National Center for Infectious Diseases

Epstein-Barr Virus and

Infectious Mononucleosis
DISEASE INFORMATION
Epstein-Barr virus, frequently referred to as EBV, is a member of the herpesvirus
family and one of the most common human viruses. The virus occurs worldwide, and
most people become infected with EBV sometime during their lives. In the United
States, as many as 95% of adults between 35 and 40 years of age have been infected.
Infants become susceptible to EBV as soon as maternal antibody protection (present at
birth) disappears. Many children become infected with EBV, and these infections
usually cause no symptoms or are indistinguishable from the other mild, brief illnesses
of childhood. In the United States and in other developed countries, many persons are
not infected with EBV in their childhood years. When infection with EBV occurs
during adolescence or young adulthood, it causes infectious mononucleosis 35% to
50% of the time.
Symptoms of infectious mononucleosis are fever, sore throat, and swollen lymph
glands. Sometimes, a swollen spleen or liver involvement may develop. Heart
problems or involvement of the central nervous system occurs only rarely, and
infectious mononucleosis is almost never fatal. There are no known associations
between active EBV infection and problems during pregnancy, such as miscarriages or
birth defects. Although the symptoms of infectious mononucleosis usually resolve in 1
or 2 months, EBV remains dormant or latent in a few cells in the throat and blood for
the rest of the person's life. Periodically, the virus can reactivate and is commonly
found in the saliva of infected persons. This reactivation usually occurs without
symptoms of illness.
EBV also establishes a lifelong dormant infection in some cells of the body's immune
system. A late event in a very few carriers of this virus is the emergence of Burkitt's
lymphoma and nasopharyngeal carcinoma, two rare cancers that are not normally
found in the United States. EBV appears to play an important role in these
malignancies, but is probably not the sole cause of disease.
Most individuals exposed to people with infectious mononucleosis have previously
been infected with EBV and are not at risk for infectious mononucleosis. In addition,
transmission of EBV requires intimate contact with the saliva (found in the mouth) of
an infected person. Transmission of this virus through the air or blood does not
normally occur. The incubation period, or the time from infection to appearance of
symptoms, ranges from 4 to 6 weeks. Persons with infectious mononucleosis may be
able to spread the infection to others for a period of weeks. However, no special
precautions or isolation procedures are recommended, since the virus is also found

frequently in the saliva of healthy people. In fact, many healthy people can carry and
spread the virus intermittently for life. These people are usually the primary reservoir
for person-to-person transmission. For this reason, transmission of the virus is almost
impossible to prevent.
The clinical diagnosis of infectious mononucleosis is suggested on the basis of the
symptoms of fever, sore throat, swollen lymph glands, and the age of the patient.
Usually, laboratory tests are needed for confirmation. Serologic results for persons
with infectious mononucleosis include an elevated white blood cell count, an increased
percentage of certain atypical white blood cells, and a positive reaction to a "mono
spot" test.
There is no specific treatment for infectious mononucleosis, other than treating the
symptoms. No antiviral drugs or vaccines are available. Some physicians have
prescribed a 5-day course of steroids to control the swelling of the throat and tonsils.
The use of steroids has also been reported to decrease the overall length and severity of
illness, but these reports have not been published.
It is important to note that symptoms related to infectious mononucleosis caused by
EBV infection seldom last for more than 4 months. When such an illness lasts more
than 6 months, it is frequently called chronic EBV infection. However, valid laboratory
evidence for continued active EBV infection is seldom found in these patients. The
illness should be investigated further to determine if it meets the criteria for chronic
fatigue syndrome, or CFS. This process includes ruling out other causes of chronic
illness or fatigue.
DIAGNOSIS OF EBV INFECTIONS
In most cases of infectious mononucleosis, the clinical diagnosis can be made from the
characteristic triad of fever, pharyngitis, and lymphadenopathy lasting for 1 to 4
weeks. Serologic test results include a normal to moderately elevated white blood cell
count, an increased total number of lymphocytes, greater than 10% atypical
lymphocytes, and a positive reaction to a "mono spot" test. In patients with symptoms
compatible with infectious mononucleosis, a positive Paul-Bunnell heterophile
antibody test result is diagnostic, and no further testing is necessary. Moderate-to-high
levels of heterophile antibodies are seen during the first month of illness and decrease
rapidly after week 4. False-positive results may be found in a small number of patients,
and false-negative results may be obtained in 10% to 15% of patients, primarily in
children younger than 10 years of age. True outbreaks of infectious mononucleosis are
extremely rare. A substantial number of pseudo-outbreaks have been linked to
laboratory error, as reported in CDC's Morbidity and Mortality Weekly Report, vol. 40,
no. 32, on August 16, 1991.
When "mono spot" or heterophile test results are negative, additional laboratory testing
may be needed to differentiate EBV infections from a mononucleosis-like illness

induced by cytomegalovirus, adenovirus, or Toxoplasma gondii. Direct detection of

EBV in blood or lymphoid tissues is a research tool and is not available for routine
diagnosis. Instead, serologic testing is the method of choice for diagnosing primary
infection.
EBV-Specific Laboratory Tests
Laboratory tests are not always foolproof. For various reasons, false-positive and falsenegative results can occur for any test. However, the laboratory tests for EBV are for
the most part accurate and specific. Because the antibody response in primary EBV
infection appears to be quite rapid, in most cases testing paired acute- and
convalescent-phase serum samples will not demonstrate a significant change in
antibody level. Effective laboratory diagnosis can be made on a single acute-phase
serum sample by testing for antibodies to several EBV-associated antigens
simultaneously. In most cases, a distinction can be made as to whether a person is
susceptible to EBV, has had a recent infection, has had infection in the past, or has a
reactivated EBV infection.
Antibodies to several antigen complexes may be measured. These antigens are the viral
capsid antigen, the early antigen, and the EBV nuclear antigen (EBNA). In addition,
differentiation of immunoglobulin G and M subclasses to the viral capsid antigen can
often be helpful for confirmation. When the "mono spot" test is negative, the optimal
combination of EBV serologic testing consists of the antibody titration of four
markers: IgM and IgG to the viral capsid antigen, IgM to the early antigen, and
antibody to EBNA.
IgM to the viral capsid antigen appears early in infection and disappears within 4 to 6
weeks. IgG to the viral capsid antigen appears in the acute phase, peaks at 2 to 4 weeks
after onset, declines slightly, and then persists for life. IgG to the early antigen appears
in the acute phase and generally falls to undetectable levels after 3 to 6 months. In
many people, detection of antibody to the early antigen is a sign of active infection, but
20% of healthy people may have this antibody for years.
Antibody to EBNA determined by the standard immunofluorescent test is not seen in
the acute phase, but slowly appears 2 to 4 months after onset, and persists for life. This
is not true for some EBNA enzyme immunoassays, which detect antibody within a few
weeks of onset.
Finally, even when EBV antibody tests, such as the early antigen test, suggest that
reactivated infection is present, this result does not necessarily indicate that a patient's
current medical condition is caused by EBV infection. A number of healthy people
with no symptoms have antibodies to the EBV early antigen for years after their initial
EBV infection.
Therefore, interpretation of laboratory results is somewhat complex and should be left
to physicians who are familiar with EBV testing and who have access to the entire

clinical picture of a person. To determine if EBV infection is associated with a current

illness, consult with an experienced physician.
Additional Information about EBV Antibody Tests and Interpretation
Antibody tests for EBV can measure the presence and/or the concentration of at least
six specific EBV antibodies. By evaluating the results of these different tests, the stage
of EBV infection can be determined. However, these tests are expensive and not
usually needed for the diagnosis of infectious mononucleosis.
It is not appropriate for CDC to interpret test results or to handle counseling for the
public. We suggest that questions be directed to a local physician who is familiar with
the patient's history and laboratory test results. In addition, CDC cannot recommend
specific physicians for referral. Our general recommendation is for patients to consult
with an infectious disease specialist or their local or state public health department.
SUMMARY OF INTERPRETATION
The diagnosis of EBV infection is summarized as follows:
Susceptibility
If antibodies to the viral capsid antigen are not detected, the patient is susceptible to
EBV infection.
Primary Infection
Primary EBV infection is indicated if IgM antibody to the viral capsid antigen is
present and antibody to EBV nuclear antigen, or EBNA, is absent. A rising or high IgG
antibody to the viral capsid antigen and negative antibody to EBNA after at least 4
weeks of illness is also strongly suggestive of primary infection. In addition, 80% of
patients with active EBV infection produce antibody to early antigen.
Past Infection
If antibodies to both the viral capsid antigen and EBNA are present, then past infection
(from 4 to 6 months to years earlier) is indicated. Since 95% of adults have been
infected with EBV, most adults will show antibodies to EBV from infection years
earlier. High or elevated antibody levels may be present for years and are not
diagnostic of recent infection.
Reactivation
In the presence of antibodies to EBNA, an elevation of antibodies to early antigen
suggests reactivation. However, when EBV antibody to the early antigen test is
present, this result does not automatically indicate that a patient's current medical
condition is caused by EBV. A number of healthy people with no symptoms have
antibodies to the EBV early antigen for years after their initial EBV infection. Many
times reactivation occurs subclinically.

Chronic EBV Infection

Reliable laboratory evidence for continued active EBV infection is very seldom found
in patients who have been ill for more than 4 months. When the illness lasts more than
6 months, it should be investigated to see if other causes of chronic illness or CFS are
present.

Detection of Epstein-Barr Virus in

Metastatic Lymph
Nodes of Patients With
Nasopharyngeal Carcinoma
and a Primary Unknown Carcinoma
Kazunari Nakao, MD; Tadashi Yuge, MD; Masato Mochiki, MD; Ken-ichi Nibu, MD; Masashi
Sugasawa, MD
Background: Nasopharyngeal carcinoma is often associated
with neck lymph node (LN) metastases, which
in many cases is the only manifestation of this disease.
The submucosal and infiltrative characteristics of nasopharyngeal
carcinoma make this type of cancer difficult
to diagnose. Nasopharyngeal carcinoma has also been
reported to be strongly associated with the Epstein-Barr
virus.
Methods: We examined 36 nasopharyngeal carcinomas
(from 30 primary sites and from 6metastasized LNs),
13 metastasized LNs of other head and neck cancers, and
12 primary unknown neck metastases using an in situ
hybridization technique.
Results: In the nasopharyngeal carcinomas, in situ hybridization
with an Epstein-Barr virusencoded smallRNA
identified the Epstein-Barr virus in 20 (67%) of the 30
primary sites and in 3 (50%) of the 6 metastasized LNs.
Epstein-Barr virus was not detected in metastasized
LNs of other head and neck cancers, but was detected in
1 of the primary unknown neck metastases.
Conclusion: In situ hybridization using a digoxigeninlabeled
Epstein-Barr virusencoded small RNA probe is
useful for the differential diagnosis of metastasized LNs
when the primary site is unknown.
Arch Otolaryngol Head Neck Surg. 2003;129:338-340

ASOPHARYNGEAL

carcinoma

(NPC) is one of the

most common malignancies
in southern China
and Taiwan, accounting
for up to 18% of all cancers.1 However, it
is rare in Japan, North America, and northern
Europe. Nasopharyngeal carcinoma affects
women more than other head and
neck malignancies, and also tends to affect
younger age groups compared with most
other cancers. Five-year survival rates for
NPC range from 30% to 48%. Nasopharyngeal
carcinoma is frequently accompanied
by neck lymph node (LN) metastasis,
which in many instances is the only
manifestation of this disease. An endoscopic
examination or a biopsy of the nasopharynx
often fails to detect the primary
site because of the submucosal and
infiltrative characteristics of NPC.2 This
difficulty in diagnosing the primary site
may result in suboptimal therapy. Thus,
development of a sensitive, specific, and
reliable procedure to identify NPC inmetastasized
LNs will allow early diagnosis
of NPC and optimal therapeutic treatment
of this tumor.
The strong association of NPC with
the Epstein-Barr virus (EBV) is well documented.
3-6 The significant relationship between
the presence of EBV in neck metastasis
and primary NPC has been shown
by in situ hybridization (ISH) and the polymerase
chain reaction.7-12 Previous studies
have detected EBV in metastatic LNs
of NPC, but not in other primary sites. Encouraged
by these reports, we attempted
to detect the presence of EBV using ISH
with an EBV-encoded small RNA (EBER)
in the neck metastases of patients with primary
unknown squamous cell carcinoma
(SCC). This study tests the efficacy
of this method for identifying the nasopharyngeal
histogenesis of metastases of
unknown primary tumors.
METHODS
MATERIALS

Paraffin-embedded specimens of NPC and primary

unknown neck LN metastases were randomly
selected from patients treated at the Department
of OtolaryngologyHead and Neck
Surgery, University of Tokyo Hospital, Tokyo,
Japan, from January 1, 1987, to December 31,
1999. Thirty-six of the specimens were NPCs
(from 30 primary tissues and 6 LNs) and 12
were neck metastases of primary unknown
SCCs. Thirteen specimens of metastases from

ORIGINAL ARTICLE
From the Departments of
OtolaryngologyHead and
Neck Surgery, Graduate School
of Medicine, The University of
Tokyo, Tokyo (Drs Nakao,
Yuge, Mochiki, and Sugasawa),
and Kobe University Graduate
School of Medicine, Kobe
(Dr Nibu), Japan.
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other head and neck cancers were also used as negative controls.
The pathological diagnoses of these specimens according
to Union Internationale Contre le Cancer criteria are
summarized in the Table.
ISH TECHNIQUE
Serial sections (5 m) were cut from formaldehyde-fixed paraffinembedded specimens and mounted on silane-coated slides
(Dako Japan, Tokyo). Paraffin was removed from the sections,
and they were rehydrated by means of a xylene and alcohol
series. In situ hybridization was performed using an ISH
kit (Rembrandt; Kreatech, Amsterdam, the Netherlands) according
to the manufacturers protocol. Briefly, sections were
first predigested with pepsin solution at 37C for 30 minutes.
Sections were then hybridized with either EBV nuclear antigen
1 or an EBER oligonucleotide probe at 37C for 2 hours to
detect EBV DNA or messenger RNA (mRNA), respectively. Alkaline
phosphataseconjugated digoxigenin was applied for
30 minutes at 37C for detection, and nitroblue tetrazolium/
5-bromo-4-chloro-3-indolyl phosphate, p-toluidine salt substrate
was applied for 10 minutes at 37C and used as a chromogen.
Sections were counterstained with nuclear fast red.
STATISTICAL ANALYSIS
The Fisher exact test was used to evaluate the statistical
significance in the frequencies of EBV according to the pathological
classifications.
RESULTS

In this study, we first performed ISH using an EBV nuclear

antigen 1 probe to identify EBVDNAon 22 primary specimens
of NPCs (18 undifferentiated and 4 poorly differentiated
carcinomas) as a pilot study. However, an EBV
nuclear antigen 1 signal was identified in only 1 (5%) of
the undifferentiated carcinomas (UNPs). Because this frequency
was significantly lower than we expected, we instead
performed EBER ISH. The EBER signals were identified
in 20 of the 30 primary sites (Table). In the primary
lesions, the association of EBV was significantly higher
in the UNPs, 15 of 18, than in the poorly differentiated

carcinomas, 3 of 8 (P=.06). As expected, an EBER signal

was not detected in an adenoid cystic carcinoma.
A similar tendency toward a higher EBV association
with UNPs was observed in neck metastases of NPC. An
EBER signal was detected in 3 of 4 UNPs, but not in a
poorly differentiated SCC (Table). The EBER-ISH signal
in metastatic LNs was localized in the nuclei of most
of the tumor cells, but was not seen in adjacent lymphocytes
(Figure, A).
As for the neck metastases of primary unknown
SCCs, an EBER signal was detected in only 1 of 12 specimens
(Figure, B). This patient presented to our clinic with
a single metastasis on the left side of the neck. An excisional
biopsy was performed, and the pathological diagnosis
of the tumor was poorly differentiated SCC. There
were no abnormal findings on an endoscopic examination,
and a computed tomographic scan did not show any
additional LN metastases. Because we were unable to detect
the primary site, the patient was treated with radiotherapy.
She has been followed up for 2 years, with no
evidence of disease. No EBER signals were detected in
any of the neck metastases of the other head and neck
cancers.
In Situ Hybridization With an EBER in Patients
With NPC and Primary Unknown Carcinoma

Type of Ca
No. of
Patients
EBER
Signal Detected*
NPC
Primary lesion 30 20 (67)
Undifferentiated Ca 18 15 (83)
Nonkeratinizing Ca 8 3 (38)
Keratinizing SCC 3 2 (67)
Adenoid cystic Ca 1 0
LN 6 3 (50)
Undifferentiated Ca 4 3 (75)
Poorly differentiated SCC 2 0
Primary unknown SCCs (LN) 12 1 (8)
Oropharyngeal SCC (LN) 8 0
Poorly differentiated SCC
of the tongue (LN)
10
Cancer of the nasal cavity (LN) 2 0
Transitional cell Ca 1 0
Olfactory neuroblastoma 1 0
Hodgkin disease (LN) 1 0
Abbreviations: Ca, carcinoma; EBER, Epstein-Barr virusencoded small
RNA; LN, neck lymph node metastasis; NPC, nasopharyngeal carcinoma;
SCC, squamous cell carcinoma.
*Data are given as number (percentage) of patients.
A
B

In situ hybridization of neck metastases with an Epstein-Barr virusencoded

small RNA. Positive signals are seen in the nuclei of tumor cells, but not in
adjacent lymphocytes. A, Neck metastasis of an undifferentiated carcinoma
of the nasopharynx (hematoxylin, original magnification _100). B, Neck
metastasis of a primary unknown squamous cell carcinoma (hematoxylin,
original magnification _400).
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COMMENT

The strong association of NPC with EBV has been shown

by various methods, including immunoblotting, nucleic
acid hybridization, ISH, and polymerase chain reaction.
3-6 Among them, polymerase chain reaction and ISH
have been reported as sensitive methods for detecting EBV
in NPCs. Of these 2 methods, ISH has the advantage of
being able to precisely localize EBV in tumor cells. 11 In
our study, ISH signals were observed in the nuclei of tumor
cells, but not in adjacent lymphocytes. Recently, the
trend for using ISH to detect EBV has changed from EBV
DNA to EBERs.10,11,13,14 The abundance and stability of
these mRNAs provide a higher sensitivity than DNA ISH,
as shown by the results of the present study. DNA ISH
using an EBV nuclear antigen 1 probe detected EBV in
only 1 of 22 specimens, whereas RNA ISH using an EBER
probe detected 22 of 30 specimens in primary sites, suggesting
the significantly higher sensitivity of RNA ISH.
The close association of nonkeratinizing carcinoma
and UNP with EBV is widely accepted,15 but the
association of keratinizing SCC with EBV is still controversial.
7,8,10,16,17 In this study, we detected EBV in 2 of 3
keratinizing SCCs. On the other hand, a marginally significant
difference (P=.06) was found in the frequency
of EBV between nonkeratinizing carcinoma (3 [38%] of
8) and UNP (15 [83%] of 18). This variation of EBV association
with NPC may reflect technical factors and real
geographic variations. Further studies with a larger population
are needed to examine this issue.
This study tested the efficacy of the EBER-ISH technique
in identifying the nasopharyngeal histogenesis of
neck metastases of unknown primary tumors. The EBER
signals were found in the nuclei of LN metastases, but
not in host lymphocytes, in accordance with previous reports.
In addition, an EBER signal was detected in 1 of
the metastatic SCCs of unknown origin, but in none of
the 13 LNs with other types of cancer. These observations
suggest that EBER ISH may be used as a supplemental
tool for the differential diagnosis of a neck
mass when the primary site is unknown. Recently,
Lee et al14 succeeded in detecting EBV by the EBER-ISH
technique in fine-needle aspiration cytologic specimens
from neck metastases of NPCs. This assay is a specific,
sensitive, and nonradioisotopic way to confirm nasopharyngeal
origin and should be routinely performed on neck
metastases with an unknown primary site.
Accepted for publication August 7, 2002.
This study was supported in part by grants 11671671
and 14370543 from the Japanese Ministry of Education, Culture,
Sports, Science, and Technology, Tokyo; and by a Health
Science Research Grant on Comprehensive Research on
Aging and Welfare 13-012 from the Japanese Ministry of
Health, Labour, and Welfare, Tokyo.
This study was presented at the Fifth International
Conference on Head and Neck Cancer, San Francisco, Calif,
July 30, 2000.

Corresponding author and reprints: Ken-ichi Nibu, MD,

Department of OtolaryngologyHead and Neck Surgery,
Kobe University Graduate School of Medicine, 7-5-1
Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan (e-mail:
nibu@med.kobe-u.ac.jp).
REFERENCES

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2003

The oncogenic properties of the principal EBV oncoprotein, Latent Membrane Protein 1 (LMP1), include the ability to induce invasiveness and metastasis factors. We have shown that LMP-1
induces matrix metalloproteinase 9 (MMP-9), a type IV collagenase that disrupts basement
membrane. Also, cyclooxygenase-2 (COX-2), which is overexpressed in diverse malignancies, is
induced by LMP-1; the enzyme is functional, and co-expressed with LMP-1 in NPC. Inhibitors
of the NF kappa B signaling pathway, which is activated by LMP-1, including I kappa B superrepressor and aspirin reduce or cancel induction of MMP-9, COX-2 and invasiveness of LMP-1expressing cells. Production of VEGF, also induced by LMP-1, is decreased by a COX-2-specific
inhibitor. We now show that LMP-1 induces expression of the angiogenic Fibroblast Growth
Factor-2 (FGF-2). Furthermore, LMP-1 also causes secretion of the 18 kDa isoform of this
protein--a newly identified function for LMP-1. Secretion of FGF-2 is independently signaled

through the NF-kappa B pathway. Release of the protein is not dependent on the classical
ER/Golgi secretory pathway, but secretion of FGF-2 is suppressed by ouabain, an inhibitor of the
Na+/K(+)-ATPase alpha 1 subunit. Finally LMP-1 induces expression of Hypoxia-Inducible
Factor-1 alpha (HIF-1 alpha), which mediates adaptation of cells to O2-depleted states. Thus
LMP-1 is not only directly oncogenic, it can induce a constellation of factors that reveal the
additional role of EBV in invasive cancers such as NPC. Alteration of cellular phenotype
independent of transforming effects may be a property of other tumor viruses.
PMID:
12894587
[PubMed - indexed for MEDLINE]