Yvonne Barr and Bert
EBV is the most common and father of germ theory, is also responsible for Achong, decided to attempt to culture the
persistent virus infection in humans, with a well-known aphorism — “chance favours BL cells. Epstein knew that certain chicken
approximately 95% of the world’s population the prepared mind” (REF. 5). For it was the tumour viruses only became evident when
sustaining an asymptomatic life-long ‘prepared mind’ of a young pathologist, they reproduced as a consequence of
infection3. This persistence is the result Anthony Epstein, and his ‘chance’ attendance reactivation induced by in vitro culture6.
of EBV’s unique interaction with B cells, at a talk by Denis Burkitt, a surgeon working However, in the early 1960s no one had
whereby the infection resides in the memory in Uganda, that led to the discovery of succeeded in growing any type of human
B cell pool in normal, healthy individuals. EBV6. From 1948 Epstein had worked on lymphocyte, despite innumerable attempts
Disruption of this finely regulated B cell Rous sarcoma virus (RSV), the transmissible using a variety of different approaches.

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PERSPECTIVES

• VCA IgA levels detected in mass • EBV genome

serological screening of NPC patients detected in
ACIF developed to in China, confirming NPC risk166 ENK/T cell
detect EBNA1 protein • EBNA1 maintains the EBV genome212 lymphoma155
in BL and NPC cells56 • LMP1 is a classic oncogene221 • Latent gene
EBV particles EBV transformed EBV genome expression
discovered in lymphocytes in detected in EBV antibody and genome EBV genome in NPC
BL cells7 culture10,11 NPC cells169 detected in PTLD106 sequenced16 identified32

1964 1966 1967 1968 1970 1973 1974 1975 1978 1980 1982 1984 1985 1987 1988

• High EBV EBV EBV genome XLP1 was first described121 • HIV is a co-factor for • EBV DNA
antibody levels causes detected in EBV-positive BL149 detected in
in BL and NPC40 IM78 BL cells and Epidemiological study confirms • EBNA2 is essential for HL biopsies132
• EBV found in extracts link between EBV and BL44 B cell transformation215 • Latent gene
all human from NPC expression in
populations41 tumours55 • BZLF1 is the switch from latent to lytic cycle36 BL identified30
• EBV replication observed in oral ‘hairy’ leukoplakia80
• EBNA protein detected in HRS cells of a single HL patient131

Figure 1 | Timeline showing the major milestones in EBV research. ACIF, anti-complement immunofluorescence; BL, Burkitt lymphoma; EBER ISH,
Epstein–Barr virus-encoded RNA in situ hybridization; EBNA, Epstein–Barr nuclear antigen; EBV, Epstein–Barr virus; ENK, extranodal natural killer; HIV,
human immunodeficiency virus; HL, Hodgkin lymphoma; HRS, Hodgkin–Reed–Sternberg; IgA, immunoglobulin A; IM, infectious mononucleosis; LMP,
latent membrane protein; NPC, nasopharyngeal carcinoma; PTLD, post-transplant lympho­proliferative disease; Q‑PCR, quantitative PCR; TNFR, tumour
necrosis factor receptor; VCA, viral capsid antigen; XLP1, X‑linked lymphoproliferative disease type 1.

It is here that serendipity played a part:
a delayed and rerouted flight from Kampala
in December 1963 resulted in a BL biopsy
reaching the London laboratory later than
expected. The transit medium in which the
BL biopsy was shipped was unusually cloudy
but, rather than the suspected contamination
with bacteria, microscopic examination
revealed viable tumour cells floating free of
the main lymphoma mass. These cells grew
when suspended in fresh culture medium,
requiring the culture to be split into new
culture bottles. And so the first cell line
from a human lymphoma was established,
and early in 1964, Epstein prepared these
BL cells (now named the EB1 cell line after
Epstein and Barr) for electron microscopy 3,6.
He was “exhilarated to observe unequivocal
virus particles in a cultured BL cell” (REF. 6)
and it was clear from the morphology of the
virus particles that this was a herpesvirus6.
Unlike the three other human herpesviruses
known at that time, this virus was inert as
it did not induce the characteristic lytic
infection usually associated with herpesvirus
replication. These observations were
published in a Lancet paper 7 that became
a citation classic in 1979 (REF. 8).
The first human tumour virus
Discovering the potent transforming
ability of EBV. As more BL cell lines were
established9, the next key objective was to
determine whether EBV could transform
normal B lymphocytes. In the absence of
a BL cell line that produced large amounts
of EBV, Volker Diehl, working in the
laboratories of Werner and Gertrude
Henle, used an irradiated EBV producer
BL cell line (Jijoye) or a non-producer BL
cell line control (Raji) co‑cultivated with
sex-mismatched cord blood lymphocytes.
Within weeks, growth of the lymphocytes
was observed only after co‑culture with Jijoye
cells, confirming that this effect was most
likely due to EBV infection10. This study
was published in 1967, the same year that
John Pope in Brisbane had observed that cell
lines could readily be established from the
peripheral blood lymphocytes of patients
with infectious mononucleosis (IM)11. The
cell lines from patients with IM are the result
of spontaneous transformation — the
occasional growth of EBV-transformed
B cells from the blood of EBV-infected
individuals. Pope then took filtrates from one
of these B cell lines and demonstrated that
they could efficiently transform fetal human
lymphocytes12. These experiments confirmed
that EBV was indeed a potent transforming
virus in cell culture. EBV remains the
most efficient transforming agent, rapidly
converting approximately 3–10% of all
target B cells into permanently growing
lymphoblastoid cell lines (LCLs)13–15.
The publication in 1984 of the complete
sequence of an EBV strain (the B95‑8
strain)16 was the stimulus for a series of
studies that identified individual viral
genes, including the so‑called latent
genes, the coordinated expression of
which is responsible for in vitro B cell
immortalization (FIG. 2). Subsequent genetic
studies revealed the existence of two EBV
types17,18 (BOX 1) as well as substantial genetic
variation, affecting mainly the latent genes,
within each type19–29. The impact of genetic
variation on the biology of EBV infection
remains only poorly understood.
The EBV genome within LCLs usually
exists in multiple copies of extrachromo-
somal circular genetic material known as
episomes and expresses all latent genes
(referred to as latency III or the ‘growth
programme’), including six Epstein–Barr
nuclear antigens (EBNAs 1, 2, 3A, 3B and
3C and EBNA leader protein (EBNA‑LP)),
latent membrane proteins LMP1 and LMP2
(which encodes two isoforms, LMP2A and
LMP2B) and the non-coding EBV-encoded
RNAs (EBER1 and EBER2) and viral
microRNA (miRNA) (FIG. 2). During various
stages of B cell differentiation in vivo, EBV
expresses either the latency III programme,
or one of two alternative forms of virus
latency (known as latency I and latency II).
EBV-associated B cell lymphomas express
either latency I, II or III depending, at
least partly, on the B cell stage from which
each tumour is derived30,31. In contrast, all
EBV-associated epithelial cancers express
a latency II programme, or a variant
thereof 32,33. The contribution of these latent
genes to the oncogenic process remains the
subject of intense study (BOX 2).
EBV is also able to induce its lytic cycle,
leading to virus replication. Although the
lytic cycle was known to be activated by

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 PERSPECTIVES

• NPC tumour cells carry

clonal EBV genomes170 • Complete
• LMP1 is essential • LMP1 is a constitutively sequences of 71
• EBER ISH for B cell activated member of Use of Q-PCR EBV strains
assays transformation222 the TNFR superfamily145 to detect Wild-type First controlled published29
developed134 • EBNA3A and 3C are • First successful use of cell-free EBV EBV trial of an EBV • Further
• LMP2 essential for B cell EBV-specific T cell DNA as NPC sequence vaccine (gp350 development of
identified224 transformation217 therapy in PTLD117 biomarker168 released19 vaccine)103 gp350 vaccine101

1989 1990 1992 1993 1994 1995 1998 1999 2002 2003 2004 2007 2012 2015

EBV genome • Identification of three HL is a B cell EBV Wp-restricted EBV EBNA3B is a

detected in forms of latency31 lymphoma135 persistence latency microRNAs tumour
HRS cells of • First demonstration of in memory identified in BL73 discovered232 suppressor219
HL biopsies133 EBV in typical gastric B cells in
adenocarcinomas180 vivo81

Nature Reviews | Cancer

diverse stimuli, including the phorbol ester
12‑O-tetra-decanoylphorbol‑13‑acetate
(TPA) and sodium butyrate34,35, in 1985
George Miller showed that the gene
responsible for the latent to lytic cycle switch
was BZLF1 (REF. 36). Since then, the functions
of many of the virus genes expressed during
the lytic cycle have been characterized
(BOX 3). Although the precise in vivo
conditions responsible for activating the EBV
lytic cycle remain unknown, this process is
intimately related to the differentiation of
both B cells and epithelial cells37–39.
Defining the aetiological role of EBV in
BL. Only 2 years after the discovery of
EBV, Lloyd Old and colleagues40 described
antibodies in the serum of African patients
with BL that recognized antigens from
BL cell lines. However, the first definitive
clues of a pathogenic role for EBV in
BL came from sero-epidemiological
studies that reported higher titres of EBV
antibodies in the serum of patients with
BL compared with age-matched controls
living in the same area41–43. The definitive
epidemiological study published in 1978
was performed by Guy de Thé who
tested pre‑BL serum from around 42,000
Ugandan children44. Of the 14 children who
subsequently developed BL, most had raised
EBV antibody titres long before diagnosis,
leading to the conclusion that “BL develops
in children who have had a long and heavy
exposure to EBV” (REF. 44). Although this
study failed to find marked differences in
the burden of malaria in BL cases compared
with controls, the authors still regarded
malaria as an important cofactor, on the
basis of the tumour’s unique geographical
distribution44. Malaria probably induces
an intense polyclonal B cell activation and
loss of T cell control of EBV, both of which
could increase the pool of EBV-infected
BL progenitors45–48.
Although malaria impairs T cell
immunity to EBV, this impairment may be
transient, whereas the increased EBV loads
in circulating memory B cells associated
with malarial infection are long-lived45,49,50.
Plasmodium falciparum, the parasite that
causes malaria, may also act more directly
to induce BL. For example, it has been
shown that P. falciparum can increase
expression of the enzyme activation-­
induced cytidine deaminase (AID), which
induces BL‑causing mutations, including
the characteristic translocations51,52. Other
environmental factors may also be involved.
For example, in areas endemic for BL,
extracts of the plant Euphorbia tirucalli have
many traditional uses; these extracts have
been shown to induce the EBV lytic cycle
and to induce chromosomal abnormalities
in B cell lines53,54.
More direct evidence of a causal
relationship between EBV and BL was
provided in 1970 by Harald zur Hausen55,
who used DNA hybridization to identify
the EBV genome in BL (and also in NPC;
see below). In 1974, Beverley Reedman56
used anti-complement immunofluores-
cence (ACIF) to show, for the first time,
expression of an EBV protein in BL cell
lines; initially named ‘EBNA’, it is now
known as EBNA1 (REFS 57,58).
Work in the laboratories of George
and Eva Klein showed that BL was
characterized by abnormalities of
chromosome 14 (REF. 59), later shown to
be the result of translocations involving
chromosomes 14 and 8 (REF. 60). There are
two less-common variant translocations61;
all three deregulate expression of the
MYC oncogene on chromosome 8, placing
it under the transcriptional control of
an immunoglobulin locus62. The MYC
translocations are also found in the
EBV-negative forms of BL that occur
sporadically worldwide, and because they
are required to sustain the high rate of
proliferation characteristic of BL, MYC
abnormalities are now considered the
hallmark of BL63,64.
The demonstration that Akata BL cells
that had lost EBV episomes in culture
were more sensitive to apoptosis than the
parental virus-positive cells provided
the first evidence that EBV might contribute
an anti-apoptotic function that counteracts
MYC-driven apoptosis65–67. EBNA1 was
initially shown to mediate some of the
anti-apoptotic effects of EBV in BL cells68.
However, EBNA1 alone is not sufficient
to reconstitute complete protection from
cell death and other groups have shown an
anti-apoptotic function for the EBERs in
BL69,70. The restricted pattern of virus latency
observed in BL is also apparently important
for the BL phenotype, as forced expression of
the full spectrum of latent genes in BL cells
antagonizes MYC-driven oncogenesis71,72.
Although most BLs express a so‑called
type I form of latency probably restricted
to protein expression of EBNA1, Alan
Rickinson’s group73,74 showed that some BLs
express additional virus genes, including
EBNA3A, EBNA3B, EBNA3C and EBNA‑LP,
together with an EBNA2 deletion that
suppresses the full latency programme.
These are often referred to as Wp‑restricted
BL because virus gene expression is driven
from the Wp, rather than from the Qp,
promoter (FIG. 2a). Wp‑restricted BL cell
lines are less sensitive to apoptosis than
conventional EBV-positive BL lines, an

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a EBV genome: latent genes EBER1 effect attributed to downregulation of the

EBER2 pro-apoptotic molecule BIM (also known
LMP2A Wp
as BCL2L11) by EBNA3A and EBNA3C
LMP2B and to the overexpression of the viral BCL‑2
BARTs Cp
BARF1 homologue, BamHI fragment H rightward
open reading frame 1 (BHRF1), a gene
TR
LMP1
OriP originally thought to be expressed only in
the virus lytic cycle75,76. Recent global virus
gene expression studies indicate that BHRF1
is not the only lytic gene expressed in BL, at
Double-stranded
DNA episome least at the RNA level77.
EBNA-LP In this section we have described how the
EBNA2 discovery of EBV in BL eventually led to a
BHRF1 better understanding of the molecular basis
EBNA1
of this tumour. Below, we discuss how the
EBNA2 detection of EBV in other forms of cancer
deletion
led to the discovery of more unexpected
virus associations and of additional
Qp
EBNA3C mechanisms of virus-driven oncogenesis.
EBNA3B
EBNA3A Infections in asymptomatic hosts
Although the detection of antibodies to
EBV antigens in the sera of patients with
b Position of latent gene products on a linear genome BL provided the first evidence that EBV
Nhet OriP a S e1–e3 Z R c bT d might be causally linked to BL, antibodies
Nhet to EBV were also found to be present, albeit
CW W W W W W Y H F QU P O M L E K B G D XV I A
at lower levels, in normal healthy people,
TR W WWW W W EBNA1 TR including children41. A breakthrough in our
EBNA3A EBNA3C understanding of primary infection came
EBNA-LP EBNA3B LMP1 from another chance observation, this time
from the Henle laboratory, where a young
EBNA2 BHRF1
BARF1 technician, who was regularly bled as a
negative control in serological assays for EBV,
miR-BHRF1-1 miR-BHRF1-2 miR-BART-3 miR-BART-21 miR-BART-2
miR-BHRF1-3 miR-BART-4 miR-BART-18 became seropositive after developing IM,
miR-BART-1 miR-BART-7 at that time a well-recognized condition of
miR-BART-15 miR-BART-8 unknown aetiology. The link between EBV
miR-BART-5 miR-BART-9
miR-BART-16 miR-BART-22 and IM was confirmed in a study of serum
miR-BART-17 miR-BART-10 from Yale University students before and
miR-BART-6 miR-BART-11 after a diagnosis of IM78. The detection of
miR-BART-12
miR-BART-19 EBV in saliva from IM patients suggested
miR-BART-20 that oral transfer might be the route of
miR-BART-13
miR-BART-14 transmission79. In 1985, EBV replication
was observed in oral ‘hairy’ leukoplakia —
Figure 2 | EBV and its latent genes in 2016. a | Location and transcription of Epstein–Barr virus (EBV) white plaques on the tongue and oral tissues
Nature Reviews | Cancer
latent genes on the double-stranded viral DNA episome updated from the original figure shown in an commonly seen in male homosexuals with
Oncogene article in 2003 (REF. 265). The origin of plasmid replication (OriP) is shown in orange. The
human immunodeficiency virus (HIV)
short thick green arrows represent exons encoding latent proteins: six Epstein–Barr nuclear antigens
(EBNAs 1, 2, 3A, 3B and 3C, and EBNA‑LP), latent membrane proteins (LMPs 1, 2A and 2B), BamHI — suggesting that primary EBV infection
fragment H rightward open reading frame (BHRF1) and BamHI-A fragment rightward reading frame might involve virus replication in oral
(BARF1). The short blue arrows at the top represent the highly transcribed non-polyadenylated EBV- epithelial cells; however, it is still uncertain
encoded RNAs (EBER1 and EBER2). The middle long green arrow represents EBV transcription during whether this is the case80. Recent studies in
latency III, in which all the EBNAs are transcribed from either the Cp or Wp promoter; the different an organotypic keratinocyte culture system
EBNAs are encoded by individual mRNAs that are generated by differential splicing of the same long support a role for epithelial cells in the
primary transcript. The inner red arrow represents the EBNA1 transcript, which originates from the Qp replication and spread of EBV39.
promoter during latency I and latency II. Latency II is characterized by expression of EBNA1 together In 1998, David Thorley-Lawson81 showed
with the LMPs. Wp-restricted latency is initiated from the Wp promoter and there is expression of all that EBV-positive cells in the blood of
the EBNAs, except EBNA2, which is deleted in this form of latency (outer long blue arrow). b | Location
asymptomatic carriers are memory B cells.
of open reading frames for EBV latent proteins on the BamHI restriction map of the prototype EBV
B95‑8 genome and including the viral microRNAs (miRs) in sequence order. The BamHI fragments are How the virus gets into memory B cells
named according to size, with A being the largest. Lower-case letters indicate the smallest fragments. remains controversial. One model proposes
TR refers to the terminal repeats at each end of the genome. This region was often referred to as Nhet that memory B cells are directly infected
to indicate heterogeneity in this region according to the number of TRs within different virus isolates. with EBV82,83 (FIG. 3). Another, the germinal
BART, BamHI‑A rightward transcript. Adapted from REF. 265, Nature Publishing Group. centre model, proposes that EBV initially

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 PERSPECTIVES

infects naive B cells and expresses its growth surveillance in preventing lymphoma with intense immunosuppression, similar
programme (latency III), which drives B cell development was fully appreciated, when tumours, not all of which are EBV positive,
proliferation84. EBV-infected B cells then several reports of lymphoma arising in renal occur several years or more after solid organ
enter a germinal centre reaction and express allograft recipients immunosuppressed to transplantation in patients on low-level
the default programme (latency II); LMP1 prevent graft rejection were published104,105. immunosuppression for life112. Collectively,
and LMP2, as mimics of CD40 (also known The first hints that EBV might the term post-transplant lymphoproliferative
as TNFRSF5) and B cell receptor (BCR) be involved in the development of disease (PTLD) has been used to describe
signalling, respectively, probably mediate post-transplant lymphomas came from this heterogeneous group of conditions.
the survival of EBV-infected B cells in the a report in 1980 of raised antibody titres Many cases of early-onset PTLD are
germinal centre and their subsequent exit to EBV viral capsid antigen (VCA) oligoclonal and are regarded as outgrowths
as memory cells84–93. EBV-infected memory in transplant patients treated with of EBV-infected B cells, whereas others
B cells downregulate virus gene expression cyclosporin A106. Later that same year, resemble monoclonal lymphomas,
(latency 0) to avoid immune recognition. Dorothy Crawford107 identified EBNA commonly diffuse large B cell lymphoma
EBNA1 is expressed (latency I) during the protein in post-transplant lymphoma tissues. (DLBCL)110. Most PTLDs display an
proliferation of memory B cells. Activation This report was quickly followed by others unrestricted pattern of virus latency in which
of EBV-infected memory B cells can lead documenting EBV in lymphomas arising in EBV’s transforming proteins, EBNA2 and
to their differentiation into plasma cells, a other solid organ transplant recipients, and LMP1, are expressed. The EBNA3 family
process that has been shown to switch on the in bone marrow transplant patients108–110. members, which are highly immunogenic,
EBV lytic cycle with subsequent production Most lymphomas in bone marrow transplant are also expressed. In these cases the
of new virions94 (FIG. 3). recipients are of donor origin111. As well as virus growth transformation programme
The identification that humans evoke these early-onset forms of post-transplant proceeds unchecked113. However, many
humoral responses to EBV proteins provided lymphoma, occurring within weeks or of these tumours remain sensitive to
some of the key evidence linking the virus to months after transplantation and associated control by EBV-specific T lymphocytes114.
tumour development 40–44,78. However, in the
period after the initial discovery of EBV, little
was known about the nature of cell-mediated Box 1 | Strain variation
immunity to EBV. After it was shown Publication of the complete B95‑8 Epstein–Barr virus (EBV) sequence in 1984 represented the
that the high lymphocyte count that was largest DNA sequence to be determined16. In 2003, the wild-type EBV sequence was constructed
characteristic of IM was dominated mainly using B95‑8 as a backbone and Raji-derived sequences to replace the 12‑kb deletion in B95‑8
by T lymphocytes and not, as expected, (REF. 19). The current reference EBV sequence was released in 2010 (GenBank accession number
transformed B cells95,96, Denis Moss and NC_007605).
Rickinson set about unravelling the possible There are two EBV types, type 1 and type 2, which differ mainly in the sequences of the
contribution of T cells to EBV-specific Epstein–Barr nuclear antigen 2 (EBNA2) and EBNA3 genes17,18. Type 1 strains are more prevalent
immunity. They showed that in vitro worldwide and have greater transforming potential210,211.
Although there is a high level of similarity between EBV strains, variations exist in some viral genes,
infection of blood lymphocytes with EBV
mainly in the latent genes, that give rise to functional differences.
led to an initial phase of B cell proliferation, Complete genomes of EBV strains published to date are shown in the table.
but after a week or two the EBV-infected
B cells died — a phenomenon they termed EBV strain Type Origin Disease Year Refs
regression. They showed that regression was
B95‑8/Raji 1 USA and Nigeria IM and BL 2003 19
mediated by EBV-specific T cells present in
the blood of all healthy EBV-seropositive GD1 1 China NPC 2005 20
individuals97,98, and they were able to isolate AG876 2 Ghana BL 2006 21
CD4+ and CD8+ cytotoxic T cell (CTL) GD2 1 China NPC 2011 22
clones that recognized autologous LCLs99.
Using these clones to screen a series of HKNPC1 1 Hong Kong NPC 2012 23
peptides, they identified the first EBV CTL Akata 1 Japan BL 2013 24
epitope in 1990 (REF. 100), heralding a new Mutu 1 Kenya BL 2013 24
era in our understanding of the nature of M81 1 Hong Kong NPC 2013 25
T cell-mediated responses to EBV proteins
during virus infection, as well as stimulating K4123‑Mi and 1 USA LCLs 2013 26
K4413‑Mi
novel approaches to the rational design of
both prophylactic and therapeutic vaccines HKNPC2–HKNPC9 1 Hong Kong NPC 2014 27
against EBV101–103. NA19114, NA19315 1 USA LCLs 2014 28
and NA19384
EBV and other lymphomas 71 EBV strains 60 type 1 UK, Germany, Healthy 2015 29
Post-transplant lymphoproliferative and Hong Kong, China, Japan, saliva, LCLs,
disease. Although it was known that patients 11 type 2 S. Korea, Australia, Kenya PTLD, HL,
and Nigeria BL, NPC and
with various deficiencies of humoral or EBV-GC
cell-mediated immunity had an increased
BL, Burkitt lymphoma; EBV-GC, EBV-associated gastric carcinoma; HL, Hodgkin lymphoma; IM, infectious
lymphoma risk, it was not until the late mononucleosis; LCLs, lymphoblastoid cell lines; NPC, nasopharyngeal carcinoma; PTLD, post-transplant
1960s that the importance of immune lymphoproliferative disease.

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Box 2 | Function of EBV latent genes LMPs as adjuvant therapy remained in

remission for a median of 3.1 years following
Latent proteins CTL infusion; of 21 patients with relapsed or
• Epstein–Barr nuclear antigen 1 (EBNA1) resistant disease at the time of CTL infusion,
-- Essential for episomal maintenance and replication212 13 had clinical responses, including 11
-- Induces B cell lymphomas in transgenic mice213 complete responses119.
-- Enhances cell survival68 It should be noted that EBV-positive
-- Induces genetic instability214 DLBCL also arises in patients, including
• EBNA2 children, without any previous history of
-- Discovered when a laboratory-derived Epstein–Barr virus (EBV) strain, P3HR‑1, with an EBNA2 immunosuppression; in older patients it
deletion was unable to transform B cells in vitro215
is assumed that a decline in EBV-specific
-- Mimics a constitutively activated NOTCH receptor216
immunity associated with ageing is
• EBNA3A and EBNA3C
responsible120.
-- Essential for in vitro B cell transformation217
-- Cooperate with oncogenic HRAS in transforming primary rodent fibroblasts218
X‑linked lymphoproliferative disease
• EBNA3B
type 1. David Purtilo121 provided the first
-- May function as a tumour suppressor in diffuse large B cell lymphoma (DLBCL)219
detailed characterization of ‘Duncan’s
• EBNA leader protein (EBNA‑LP)
syndrome’, later to be called X‑linked
-- Required for the efficient outgrowth of lymphoblastoid cell lines (LCLs)220
lympho­proliferative disease type 1 (XLP1).
• Latent membrane protein 1 (LMP1)
Boys with XLP1 develop a severe, often
-- Classic oncogene221
-- Essential for in vitro B cell transformation222
fatal, IM within weeks after primary EBV
-- Functions as a constitutively activated tumour necrosis factor receptor (TNFR)145 and resembles infection. This may be accompanied by
CD40 receptor in vivo85 a condition known as haemophagocytic
-- Activates signalling pathways that contribute to its oncogenic effects223 lymphohistiocytosis, in which there is
• LMP2A and LMP2B uncontrolled proliferation of activated
-- Identified in 1990 (REF. 224) lymphocytes and macrophages that secrete
-- LMP2A is a B cell receptor (BCR) mimic that can rescue BCR-deficient B cells from apoptosis86 high levels of inflammatory cytokines.
• BamHI fragment H rightward open reading frame 1 (BHRF1) Survivors have multiple immune defects,
-- BCL‑2 homologue originally thought to be a lytic gene225,226 including reduced levels of antibodies
-- Important for apoptotic protection in Wp‑restricted Burkitt lymphoma227 in the blood and a propensity to develop
• BamHI‑A fragment rightward reading frame 1 (BARF1) lymphoma121,122. In 1998, an international
-- Induces transformation of rodent fibroblasts and a human B cell line228,229 consortium of scientists published three
-- Reported as an early lytic gene230 papers describing the genetic defect in
-- Expressed in nasopharyngeal carcinoma (NPC) and EBV-gastric cancer in the absence of lytic XLP1 as a mutation in the SH2 domain
gene expression231 containing 1A (SH2D1A) gene, which
Latent non-coding RNA encodes a defective signalling lymphocyte
EBV was the first human virus found to encode microRNAs (mi­RNAs)232. activation molecule (SLAM)-associated
• BamHI‑A rightward transcript mi­RNAs (BART mi­RNAs) protein (SAP)123–125. Thus, patients with
-- 44 mature BART mi­RNAs conclusively verified to date233 XLP1 have impaired T cell and natural killer
-- Maintain latency by targeting EBV lytic genes, modulating LMP1 expression, targeting (NK) cell engagement of B cells rather than
pro-apoptotic proteins, impairing host immune responses and inactivating tumour a specific inability to cope with EBV itself 126.
suppressor genes234–240
-- Can drive tumour growth in vivo241,242
Hodgkin lymphoma: another addition to
-- Potential serum biomarkers in patients with NPC201,243
the EBV-positive B cell lymphoma family.
• BHRF1 mi­RNAs
Hodgkin lymphoma (HL) is an unusual
-- Three BHRF1 mi­RNAs are encoded232
-- Important for B cell transformation244 tumour characterized by the presence of
rare, large, often binucleated, tumour cells,
• EBV-encoded RNA 1 (EBER1) and EBER2
-- Are highly abundant non-coding RNAs245
known as Hodgkin–Reed–Sternberg (HRS)
-- Form stem–loop structures giving rise to double-stranded RNA-like molecules246 cells, which are surrounded by a prominent
-- Interact with a number of proteins and contribute to the activation of innate immunity247 microenvironment composed of various
non-malignant inflammatory cells, including
T cells, macrophages and fibroblasts.
Early therapies for PTLD relied on surgery to EBV has led to the development of HL displays a bimodal age distribution in
with irradiation, chemotherapy and, in adoptive CTL therapy for PTLD and other developed countries: the first peak occurs
some cases, treatment with antiviral drugs, EBV-related tumours. This approach, between the ages of 15 and 34 years, and the
but mortality was high115. Thomas Starzl pioneered by Cliona Rooney, is especially second at older than 50 years127. In contrast,
and colleagues116 were the first to show that successful against EBV-positive lymphomas in developing countries, the young adult
reduction or discontinuance of immuno- expressing the latency III programme117,118. peak is less conspicuous and childhood
suppression caused regression of the lesions, For example, in a recent clinical trial, 28 of rates are higher 128. It has been suggested
often without subsequent rejection of the 29 high-risk or multiple-relapse patients that the increased HL risk in young adults
grafts. A better understanding of immunity with lymphoma receiving CTLs against in developed countries might be due to

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late exposure to a common infectious
agent. In 1974, two studies suggested that
EBV might be this infectious agent; both
reported a significantly increased risk of
HL in individuals with a previous history
of IM129,130. In 1985, Sibrand Poppema131
provided the first direct evidence of EBV’s
involvement when he detected EBNA
protein in the HRS cells of a single patient.
EBV DNA was then detected in whole
HL biopsies and later in HRS cells132,133.
In 1990, Richard Ambinder 134 used in situ
hybridization to detect expression of the
EBERs in HRS cells; this is now the gold
standard approach for latent EBV detection
in pathology samples.
Despite the strong linkage to EBV, the
B cell origin of HL was demonstrated only
in 1994 when Ralf Küppers showed that HRS
cells harbouring clonal immunoglobulin
rearrangements are somatically
hypermutated, and in some cases have
mutations that destroy the coding capacity of
originally functional immunoglobulin genes
(crippling mutations)135. Thus, HRS cells are
thought to be derived from germinal centre
B cells that lack a functional BCR and that
must have escaped apoptosis, which would
otherwise have been their normal fate.
Two pieces of evidence suggest that EBV
can provide this anti-apoptotic function:
crippling immunoglobulin mutations are
found almost exclusively in EBV-positive
patients136, and EBV can efficiently
immortalize BCR-negative germinal centre
B cells in vitro137–139. LMP1 and LMP2A,
which are expressed in EBV-positive
HRS cells, are thought to provide these
anti-apoptotic signals by mimicking
CD40 receptor and BCR functions,
respectively 140–145. Thus, the mechanism
of protection in HL is distinct from that
observed in BL, probably reflecting the
different apoptotic stimuli that must be
overcome in each tumour.
Although the epidemiology had initially
provided the impetus for the search for
EBV in HL, contrary to expectations it was
shown that in developed countries, EBV
is present mostly in a histological variant
of HL known as mixed cellularity HL that
occurs mainly in children and older adults,
and is found less frequently in the other
major histological variant, known as nodular
sclerosis HL, which makes up most of the
young adult peak. Nevertheless, a small
fraction of young adult HLs are EBV positive
and some are preceded by IM; we now know
that a prior diagnosis of IM is associated
with an increased risk of developing
EBV-positive, but not EBV-negative, HL146,147.
Genetic studies also show that different this stage of HIV infection152,153. Thus, like
alleles of the human leukocyte antigen A malaria, HIV probably acts as a chronic
(HLA‑A) gene are associated with different B cell stimulator that increases the pool of
susceptibilities to both EBV-positive HL and EBV-infected B cells carrying accidental
IM. Most notably, individuals possessing the MYC translocations. The incidence of HL
HLA‑A*01 variant have an increased risk of in patients with HIV also increases as CD4+
both EBV-positive HL and IM, and those T cell counts begin to fall but reaches a
with the HLA‑A*02 variant have a decreased peak with intermediate levels of immune
risk of both conditions147,148. Because HLA‑A impairment and declines thereafter in
proteins are critical for antigen presentation severely immunocompromised patients154,
to CTLs, these observations point to the suggesting that CD4+ T cells contribute to
importance of immune responses to EBV HL pathogenesis.
as potentially shared pathogenic triggers in
EBV-positive HL and IM147,148. More surprises: EBV-associated diseases
of NK cells and T cells. Although it was
HIV and EBV-associated B cell lymphomas. originally thought that EBV could only
Early in the 1980s, reports appeared of infect lymphocytes of the B cell lineage,
the increased incidence of lymphomas in in 1988, James Jones and colleagues155
patients with the newly described acquired found EBV genomes in the CD4+ T cells
immunodeficiency syndrome (AIDS). The of three patients with T cell lymphoma.
first report was of a case in a homosexual Subsequently, EBV was found in the CD4+
man, who had raised antibody titres to T cells of a boy with chronic active EBV
VCA149. Soon after, John Ziegler 150 reported infection (CAEBV), a life-­threatening
four cases of BL in homosexual men; of the condition characterized by severe persistent
three tumours with available tissue, two or progressive IM156. Two years later,
were EBNA positive as determined by ACIF. extranodal natural killer (ENK)/T cell
Thereafter, it was shown that BL in AIDS lymphoma, an aggressive form of lymphoma
carries the typical MYC translocations151. that expresses some T cell markers and is
Unlike DLBCL, which is also increased in usually of NK cell origin, was also found
HIV infection but only when the patient is to be consistently EBV positive157. We
severely immunocompromised, BL occurs now know that CAEBV, haemophagocytic
early in HIV infection when circulating lympho­histiocytosis (discussed above)
CD4+ T cell numbers are relatively normal; and ENK/T cell lymphoma represent a
this is probably the result of expanded spectrum of disease often characterized by
germinal centre activity characteristic of EBV infection of T or NK cells; CAEBV
Box 3 | Functions of EBV lytic genes
Two Epstein–Barr virus (EBV) genes, BZLF1 and BRLF1, synergistically induce the expression
of multiple early lytic genes, including those that encode the six viral proteins required to
assemble the replication machinery; BALF5 (the DNA polymerase), BALF2 (the single-stranded
DNA-binding protein homologue), BMRF1 (the DNA polymerase processivity factor), BSLF1
(the primase homologue), BBLF4 (the helicase homologue) and BBLF2/3 (formed from the splicing
of BBLF2 and BBLF3 open reading frames; a potential homologue of the third component of the
helicase–primase complex)248. In addition to its function as a transactivator, direct interactions of
the BZLF1 protein with the lytic origin of DNA replication (oriLyt), and the viral replication
proteins, were found to be essential for EBV DNA replication249–251. In 2007, it was shown that lytic
cycle transcription occurs following primary infection of B cells252, a phenomenon that was
subsequently confirmed in epithelial cells253. However, a full lytic cycle immediately post-infection
cannot be induced because BZLF1 does not bind to a viral genome that is devoid of CpG
methylation254. Thus, an abortive lytic cycle has been proposed to be the initial mode of primary
EBV infection that is considered essential for the transformation of resting B cells255. Several lytic
genes encode immune evasion proteins, including BNLF2a (inhibits the transporter of antigen
processing)256, BILF1 (induces major histocompatibility complex (MHC) class I internalization and
degradation)257 and BGLF5 (mediates host shut-off)258.
Evidence for a contribution of the lytic cycle to EBV-induced oncogenesis has emerged only in
recent years. For example, the presence of low numbers of lytically infected cells could enhance
tumour growth through the release of growth factors and immunosuppressive cytokines259,260.
EBV lytic genes BZLF1, BGLF4 and BGLF5 can also induce genome instability261–263. Recent RNA
sequencing and high-throughput PCR array analyses have revealed high-level expression of lytic
genes, particularly LF1, LF2, LF3, BILF1, BALF4 and BHLF1, in cell lines and tumour biopsies,
although it is not clear whether these genes are expressed in latently infected cells77,264.

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often develops into either ENK/T cell contributes to the excessive production of lytic cycle proteins163. Further serological
lymphoma155 or haemophagocytic lympho­ pro-­inflammatory cytokines characteristic analysis showed an association between
histiocytosis158. ENK/T cell lymphomas of ENK/T cell lymphomas162. EBV antibody titres and NPC tumour
are more common in Asian populations159 stage164, and identified VCA-specific
and express a virus latency II pattern with EBV and epithelial cancers immunoglobulin A (IgA) levels as a
variable LMP1 expression160. Recent work NPC. A surprising early observation made potential prognostic indicator 165. The
demonstrates that EBV type 2 strains in 1966 was that the BL antigen-specific apparent specificity of the association
(BOX 1) preferentially infect primary T cells antibodies described by Old et al.40 were between VCA IgA levels and NPC led
in vitro, resulting in expression of latent also detectable in the serum of patients Zeng Yi to embark on mass serological
genes and leading to T cell activation and with “post-nasal space carcinomas” or, screening programmes in Wuzhou City,
proliferation, with alteration of cytokine as it later became known, NPC. Patients China. These studies not only confirmed
profiles161. These preliminary observations with NPC were subsequently shown to that IgA responses to VCA and early
are consistent with data showing that LMP1 have elevated titres of antibodies to several antigen were detectable in patients with
NPC, but also revealed that they were
present in some patients with hitherto
Germinal centre model
unrecognized NPC166. These data suggest
EBV that EBV-based screening programmes
Germinal centre could reduce mortality from NPC through
Plasma cell EBV replication
early detection and treatment. The approach
to screening for early NPC has been greatly
enhanced recently by the use of quantitative
Naive Latency II
B cell PCR to detect circulating EBV DNA167.
(EBNA1, LMP1 and LMP2)
Latency III In fact, circulating cell-free EBV DNA has
(all EBNAs and LMPs) become a gold standard biomarker that
Latency 0 Latency I
(no protein expression) (EBNA1) can be used to stage patients and provide
prognostic information168.
Direct infection model Although zur Hausen55 had already
shown that extracts of NPC biopsies
contained EBV DNA, crucially, in 1973, EBV
? DNA was detected by in situ hybridization in
NPC tumour cells and not in the infiltrating
Memory lymphoid cells as had been expected169.
B cell Normal persistence Subsequent studies showed that NPC
? ?
Latency III tumour cells carry clonal EBV genomes170
B cell lymphomas and express a virus latency II pattern
with variable LMP1 expression32,171–173.
It is the most undifferentiated type of NPC
Burkitt lymphoma Hodgkin lymphoma PTLD and DLBCL (so‑called non-keratinizing type II and
Latency I Latency II Latency III type III tumours) that are predominantly
EBV positive174. Tumours histologically
Figure 3 | Virus persistence in the B cells of the human host and the origin of EBV-associated similar to undifferentiated NPC can be
Nature Reviews | Cancer
B cell lymphomas. Upper panel: normal persistence. Epstein–Barr virus (EBV) resides in found at other sites, such as the stomach
memory B cells of asymptomatic hosts. There are two models to explain how the virus enters mem‑ and salivary glands, and are also frequently
ory B cells. In the germinal centre model, EBV infection of naive B cells leads to a latency III growth EBV positive175.
programme, in which the proliferation and expansion of the infected B cell pool is driven by expres‑ A scheme for NPC pathogenesis has been
sion of all EBV latent genes. Cells then enter the germinal centre and express latency II (default
proposed whereby loss of heterozygosity
programme), characterized by expression of Epstein–Barr nuclear antigen 1 (EBNA1), latent mem‑
brane protein 1 (LMP1) and LMP2. LMP1 provides a CD40‑like signal and LMP2 a surrogate B cell (LOH) at chromosomes 3p and 9p occurs
receptor-like signal — mimicking the same signals as those provided to the antigen-specific as an early event 176, possibly as a result of
B cell in the normal germinal centre. EBV-infected germinal centre B cells leave and enter the mem‑ exposure to environmental cofactors such
ory B cell pool. Here, EBV protein expression is silenced (latency 0); these cells are replenished by as dietary components (that is, carcinogenic
the same signals that induce the proliferation of normal memory B cells. Proliferating EBV-infected volatile nitrosamines in salted fish or other
memory B cells require EBNA1 expression (latency I) for viral episome segregation. Memory B cells preserved food) and smoking 177, creating
can terminally differentiate into plasma cells (solid arrow), which triggers virus replication. EBV- low-grade pre-invasive lesions that, after
infected germinal centre B cells might also differentiate directly into plasma cells (dashed arrow). additional genetic and epigenetic events,
In the direct infection model (shown below), EBV accesses memory B cells following the direct become susceptible to EBV infection4,178
infection of these cells, which may involve a latency III intermediary. Lower panel: the origin of the
(FIG. 4). Once infected, EBV latent genes
EBV-associated B cell lymphomas. There is uncertainty about the exact stages of differentiation
from which the EBV-positive B cell lymphomas arise (as indicated by the black dotted lines), as it provide growth and survival benefits,
cannot be assumed that the pattern of latency observed in the progenitor cell is recapitulated in resulting in the development of NPC.
the corresponding tumour. The figure illustrates the presumed (question marks indicate this uncer‑ Additional genetic and epigenetic changes
tainty) cell of origin based on current evidence for post-transplant lymphoproliferative disease occur after EBV infection. Detailed analysis
(PTLD), diffuse large B cell lymphoma (DLBCL), Hodgkin lymphoma and Burkitt lymphoma. of the genomic landscape of NPC has

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Normal epithelium Pre-invasive lesions NPC

CIS?

Low grade High grade

Epithelium

Superficial zone

Midzone

Basal layer
Basement
membrane Dermis

LOH on chromosomes 3p and 9p

Overexpression of CCND1 and BCL-2; increased telomerase activity

Inactivation of RASSF1A, RARB2, CDKN2A and DAPK1

EBV latent infection

LOH on chromosomes 11q, 13q, 14q and 16q

Other genetic changes (for example inactivation of EDNRB)

Figure 4 | Schematic representation of the pathogenesis of NPC.
In nasopharyngeal carcinoma (NPC), the tumour cells carry monoclonal
viral genomes indicating that Epstein–Barr virus (EBV) infection must have
occurred before expansion of the malignant cell clone170. EBV infection is
detected in high-grade pre-invasive lesions in NPC but not in low-grade
disease170. Multiple genetic changes have been found in NPC, with fre‑
quent chromosomal deletions and promoter hypermethylation of specific
genes on chromosomes 3p, 9p and 11q266. Deletions in both 3p and 9p
have been identified in low-grade EBV-negative dysplastic lesions and in
normal nasopharyngeal epithelium from individuals who are at high risk
of developing NPC. In vitro studies demonstrate that cyclin D1 (CCND1)
overexpression (a consequence of cyclin-dependent kinase inhibitor 2A
(CDKN2A, which encodes p16INK4A) deletion on chromosome 9p and ampli‑
fication of the CCND1 locus on chromosome 11q) facilitates persistent
EBV infection of immortalized nasopharyngeal epithelial cells 267.
Increased expression of the anti-apoptotic protein BCL‑2 (REF. 268) and
elevated telomerase activity269 in low-grade dysplastic lesions may also
promote the establishment of latent EBV infection. A scheme has been
proposed whereby loss of heterozygosity (LOH) occurs early in the patho‑
genesis of NPC, possibly as a result of exposure to environmental
cofactors such as dietary components (for example, salted fish), creating
low-grade pre-invasive lesions that after additional genetic and epi­
genetic events become susceptible to stable Nature Reviews | Cancer
EBV infection. Once
infected, EBV latent genes provide growth and survival benefits that result
in the development of NPC. Additional genetic (for example, LOH on
chromosomes 11q, 13q, 14q and 16q) and epigenetic changes occur after
EBV infection and contribute to metastatic disease176,270. The precise tim‑
ing of epigenetic events in the pathway of NPC development is unknown,
although some changes such as RAS association domain family member 1
(RASSF1A) inactivation may occur early, whereas others (for example,
inactivation of retinoic acid receptor β2 (RARB2), CDKN2A and death-­
associated protein kinase 1 (DAPK1)) may be influenced by the ability of
EBV to enhance the activity of the methylation machinery271. Recent
analy­sis of the genomic landscape in NPC has confirmed a role for chro‑
matin modification in the carcinogenic process and identified additional
somatic events, including mutations in the ERBB–PI3K signalling path‑
way179. The pathogenesis of EBV-associated gastric cancer is similar to
that of NPC, with premalignant lesions occurring before EBV infection190
and a distinctive hypermethylation phenotype188. CIS, carcinoma in situ;
EDNRB, endothelin receptor B.

revealed particular pathways (for example,
chromatin modification and ERBB–PI3K
signalling) that may work in concert
with EBV to drive the development of
this tumour 179.
Gastric cancer. In 1992, EBV was detected
in the tumour cells of typical gastric
adenocarcinoma180. Subsequent studies
confirmed that approximately 10% of
these tumours are EBV positive4. Given
the worldwide prevalence of gastric cancer
(GC) (952,000 cases in 2012)181, EBV‑GC
may represent the most common form of
EBV-associated malignancy 182,183. EBV‑GC
displays a viral latency pattern similar to that
of NPC33. There is substantial geographical
variation in the association of EBV with GC
that may be attributed to ethnic and genetic
differences182,183. EBV-GCs have distinct
phenotypic and clinical characteristics
compared with EBV-negative GC, including
loss of cyclin-dependent kinase (CDK)
inhibitor p16INK4A (encoded by CDKN2A)
expression, high CpG island methylator
phenotype (CIMP), the presence of
wild-type p53, apparent driver mutations in

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PERSPECTIVES

the PI3K catalytic subunit-α (PIK3CA) and
AT‑rich interactive domain 1A (ARID1A)
genes, and better patient survival184–189.
As in NPC, the precise role of EBV in
the pathogenesis of GC is unknown, but the
absence of EBV infection in premalignant
gastric lesions supports the contention that
virus infection is a relatively late event190.
Epidemiological studies show that EBV‑GC
is related to birth order (younger siblings
being more susceptible possibly as a result of
earlier exposure to EBV), high salt intake and
exposure to metal dust, but these factors may
vary geographically (for example, between
Japan and Colombia, which both have
high rates of GC), supporting the need for
more detailed investigation182,183. It has been
suggested that distinct EBV strains contribute
to the development of EBV‑GC, although this
could simply reflect geographical variation
whereby EBV strains common in the general
population of certain countries are more able
to promote GC development191.
Future perspectives
EBV was discovered more than 50 years ago
and remains the most common persistent
asymptomatic virus infection in humans.
The life-long relationship between EBV and
its immune host is a consequence of the
ability of the virus to persist in the memory
B cell pool of normal healthy individuals.
Such an intimate host–pathogen interaction
is reflected by the presence of EBV-like
B cell trophic viruses (lymphocrypto­
viruses) in non-human primates, suggesting
that EBV has co‑evolved with its host 192.
The ability of EBV to transform B cells
in vitro has contributed to the unequivocal
identification of EBV as oncogenic in
humans and to a deeper understanding
of the role of the virus in B cell tumours.
The precise role of EBV in the pathogenesis
of epithelial cancers, including NPC and
EBV‑GC, remains unclear. The detection
of EBV in other rarer forms of cancer,
such as leiomyosarcomas in immuno­
suppressed children, not only underlines the
epithelial cells in this process. The impact
of EBV infection on the development of
NPC and EBV‑GC may be a consequence
of the aberrant establishment of virus
latency in epithelial cells that have already
undergone premalignant changes — a
pathogenic process that is distinct from
EBV-induced lymphomagenesis in which
the virus appears to be the initiator. Unlike
receptor-mediated infection of B cells,
cell‑to‑cell infection is well recognized as
the main pathway for EBV infection in
epithelial cells195,196. Recently, a novel in‑cell
infection mechanism was described for
EBV infection of nasopharyngeal epithelial
cells through the formation of heterotypic
cell‑in‑cell structures (invasion of B cells
into epithelial cells by an as yet unknown
mechanism), a process that might be
related to the maintenance of EBV latency
in epithelial cells197. The development of
more efficient in vitro systems for studying
EBV infection and replication in different
cell types is helping to unravel the complex
interplay between the virus and the host cell.
The use of recombinant EBVs continues to
shed light on the role of viral genes in the
transformation process, on the requirements
for the efficient production of progeny virus
and on the role of EBV-encoded membrane
glycoproteins in the infection process.
The key question of the role of EBV strain
variation in oncogenesis is becoming more
tractable with the advent of more sensitive
virus genome sequencing technologies29
and the ability to clone wild-type EBV
strains for biological charac­terization25,198.
Understanding the host cell–virus interaction
will depend on the generation of appropriate
in vitro and in vivo models, particularly those
that allow an understanding of the role of the
tumour microenvironment, including the
local cytokine milieu.
A characteristic feature of many
EBV-associated cancers is the involvement
of chronic inflammation. This is exemplified
by tumours such as BL and is also apparent
in other rarer forms of EBV-positive cancer
EBV is the ultimate biomarker, and routine
application of serum testing for virus
DNA could prove useful in detecting
early disease, providing prognostic
information, and in monitoring patients
after treatment, approaches that may be
further enhanced by the development of
additional adjunctive tests (for example, to
detect EBV mi­RNAs and methylated tumour
suppressor genes)201,202. The development
of novel therapeutic approaches using virus
reactivation203, gene therapy 204, drugs that
target EBV latent protein function, such as
inhibitors of EBNA1, which is known to be
expressed in every EBV-positive tumour 205,206,
or therapeutic vaccination207,208 provides
exciting opportunities for meaningful clinical
intervention. The effectiveness of these
modalities will be further enhanced by the
molecular classification and stratification
of cancer patients with EBV-associated
tumours. Although there is renewed
interest in the possibility of a prophylactic
vaccine against EBV101, there are substantial
economic and practical hurdles in the
implementation of this approach, along
with concerns about the possibility of
unintended consequences of eradication
of EBV infection; for example, one study
has indicated that herpesvirus latency
confers symbiotic protection from bacterial
infection209. Our growing understanding
of EBV-associated oncogenesis provides
paradigms for the development of targeted
cancer therapies and diagnostics and further
confirms the far-reaching value of tumour
virology to the whole cancer field.
L.S.Y. is at Warwick Medical School, The University of
Warwick, Coventry CV4 7AL, UK.
L.F.Y. is at the Department of Oral and Craniofacial
Sciences and Oral Cancer Research Coordinating
Centre, Faculty of Dentistry, University of Malaya,
Kuala Lumpur, Malaysia.
P.G.M. is at the Institute of Cancer and Genomic
Medicine, University of Birmingham, Vincent Drive,
Edgbaston, Birmingham B15 2TT, UK.
Correspondence to L.S.Y.
L.S.Young@warwick.ac.uk

importance of disturbed immunity in viral arising at sites of long-term persistent doi:10.1038/nrc.2016.92

oncogenesis but also provides a striking
example of the harmful consequences
that can follow the aberrant infection of
other cell types193. The complex interplay
between EBV and the immune system can
also manifest itself in other unexpected
ways, as demonstrated by increasing
evidence causally implicating EBV in the
pathogenesis of multiple sclerosis194.
Little is known about the replicative
life cycle of EBV in vivo particularly with
regard to the relative role of B cells versus
inflammation, such as pyothorax-
and chronic osteomyelitis-associated
lymphomas199, in so-called ‘remnant’ GCs
that develop after partial gastrectomy 190
and in certain benign conditions such as
muco-cutaneous ulcers200. Unravelling
the interplay between virus infection and
inflammation will be an important priority
for future studies.
Whatever the role of EBV in the
oncogenic process, there is an opportunity
to exploit this association for patient benefit.
Published online 30 September 2016
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Acknowledgements
The authors are grateful to A. Bell for help with revisions to
the figures. L.S.Y. has been supported by Cancer Research
UK. L.F.Y. is supported by High Impact Research Grant
UM.C/625/1/HIR/MOHE/DENT/23 from the University of
Malaya. P.G.M. is supported by a Bloodwise Programme
Grant.
Competing interests statement
The authors declare no competing interests.
DATABASES
GenBank: http://www.ncbi.nlm.nih.gov/nuccore/NC_007605
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