Journal of Clinical Virology 102 (2018) 84–92

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Journal of Clinical Virology

journal homepage: www.elsevier.com/locate/jcv

Review

Primary Epstein-Barr virus infection T

a b a,b,⁎
Samantha K. Dunmire , Priya S. Verghese , Henry H. Balfour Jr.
a
Department of Laboratory Medicine and Pathology, USA
b
Department of Pediatrics, University of Minnesota Medical Center, Minneapolis, MN 55455, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Epstein-Barr virus (EBV) infects about 90% of adults worldwide. It is the main cause of infectious mononucleosis,
Epstein-Barr virus which is observed most frequently in adolescents. The disease can last several weeks and is characterized by
Primary infection lymphocytosis, sore throat, lymphadenopathy, and fatigue. Exposure to oral secretions during deep kissing has
Infectious mononucleosis been identified as the major source for primary EBV infection in adolescents. Oral secretions are also thought to
Mono
be the source for younger children through intimate intact or sharing food and eating utensils, although this has
Vaccine
not been confirmed. Unlike most acute viral illnesses such as influenza, the incubation period of symptomatic
Immune responses
primary EBV infection is unusually long, lasting about six weeks. Diagnosis is typically made by heterophile
antibody tests and/or EBV-specific antibody tests. Long-term consequences may result from acquisition of the
virus, including nasopharyngeal carcinoma and lymphomas. Nevertheless, there remains a surprising dearth of
knowledge regarding the establishment of an immune response to persistent EBV infection, especially during the
incubation period. This lack of knowledge has impaired our ability to develop an effective prophylactic EBV
vaccine, despite various attempts. Our greatest challenges in EBV research are to develop a prophylactic vaccine
and devise treatment strategies for persons already infected with EBV.

1. Introduction
Epstein-Barr virus (EBV) is arguably the most ubiquitous of human
viruses, infecting at least 90% of adults worldwide [1]. It is also the first
human cancer virus, having been recognized as the cause of endemic
Burkitt lymphoma in the 1960s [2–4]. In addition to Burkitt lymphoma,
EBV is implicated in the pathogenesis of Hodgkin lymphoma [5], na-
sopharyngeal carcinoma [6,7], gastric cancer [8], and a myriad of
malignancies in individuals with inherited or acquired im-
munodeficiency [9]. EBV is also considered to be a major environ-
mental risk factor for multiple sclerosis [10]. Despite the enormous
disease burden attributed to EBV infection, we do not yet have a li-
censed vaccine to prevent it, or an approved treatment for it.
2. Epidemiology
2.1. Prevalence of EBV infection
The discovery of EBV was reported in 1964 [2,3]. Shortly after that,
methods to detect EBV-specific antibodies were devised [6,11] and
applied to cross-sectional studies, which showed that the vast majority
of the world's population has been infected by the time they reach
adulthood [1,12,13]. The prevalence of EBV infection in children varies
from 10 to 90%, depending on their age, geographic location, and race/
ethnicity [1,13–15]. For example, the age-specific prevalence of EBV
antibodies among white, non-Hispanic children tested in 2011 in the
Minneapolis-St. Paul metropolitan area did not reach 50% until age 18,
whereas 50% of non-white children were antibody positive by the age
of 10 [15].
Children may be acquiring primary EBV infection at a later age in
developed countries. Among entering freshmen at the University of
Minnesota, EBV antibody prevalence declined from 64% in 2006 to
52% in 2012 [16,17]. This is consistent with data from the National
Health and Nutrition Examination Surveys (NHANES). In NHANES,
EBV antibody prevalence among US children 6–19 years old declined
from an average of 72% during 2003–2004 to 65% during 2009–2010
[13]. The decline in antibody prevalence has averaged approximately
2% annually. A decrease in primary EBV infection especially among
young children has also been observed in England and Wales [18] and
Japan [19]. The explanation for this could be improvement in socio-
economic status, since higher household income and education level
were significantly associated with a lower prevalence of EBV antibody

Abbreviations: CMV, cytomegalovirus; EBV, Epstein-Barr virus; EBNA, Epstein-Barr virus nuclear antigen; LMP, latent membrane protein; NHANES, National Health and Nutrition
Examination Surveys; VCA, viral capsid antigen
⁎
Corresponding author at: MMC 609, 420 Delaware St SE, University of Minnesota, Minneapolis, MN, 55455, USA.
E-mail address: balfo001@umn.edu (H.H. Balfour).

https://doi.org/10.1016/j.jcv.2018.03.001
Received 3 January 2018; Received in revised form 2 March 2018; Accepted 3 March 2018
1386-6532/ © 2018 Elsevier B.V. All rights reserved.
S.K. Dunmire et al.
in the NHANES study [13]. This trend is important to monitor because
if the trend continues, the number of adolescents and young adults
susceptible to infectious mononucleosis in developed countries will
increase.
2.2. Incidence of primary EBV infection and infectious mononucleosis
among adolescents and young adults
The development of EBV-specific antibody tests enabled serosurveys
to be conducted in college students and military recruits, which es-
tablished unequivocally that EBV was the cause of infectious mono-
nucleosis [20–25]. The annual incidence of primary EBV infection and
the proportion of those subjects diagnosed with infectious mono-
nucleosis varied widely, most likely because the study protocols and
populations recruited were quite different. Of interest, the investigation
of the Yale class of 1973 found that during their freshman year, 17
(74%) of 23 students who acquired primary EBV infections experienced
infectious mononucleosis [23]. This is exactly the same proportion of
infectious mononucleosis we found among our University of Minnesota
students studied some 3 decades later (described in Section 4.2 below).
2.3. Relevance of age of acquisition of primary EBV infection
There is a complex relationship between age of acquisition of pri-
mary EBV, severity of the acute illness, and subsequent risk of EBV-
spurred cancers or autoimmune diseases. Infectious mononucleosis
occurs in preadolescents, although it is less common than in adolescents
and young adults. Interestingly, younger age at the time of primary EBV
infection among Kenyan infants was associated with elevated levels of
EBV viremia throughout infancy, leading the investigators to postulate
that these infants were at higher risk for endemic Burkitt lymphoma
[26,27]. Melbye and colleagues found that Greenland Eskimo children
acquired primary EBV infection at an earlier age and had higher titers
of IgG antibody against EBV viral capsid antigen (VCA) than age-mat-
ched Danish children [28]. The authors speculated that early infection
with “a large inoculum of EBV” explained why Eskimos were at high
risk for nasopharyngeal carcinoma versus Danes who were not. Finally,
early acquisition of EBV is a significant risk for MS in childhood [29].
On the other hand, late acquisition of primary EBV infection by
adolescents and young adults is thought to result in infectious mono-
nucleosis more often than it does in children [30], although there are no
hard data to support this. Studies performed on cohorts of children in
Uganda and The Gambia suggest that young children seroconvert
asymptomatically [31,32]. Jayasooriya et al. showed that infants under
two years old who acquire the virus were observed to have activated
EBV-specific T cells and viral loads comparable to adolescents pre-
senting with infectious mononucleosis, but lacked simultaneous lym-
phocytosis at the time periods examined [31]. It is important to note,
however, that in patients with infectious mononucleosis EBV-specific T
cell activation persists for a number of months. Indeed, VCA IgM titers
are highest in the so-called “subacute” phase, after bulk CD8 T cells
have already largely contracted [16,33].
3. Routes of transmission
3.1. Deep kissing
EBV infection among adolescents and young adults is acquired
primarily by deep kissing as proposed by Hoagland's careful clinical
observations [34] and confirmed decades later by prospective studies
among undergraduate university students [16,17]. The amount of EBV
DNA in washes after primary EBV infection can be extremely high and
may persist for months, which is supportive evidence for this route of
transmission. Median EBV oral loads peaked at 4.8 log10 copies/mL
(63,000 copies/mL) a median of 2 months after onset of infection and
persisted for a median of 5.2 months [16,17]. The shortest documented
85
Journal of Clinical Virology 102 (2018) 84–92
period of continuous oral shedding was 28 days and the longest was 3.1
years.
3.2. Sexual intercourse
Sexual intercourse has been reported to enhance transmission [35].
However, our University of Minnesota prospective studies found that
subjects who engaged in deep kissing with or without penetrative
sexual intercourse had the same higher risk of primary EBV infection
throughout their undergraduate years as compared with subjects who
reported no kissing and no sex [16,17].
3.3. Blood transfusion
Blood products have been reported to transmit EBV infection, which
in some instances results in an infectious mononucleosis syndrome
[36–38]. The risk appears to be low, but the exact incidence is not
known, likely because most adults who receive blood products remain
asymptomatic if they are superinfected due to residual immunity from a
previous EBV infection.
3.4. Allograft transplantation
Transmission of EBV from donor to recipient has been documented
in hematopoietic cell transplantation [39] and solid organ transplan-
tation [40]. Shapiro et al. demonstrated donor to recipient transmission
of EBV during hematopoietic cell transplantation by comparing re-
striction fragment length polymorphisms of EBV strains in blood, bone
marrow, and tissue [39]. Verghese and colleagues used sequence var-
iation in the EBV latent membrane protein 1 (LMP-1) gene to document
transmission of EBV from a living, unrelated kidney donor to an EBV-
naïve, 16-year-old boy, who subsequently developed posttransplant
lymphoproliferative disorder [40].
3.5. Close contact with household members or caregivers?
How preadolescent children contract EBV is unknown. A reasonable
supposition is that they are infected by their parents, siblings, other
household members, or caregivers who are “carriers” of the virus and
shed EBV periodically into their oral secretions [41,42]. An example of
this is the very early acquisition of EBV among three distinct Melane-
sian populations whose infants have multiple caregivers that pre-
masticate the baby’s food [43]. Another paper studying child care in
South Africa also mentions premastication of food as a way by which
children may be exposed to saliva, in addition to cleaning the child’s
face or mouth with saliva [44]. Sharing items such as eating utensils,
drinking glasses, toothbrushes, or toys with an infected person has been
implicated but not proven to be a route of transmission.
4. Clinical features
4.1. Incubation period
The incubation period for asymptomatic primary EBV infections is
nearly impossible to define. For infectious mononucleosis, Hoagland’s
clinical records suggested an incubation period of 32–49 days, based on
the dates of kissing episodes until the onset of symptoms [34]. A well-
documented Swedish case of infectious mononucleosis occurred 38 days
after a kissing event [45]. Behavioral data from our prospectively col-
lected medical history questionnaires are consistent with an incubation
period of 42 days [16,17].
4.2. Clinical findings during the acute infection
Three prospective studies of college students have been published
since the turn of the century [16,17,35]. Crawford and colleagues found
S.K. Dunmire et al. Journal of Clinical Virology 102 (2018) 84–92

that 101 (46%) of 241 Edinburgh University students (17–24 years old Table 1
on enrollment) contracted a primary EBV infection during their first 3 Symptoms of infectious mononucleosis in 60 undergraduate students studied pro-
spectively (41 women, 19 men; median age at onset, 19; range, 18–22 years).
undergraduate years, which was an annual infection rate of 15.2% [35].
Twenty-seven subjects (25%) developed infectious mononucleosis, Symptom Number of Median duration
while the remainder were reported to have been asymptomatic. subjects (percent) (days)a
The main difference between the Scottish study and ours was the
Sore throat 59 (98%) 7.5
incidence of symptomatic infection. All laboratory-documented pri-
Swollen or tender cervical 53 (88%) 15.0
mary EBV infections in our prospective studies were assigned to one of lymphadenopathy
three clinical categories according to the following criteria. Infectious Fatigue 47 (78%) 15.5
mononucleosis was an acute illness with at least two of the following Decreased appetite 39 (65%) 10.0
findings: sore throat, cervical lymphadenopathy, fever, fatigue. Headache 38 (63%) 8.0
Felt febrile 32 (53%) 5.5
Symptomatic meant having some symptoms but not fulfilling the cri-
Body aches (myalgia) 30 (50%) 8.0
teria for infectious mononucleosis. Asymptomatic subjects were entirely Upper respiratory symptoms (cough, 29 (48%) 8.0
well during the 3 months after the laboratory diagnosis of primary EBV runny nose, nasal stuffiness)
infection. Of the 81 subjects (18–22 years old) who had primary EBV Abdominal pain 9 (15%) 16.0
infections while under surveillance in our two prospective studies, 60 a
Of symptomatic subjects.
(74%) experienced infectious mononucleosis, 11 (14%) were sympto-
matic, and 10 (12%) were entirely asymptomatic. Why the rate of
and is most likely due to transient penicillin hypersensitivity induced by
symptomatic primary EBV infection was so much higher in the
the immune dysregulation accompanying the acute stage of infection
Minnesota students (74% versus 25%) is not clear. Explanations could
[47]. Subclinical hepatitis documented by elevated levels of alanine
be that the study designs were quite different, the geographic locations
aminotransferase was documented in approximately 75% of our sub-
were different, and years of surveillance were different.
jects, and overt hepatitis with tender hepatomegaly and jaundice de-
The severity of illness (SOI) of the Minnesota students was graded
veloped in 5% of cases.
on a scale from 0 to 6 according to the subjects' reports of the degree of
The assumption has been that the majority of primary EBV infec-
pain and limitation of activities [46]. A score of 0 meant the subject was
tions before puberty are asymptomatic or mild but that is not ne-
entirely asymptomatic, while a score of 6 meant that the subject was
cessarily so. One consideration is that young children, especially those
bedridden. The distribution of maximum SOI scores (Fig. 1) emphasizes
under the age of 4 years, may not develop a positive heterophile anti-
that primary EBV infection in adolescents and young adults is not tri-
body response during primary EBV infection [48]. Unless EBV-specific
vial: 33 subjects (41%) had a maximum SOI score above the modal
assays are performed, the diagnosis will be missed.
score of 3.
A noteworthy aspect of infectious mononucleosis is its long dura-
tion. The median duration of the acute illness was 18 days (mean, 4.3. Complications of the acute illness
19 days; range, 3–54 days) in the 60 subjects with infectious mono-
nucleosis. Table 1 shows the frequency of clinical findings in these Serious complications during the acute phase of primary EBV in-
subjects. Most findings had a median duration of 8 days or less, but fection are uncommon. Complications estimated to occur in at least 1%
fatigue and cervical lymphadenopathy persisted longer. Other findings, of patients with infectious mononucleosis are: airway obstruction due
seen in fewer than 20% of cases, included hepatomegaly, splenomegaly, to oropharyngeal inflammation, meningoencephalitis, streptococcal
nausea, vomiting, palatal petechiae, periorbital and eyelid edema, and pharyngitis, hemolytic anemia, and thrombocytopenia [49–51]. Splenic
rash. Rash occurs more often in patients given penicillin derivatives, rupture is a rare but feared complication [52] that may keep athletes
out of competition for weeks. A reasonable recommendation is that
athletes with infectious mononucleosis may resume contact sports after
3 weeks of illness as long as their acute signs and symptoms have
subsided [53].
We recommend that students gradually return to their normal ex-
ercise level. If strenuous activities are resumed too quickly, clinical
symptoms, especially fatigue, may recur.
Gall bladder involvement during infectious mononucleosis is un-
derappreciated, and usually resolves as the period of generalized im-
mune upregulation subsides [54]. We are aware of patients with in-
fectious mononucleosis who underwent an unnecessary
cholecystectomy. Abdominal pain accompanying the acute illness could
be due to mesenteric lymphadenitis, pressure on the liver or splenic
capsule due to organomegaly, or acalculous cholecystitis.

4.4. Chronic infectious mononucleosis

Primary EBV infection may result in a chronic multisystem disease

Fig. 1. Distribution of severity of illness scores in 81 subjects who contracted primary
EBV infection during prospective surveillance. Subjects with a score of 0 were entirely
asymptomatic, whereas subjects with a score of 6 were bedridden. The figure illustrates
that primary EBV infection in adolescents and young adults is not trivial: 33 subjects
(41%) had a maximum SOI score above the modal score of 3.
in a yet to be determined percentage of patients who do not have a
defined genetic reason for poor control of the virus. Their persistent
antibody profile is: negative VCA IgM indices, very high VCA IgG in-
dices, and modestly elevated EBNA-1 IgG levels. There appear to be two
clinical patterns of chronic infectious mononucleosis. The first and
more common is recovery from the initial disease but persistent lin-
gering or recurring symptoms that develop months to years later. These
patients may report that they have had infectious mononucleosis more
than once, but we have not been able to detect acquisition of a different

86
S.K. Dunmire et al.
strain of EBV using LMP-1 sequence variation analysis. We suspect that
the originally acquired viral strain is the culprit. The second pattern is a
continuous “mono-like” illness that lasts indefinitely. There is no bona
vide treatment regimen for these patients, but we have had some an-
ecdotal success with a combination of antiviral drugs and an anti-in-
flammatory diet, which are posted on our website (http://z.umn.edu/
ebvdiseases).
4.5. EBV-spurred malignancies and autoimmune disease
More than 50 years after the discovery of EBV, the chronic con-
sequences of EBV infection are still incompletely understood. EBV is
clearly implicated in the etiology of lymphomas, nasopharyngeal car-
cinoma, and gastric cancer [2–8]. Also, “There is an undeniable asso-
ciation between EBV and multiple sclerosis” [55].
5. Genetic influence on acquisition and severity of primary EBV
infection
Genetics likely influence the age at which primary EBV is acquired,
and its severity. Two separate cross-sectional surveys of US children
found that early acquisition of EBV infection, as evidenced by having
IgG antibodies against EBV VCA, was a racial disparity that could not be
explained solely by socioeconomic factors [13,15]. The enormous dif-
ference in overall age- and sex-adjusted EBV antibody prevalence be-
tween non-Hispanic white (32%) and non-Hispanic black children
(62%) in the study by Condon et al. [15] is consistent with a genetic
basis for the racial clustering of EBV infection in younger black chil-
dren.
In terms of the severity of primary EBV infection, Hwang and col-
leagues found that concordance for infectious mononucleosis in
monozygotic twins was twice that of dizygotic twins [56]. They stated
that their results were “compatible with a heritable contribution to the
risk of infectious mononucleosis." Rostgaard et al. [57] extended these
findings by tracking familial clustering of hospitalized cases of in-
fectious mononucleosis. Using large Danish national databases, these
investigators reported that same-sex twins had a rate ratio of 9.3 for
infectious mononucleosis as compared with 2.3 for first-degree relatives
(opposite-sex twins, siblings and parents), 1.4 for second degree re-
latives (half-siblings, grandparents, uncles and aunts) and 1.0 for third-
degree relatives (first cousins). The 95% confidence intervals for those
four classes of relationships did not overlap, supporting the conclusion
that degree of relatedness increased the likelihood of having a
87
Journal of Clinical Virology 102 (2018) 84–92
Fig. 2. A model of primary EBV infection in the or-
opharynx and periphery. The days relative to
symptom onset were calculated from our 81 pro-
spectively followed students experiencing primary
EBV infection. Epithelial cell derived EBV (purple
hexagons) is transmitted to an EBV naïve host via
infected saliva about six weeks prior to symptom
onset. Viral particles may either infect susceptible B
cells in the crypt of the tonsil or epithelial cells which
later infect B cells. Infected B cells become trans-
formed, resulting in LatencyIII cells. These cells re-
plicate in the germinal center, creating a reservoir of
virus. Some become mature infected B cells, with the
latent membrane proteins augmenting survival sig-
nals to the cell. Latency0 cells are memory-like,
changing their trafficking patterns so they recirculate
between the periphery and oropharynx, with the
median for first detection being five days prior to
symptom onset. Around the same time, EBV specific
CD8 T cells also begin circulating, first becoming
detectable about five days post symptom onset.
Other LatencyIII cells undergo reactivation, produ-
cing virus which infects epithelial cells. Virus is then
shed into the oral cavity.
symptomatic primary EBV infection.
6. Virologic events
6.1. Exposure and acquisition
What conditions are ideal for transmission of EBV are not known. A
higher inoculum of virus would presumably be more likely to result in
transmission, but the amount likely varies based on genetic factors of
both the host and the virus itself. Once transmitted, EBV infects oral
epithelial cells, most likely squamous epithelial cells in the tonsil [58].
EBV also infects B cells in the lymphatic tissues of Waldeyer’s Ring,
which include the tonsils.
Although it is unknown which cell type is infected first, evidence
exists for a ‘switch tropism’ for host cell types depending on which cells
EBV replicates in. Surface markers including gp42 on salivary virus
suggest it is derived from oral epithelial cells, making B cells the pri-
mary targets during acquisition [59]. These B cells then become
transformed, which may result in malignancies as observed during the
discovery of EBV [2].
Four possible types of viral latency may occur in B cells after in-
fection. These states are characterized by expression of specific EBV
encoded genes. Cells in latency III are the most replicative, expressing
all EBV nuclear antigens (EBNAs), LMPs, and EBV encoded small RNAs
(EBER) [60]. In contrast, latency II cells express EBNA-1 and LMP-1 and
−2. These cells are thought to be important precursors to some EBV
related malignancies [61]. Latency I involves expression of only
EBNA1, which serves to help replicate and segregate the viral genome
during cell divisions [62]. A final latency 0 is thought to exist wherein
the virally encoded gene expression is completely silenced, allowing for
infection to persist without detection by the immune system. A model of
primary infection is shown in Fig. 2.
6.2. Incubation period
Relatively little is known about the incubation period due to the
difficulty of obtaining samples from this stage of infection. A few pro-
spective studies, however, have allowed some insight into the viral
progression during acute infectious mononucleosis. Following the in-
oculating event, virus remains undetectable for the first four to five
weeks of the incubation period. We detected EBV in the oral cavity
about a week before onset of symptoms and the peripheral blood ten to
seven days prior to symptom onset [63].
S.K. Dunmire et al.
This result fits with a model whereby latently infected B cells re-
plicated slowly, eventually entering germinal centers within the tonsil.
The B cells are then reactivated through B cell receptor signaling aug-
mented by EBV encoded proteins such as LMP [60]. The terminal dif-
ferentiation of these cells into mature plasmablasts results in robust
shedding of virus back into the basal layer of epithelial cells in the oral
cavity [64].
6.3. Acute and convalescent phases
The acute phase of infectious mononucleosis is characterized by
explosive viral replication in both the periphery and oral cavity. In the
peripheral blood, virus is primarily contained within infected B cells,
and only small amounts are released into the plasma [46]. During acute
infectious mononucleosis as many as 50% of the circulating resting
memory B cells may be infected with EBV, but as the patient con-
valesces this number declines to between 1 and 50 per 106 B cells [65].
A B cell with actively replicating virus may have thousands of copies of
the EBV genome [66]. Persistent EBV is maintained in latently infected
resting memory-like B cells at about 5–500 copies per cell [65,67].
Blood viral loads are highest within two weeks of symptom onset.
The median peak viremia of 7700 copies/mL was reached 8 days after
onset of symptoms. Blood viral loads > 100,000 copies/mL were ob-
served in some of our study participants. Virus can be found in oral
epithelial cells and in the supernatant from the oral wash, showing that
in addition to cells being infected, virus is actively being shed into the
oral cavity. Over the following weeks, the amount of virus in the per-
ipheral blood contracts, but elevated viral shedding in the oral cavity
can persist for over a year [16,63].
6.4. Prevalence and pathogenicity of EBNA types
Two EBNA types of EBV exist, differentiated from one another pri-
marily through differences in the sequence of the EBNA-3 genes [68].
Type 1 is the most common type worldwide, but type 2 has about equal
prevalence with type 1 in Africa. No difference has been described to
date regarding the pathogenicity of the two types [69]. Seventy-nine of
our subjects with primary infections had samples available for EBNA
typing. EBNA type 1 was present in 69 subjects (87%), EBNA type 2 in 8
subjects (10%) and mixtures of the 2 types in 2 subjects (2.5%). There
was no correlation between disease severity and EBNA type.
7. Immune responses
7.1. Incubation period
The initiation of the immune response to EBV occurs around the
time the viral load begins to be measurable in the oropharynx and
peripheral blood. Starting about two weeks prior to symptom onset, a
systemic type I interferon gene signature was observed in peripheral
blood mononuclear cells [63]. By the time patients begin to experience
symptoms related to the acute illness, this response had waned sig-
nificantly, transitioning to a signature more closely related to hemo-
phagocytic syndromes due to rapid CD8 T cell expansion [70].
Interestingly, the number of circulating plasmacytoid dendritic cells
were drastically reduced coincident with loss of the type I interferon
signature, although whether these cells are killed or sequestered in
tissue sites remains unknown [63,71]. Furthermore, the type I inter-
feron signature observed lacked certain typical genes such as OAS1 and
MX1. EBV is known to encode miRNAs that interfere with the host's
ability to mount certain immune responses [72]. In particular, BART16
has been shown to have a dampening effect in type I interferon sig-
naling in vitro, which may account for the loss of this subset of type I
interferon genes [73].
No significant difference in proportions of CD8 T cells, CD4 T cells,
or NK cells was observed prior to symptom onset. The activation state of
88
Journal of Clinical Virology 102 (2018) 84–92
bulk CD8 T cells, however, did appear to be affected. In the ten days
prior to symptom onset, bulk CD8 T cells showed significant upregu-
lation of activation marker CD38. By contrast, EBV tetramer specific
CD8 T cells were not activated until symptom onset, indicating the
response during the incubation period is probably induced by inter-
ferons in the circulation [63]. The lack of circulating tetramer reactive
cells until symptom onset suggests any EBV-specific cells activated in
the oropharynx must stay within local lymphatic tissue.
7.2. Acute and convalescent phases
Until recently the innate immune system was an understudied as-
pect of the overall immune response during infectious mononucleosis.
In patients presenting with infectious mononucleosis, a decrease in the
number of circulating CD56bright immature NK cells was observed and
the proportion of NK cells CD56bright remained significantly depressed
up to 50 days post symptom onset. By contrast, the proportion of NK
cells that were CD56dim NKG2A+ KIR− increases and remained ele-
vated well into convalescence [63,74,75]. It has been posited from
work in humanized mice that NK cells are important for early control of
EBV, particularly in killing lytically infected B cells [76].
Bridging the gap from innate to adaptive immunity, γd T cells have
also been implicated as having an important role in controlling EBV
during primary infection. γδ T cells exist primarily in the lymphatics of
the gut and other mucosal tissues and bear highly conserved γδ T cell
receptors [77,78]. The prevalence of EBV related nasopharyngeal car-
cinomas and gastric cancers exist as a potent reminder to the im-
portance of controlling EBV-infected epithelial cells at these sites. Re-
cent findings show that about 50% of the donors tested had γδ T cells
that responded robustly to EBV infected cells in an NKG2D dependent
manner. Interestingly, only cell lines known to maintain type I latency
such as Daudi elicited activation, which suggests the innate immune
system mounts a layered response to EBV with NK cells killing lytically
infected cells and γδ targeting latently infected cells [79].
During acute infectious mononucleosis, CD8 T cells undergo ex-
plosive expansion and comprise both EBV-specific and non-EBV-specific
cells [80]. Tetramer positive EBV specific CD8 T cells increase rapidly
in number at symptom onset and bear elevated levels of CD38+ for
several weeks. Similarly, bulk CD8 T cells are also activated and pro-
duce Granzyme B, though these cells show an increased activation state
for up to six months after symptom onset [63]. The number of CD4 T
cells is not significantly affected, but the cells do become activated and
respond to EBV MHC class II tetramers [81].
CD4 T cell responses are important for subsequent generation of
antibodies to EBV, as demonstrated by the parallel between im-
munodominant CD4 T cell epitopes and subsequent humoral response.
Work in patients with infectious mononucleosis has further suggested
there is a correlation between the kinetics and strength of the antibody
response to the patient’s overall severity of illness. Those study parti-
cipants that had more rapid generation of anti-gp350 antibody had
lower severity and duration of illness [17].
8. Diagnosis
The diagnosis of primary EBV infection relies on laboratory tests.
Point-of-care tests are almost always based on detection of heterophile
antibodies. These are IgM class antibodies directed against mammalian
erythrocytes, which are raised during the generalized immune upre-
gulation that accompanies acute primary EBV infection [82]. The het-
erophile test has been the standard clinical diagnostic tool ever since it
was developed in 1932, 32 years before the discovery of EBV. Hetero-
phile tests are a practical method for confirming the clinical diagnosis.
However, they have several limitations. Approximately 40% of children
4 years of age or younger do not develop heterophile antibodies fol-
lowing a primary EBV infection [48]. If the heterophile is the only test
ordered, the diagnosis may be missed. Second, heterophile antibodies
S.K. Dunmire et al.
by definition are not specific and may be present in infections caused by
other pathogens, malignancies, or autoimmune diseases [83,84]. Fi-
nally, heterophile antibodies can persist for a year or more and there-
fore are not always diagnostic of an acute EBV infection [85].
The most useful EBV-specific antibody tests are VCA IgM, VCA IgG
and EBNA-1 IgG usually measured by an immunoassay platform. In our
prospective studies, 79 of 81 subjects who experienced primary EBV
infections had adequate samples for EBV-specific antibody testing. VCA
IgM antibodies were detected in 68 (86%) of the 79 subjects. These
antibodies declined over the next 3 months, so this is a good test to
confirm a primary EBV infection. A caveat is that false-positive EBV
VCA IgM results have been reported especially with cytomegalovirus
(CMV) infection [86].
The heterophile test is almost as sensitive as the VCA IgM, albeit
nonspecific. It was positive in 63 (80%) of the 79 subjects. All 79 of our
subjects with primary EBV infection who were followed for at least 3
months after onset of infection developed IgG antibodies to VCA. EBV
VCA IgG antibodies persist indefinitely, so this is an excellent labora-
tory test to document a previous EBV infection. Antibodies against
EBNA-1, which are typically used in commercial diagnostic tests, de-
velop slowly and usually are not detectable until 3 months or longer
after onset of illness [16].
Subjects do not generally present with both VCA IgM and EBNA-1
antibodies due to the kinetics of these responses. If both are present,
then the person has likely been infected at least three months.
Therefore, the presence of EBNA-1 antibodies during an acute illness
rules out a recent primary EBV infection. It is important to note,
however, that some individuals may not produce EBNA-1 antibodies at
all. About 8% of students who participated in our prospective study did
not develop EBNA-1 antibodies [16]. Thus, the absence of EBNA-1
antibodies is not a definitive indicator that the patient has never been
infected before, in which case VCA IgG may be a more reliable surro-
gate.
Other assays are sometimes used. Immunoblotting may include
immediate early, early, and late EBV antigens on one test strip, which
permits evaluation of the stage of EBV infection using a single serum or
plasma sample [87]. Our virology research laboratory uses the Mik-
rogen line blot immunoassay system, which provides a choice of re-
agents for detection of IgA, IgG, and IgM class antibodies and IgG an-
tibody avidity. While valuable from a research perspective,
immunoblotting is not practical for routine diagnosis of primary EBV
infection because of cost and low throughput. Serial quantitative
measurements of EBV DNA in the blood have been used to track post-
transplant EBV infections, but are not necessary to document primary
EBV infections in the otherwise normal host.
9. Prevention
9.1. Limiting exposure to EBV
Avoiding exposure to EBV is nearly impossible, as reflected by the
very high prevalence of EBV antibodies in adults worldwide. More
needs to be learned about how children acquire EBV before appropriate
measures of prevention can be recommended for them. Frequent
handwashing and not sharing items such as eating utensils, drinking
glasses, or toothbrushes is good hygiene in general, but there are no
solid data that EBV can be transmitted by fomites.
Acquisition of primary EBV infection after transplantation of solid
organs or hematopoietic cells could be prevented, at least in part, by
selecting EBV-naïve donors for EBV-naïve recipients. Because at least
90% of adults are antibody-positive, identifying a suitably matched
antibody-negative donor is difficult. Even if an EBV-naïve donor could
be found, the virus still might be acquired by the natural route after
transplantation.
89
Journal of Clinical Virology 102 (2018) 84–92
9.2. Antiviral prophylaxis
Acyclovir, valacyclovir, ganciclovir, and valganciclovir are fre-
quently prescribed for 3–6 months after transplantation to prevent or
suppress herpesvirus infections, with the major focus being on pre-
vention of CMV disease. Whereas these drugs clearly reduce the in-
cidence and severity of posttransplant CMV disease [88,89], their role
in the prevention of posttransplant EBV disease is uncertain [90,91]. A
unique approach is to give antiviral drugs to the donor pretransplant to
prevent donor to recipient transmission of herpesviruses. This has
shown some promise in a small double-blind, placebo-controlled trial
[40].
9.3. Vaccines
Three prophylactic EBV vaccine candidates have been tested in
humans. Two are based on gp350, which is the major surface glyco-
protein of EBV that allows the virus to attach to the CD21 receptor of
human B lymphocytes and subsequently infect them.
The first candidate was a vaccinia virus construct expressing the
EBV membrane glycoprotein gp220-350 [92]. This trial included 19
EBV-naïve Chinese children 1–3 years of age. Nine subjects received the
vaccine as a single dose by scarification and 10 subjects served as
controls. The vaccine was immunogenic and during 16 months of
follow-up, 3 of 9 vaccinees versus 10 of 10 subjects in the control group
became infected with EBV evidenced by development of antibodies
against EBV VCA (not contained in the vaccine). No further develop-
ment of this vaccine has been reported, possibly because it contains live
vaccinia, which is associated with potentially serious adverse events
[93].
The second candidate was a subunit EBV gp350 vaccine originally
developed in Chinese hamster ovary (CHO) cells by Jackman and col-
leagues [94]. This vaccine has been given to humans in four clinical
trials and is still under development. Parenthetically, it was recently
demonstrated that the gp350 vaccine construct must contain a con-
formationally correct 72A1 epitope to effectively neutralize EBV [95].
The first human trial with the subunit gp350 vaccine was a phase 1
study evaluating the safety and immunogenicity of a 3-dose regimen of
vaccine given intramuscularly [96]. EBV antibody-negative and anti-
body-positive subjects 18–25 years of age were randomized to receive
the vaccine adjuvanted with 3-O-desacyl-40-monophosphoryl lipid A
and aluminum salt (AS04) or aluminum salt alone. The second trial was
a phase 1/2 study that randomized EBV-naïve subjects 18–37 years old
to unadjuvanted vaccine, vaccine adjuvanted with AS04, or vaccine
adjuvanted with aluminum salt only [96]. The combined data from 138
subjects in these two trials showed that the vaccine was safe with one
exception. Ten days after receiving the second dose of vaccine ad-
juvanted with AS04, an EBV antibody-positive subject was hospitalized
for an apparent autoimmune reaction involving the central nervous
system and multiple joints. The immunogenicity data, which included
measurement of gp350 and neutralizing antibodies, indicated that
vaccine adjuvanted with AS04 was superior to both non-adjuvanted
vaccine and vaccine adjuvanted with aluminum salt.
The third trial was a phase 2, placebo-controlled, double-blind
evaluation of the safety, immunogenicity, and efficacy of gp350 vaccine
in EBV-naïve Belgian students ages 16–25 [97]. There were no sig-
nificant adverse events and 76 (98.7%) of 77 vaccine recipients who
were not subsequently infected by wildtype EBV developed gp350 an-
tibodies. The vaccine did not prevent infection: 13 (14%) of 90 vaccine
recipients became infected versus 18 (20%) of 91 placebo subjects.
However, it had a significant effect on clinical disease. In the intent-to-
treat population, infectious mononucleosis developed in 2 (2%) of 90
vaccinees as compared with 9 (10%) of 91 placebo recipients (P = 0.03,
Fisher exact test, 1-sided).
The fourth trial of the gp350 subunit vaccine was a phase 1 study of
gp350 with an aluminum hydroxide adjuvant conducted in 16 pediatric
S.K. Dunmire et al.
renal transplant candidates [98]. All 13 evaluable subjects mounted an
antibody response to gp350 but only four made neutralizing antibody.
Because there was no control group, vaccine efficacy could not be as-
sessed. However, this small phase 1 trial did show that immunization of
children awaiting transplantation for chronic renal disease is safe and
feasible.
The third prophylactic EBV vaccine candidate given to humans was
an adjuvanted EBNA-3A peptide restricted by HLA B8, which was de-
signed to control expansion of EBV-infected B cells by generating CD8
T-cell immunity to EBNAs [99]. EBV-naïve individuals were immunized
on a two-month interval schedule. Of the 14 enrolled subjects, four
received placebo, two were immunized with 50 μg dose of peptide and
the remaining 8 individuals were immunized with a 5 μg dose of pep-
tide. This strategy was effective at generating a peptide-specific CD8+
T-cell response in most individuals as measured by ex vivo peptide-
specific interferon gamma production. Among subjects who subse-
quently acquired wild-type EBV, infectious mononucleosis occurred in
1 of 2 subjects in the placebo group versus 0 of 4 in the vaccinated
cohort, a trend suggesting that this vaccine might prevent symptomatic
EBV infection. The general utility of epitope vaccines is limited by the
fact that they only target specific HLA types. Nonetheless epitope vac-
cines might be employed to prevent posttransplant lymphoproliferative
disorder (PTLD), because the HLA type of transplant patients is known.
10. Treatment
10.1. Otherwise normal host
The use of corticosteroids in acute infectious mononucleosis is
somewhat controversial [100]. Most clinicians would prescribe a short
course of corticosteroids for patients with any degree of airway ob-
struction, or autoimmune phenomena such as anemia and/or throm-
bocytopenia [51].
Several nucleoside analogs have in vitro activity against EBV [101]
but a clinical benefit has not yet been proven for any of them. Vala-
cyclovir is worth mentioning because it is generic and has very few side
effects. We evaluated valacyclovir (3 g/day for 14 days) in 20 Uni-
versity of Minnesota undergraduates with infectious mononucleosis due
to laboratory-confirmed primary EBV infection [102]. Ten subjects re-
ceived valacyclovir and 10 received no antiviral drugs. There was a
significant reduction in viral load in the oral compartment but not in
the blood. The number of reported symptoms and the severity of illness
were reduced significantly among the valacyclovir recipients as com-
pared with the control subjects. Limitations of our study were the small
number of subjects enrolled and the lack of a placebo control. These
results are promising but need to be confirmed in a larger, double-blind,
placebo-controlled trial.
10.2. Posttransplant lymphoproliferative disorder
PTLD, a heterogeneous spectrum of disorders including lymphomas,
is a well-recognized complication of both solid organ transplantation
(SOT) and hematopoietic cell transplantation (HCT). PTLD is char-
acterized by the abnormal proliferation of lymphoid immune cells in
extrinsically immunosuppressed transplant recipients due to impaired
immune surveillance. EBV is a key pathogenic driver in many cases of
PTLD, through known and unknown mechanisms.
After initial infection and lytic cycling, EBV transitions to a latency
gene program in the germinal center to avoid host recognition. Some of
these genes co-stimulate host B cells to proliferate and differentiate. In
immunocompetent hosts, EBV-specific CD8+ effector and memory T
cells control the abnormal proliferation of EBV- infected B cells, but this
ability is lost in immunosuppressed transplant recipients with resulting
B cell proliferation and PTLD [103]. In HCT, the PTLD is most often of
donor cell origin and is usually associated with EBV of recipient cell
origin. The opposite is usually the case in SOT and we were unable to
90
Journal of Clinical Virology 102 (2018) 84–92
prove that active donor EBV replication at time of organ donation in-
creased risk of PTLD [104].
The clinical features of PTLD can be non-specific and usually in-
clude fever, weight loss, fatigue, and malaise. PTLD may also present as
lymph node enlargement anywhere in the body including in the gas-
trointestinal tract, brain, liver, kidney or in SOT recipients, it may be a
mass in the graft itself. Extra-nodal PTLD is also common. Computed
tomography and positron emission tomography imaging can reveal
masses in the abdomen, thorax, and central nervous system, but a tissue
diagnosis is required for confirmation of PTLD. Therapy is determined
by World Health Organization classification: early lesions; polymorphic
PTLD; monomorphic PTLD (which includes B and T cell non-Hodgkin
lymphoma); and classical Hodgkin lymphoma [105]. Treatment stra-
tegies can include reduction of immunosuppression, conversion of im-
munosuppression to sirolimus inclusive regimens, rituximab, che-
motherapy, surgery or radiation, or combinations thereof. There is no
proven role for antiviral drugs in PTLD, although some centers still use
them as additional therapy.
Re-transplantation in PTLD survivors is successful with only one
reported case of PTLD recurrence [106]. Screening for PTLD has fo-
cused on measuring quantitative peripheral blood loads of EBV. In
general, higher EBV loads are present in PTLD versus non-PTLD cases
[107], although we reported a case without any detectable EBV [40].
Unfortunately, quantitative PCR assays for EBV cannot be used to de-
velop threshold values or patterns for intervention because results vary
widely between centers and assay platforms. Rituximab and cytotoxic T
cell infusions have been attempted as preventive measures in HCT but
their role in PTLD prevention remains debatable. Administration of
antiviral drugs to prevent PTLD is also of questionable value [108].
11. Future challenges
Despite being discovered more than 50 years ago, there remain gaps
in our knowledge base about EBV. These gaps urgently need to be filled,
especially regarding the epidemiology and pathogenesis of the virus
since more than 90% of the population eventually becomes infected. Of
particular significance is learning more about EBV infection in children.
Relatively little is known regarding transmission of the virus in children
under twelve years of age. While it is purported in the medical com-
munity that primary infection in this age group is usually asympto-
matic, more research is needed to fully address this issue. Likewise,
further investigation into asymptomatic primary infection in adoles-
cents and young adults warrants attention. Finally, a perennial sticking
point is the lack of a licensed prophylactic vaccine for EBV or, indeed,
an approved specific treatment following infection. Although we have
described both vaccine development and antiviral regimens in this re-
view, the importance of forging ahead in both areas cannot be over-
stated.
Conflicts of interest
The authors, Henry H Balfour, Priya S. Verghese, and Samantha K.
Dunmire, have no potential conflicts of interest or conflicts of interest to
declare.
Disclosure
The authors of this manuscript have no conflicts of interest to de-
clare.
Funding
This article was supported by grants from the University of
Minnesota International Center for Antiviral Research and
Epidemiology, the Richard M. Schulze Family Foundation, the Randy
Shaver Cancer Research and Community Fund, and the University of
S.K. Dunmire et al.
Minnesota Foundation.
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