1. Epstein-Barr virus (EBV) is a human herpesvirus that can establish lifelong latent infections in B cells. EBV is associated with several cancers and diseases including Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma, and infectious mononucleosis. 2. The document discusses the history and discovery of EBV and the diseases it causes. It describes infectious mononucleosis caused by primary EBV infection and provides details on its symptoms, diagnosis using heterophile antibodies, and typical laboratory findings. 3. Burkitt's lymphoma and nasopharyngeal carcinoma are also covered, noting their association with EBV and key characteristics. Molecular details of EBV replication and

1. Dr.T.V.Rao MD
EPSTEIN-BARR VIRUS
DR.T.V.RAO MD 1

2. EPSTEIN-BARR VIRUS : HISTORY
• In 1889, German physician “Pfeiffer”
• fever
• lymphadenopathy
• malaise
• Hepatosplenomegaly
• abdominal discomfort in adolescents and young adults
• In England, “DrÜsenfieber,” or glandular fever
• In the early 1900s
• numerous case descriptions of illnesses
epidemiologically and clinically compatible with IM.
FEIGIN et al. Textbook of Pediatric Infectious Diseases5th ed;2004:1952-1957.
DR.T.V.RAO MD 2

3. EPSTEIN-BARR VIRUS : HISTORY
• In 1920, Sprunt and Evans
• published cases of spontaneously resolving acute
leukemia associated with blast-like cells in the
blood
• In 1923, Downey and McKinlay
• detailed description of the lymphocyte morphology.
• In 1932, Paul and Bunnell
• Identified heterophile antibodies in serum during
acute IM.
FEIGIN et al. Textbook of Pediatric Infectious Diseases5th ed;2004:1952-1957.
DR.T.V.RAO MD 3

4. • Epstein-Barr virus (EBV) is a
human γ-herpesvirus that is
able to establish a long-
term, latent infection in human
B cells for the life of the host.
EBV infection is associated
with a range of human
cancers, including Burkitt’s
lymphoma (BL) and Hodgkin’s
disease, as well as several
AIDS-associated cancers of
which the most prevalent is
diffuse large B-cell lymphoma
(DLBCL).
IMPORTANCE OF EPSTEIN BARR VIRUS
DR.T.V.RAO MD 4

5. EBV in B Cell
Infectious mononucleosis
X-Linked Lymphoproliferative Disease
Chronic active EBV
Hodgkin Disease
Burkitt Lymphoma
Lymphoproliferative disease
EBV in Other Cells
Nasopharyngeal carcinoma
Gastric carcinoma
Nasal T/NK cell lymphomas
Peripheral T cell lymphomas
Oral hairy leukoplakia
Smooth muscle tumors in transplant patients
Diseases Associated with EBV
DR.T.V.RAO MD 5

6. Diseases Driven by Epstein-Barr Virus
Infectious mononucleosis
Chronic Active EBV
X-linked lymphoproliferative disease
Lymphoproliferative disease
Oral hairy leukoplakia
Hodgkin disease EBV EBV-Driven
Nasopharyngeal carcinoma Gene Cell
T cell lymphoma Expression Proliferation
Burkitt lymphoma
DR.T.V.RAO MD 6

7. EPSTEIN BARR VIRUS INFECTION ALSO KNOWN AS
‘THE KISSING DISEASE"
DR.T.V.RAO MD 7

8. BURKITTS LYMPHOMA
DR.T.V.RAO MD 8

9. NASOPHARYNGEAL CARCINOMA
DR.T.V.RAO MD 9

10. EPSTEIN-BARR VIRUS (EBV)
• Belong to the gammaherpesvirus subfamily of herpes viruses
• Nucleocapsid 100 nm in diameter, with 162 capsomers
• Membrane is derived by budding of immature particles
through cell membrane and is required for infectivity.
• Genome is a linear double stranded DNA molecule with 172
kbp
• The viral genome does not normally integrate into the
cellular DNA but forms circular episomes which reside in the
nucleus.
• The genome is large enough to code for 100 - 200 proteins
but only a few have been identified.
DR.T.V.RAO MD 10

11. PATHOGENESIS
• Once infected, a lifelong carrier state develops whereby a low
grade infection is kept in check by the immune defenses.
• Low grade virus replication and shedding can be demonstrated in
the epithelial cells of the pharynx of all seropositive individuals.
• EBV is able to immortalize B-lymphocytes in vitro and in vivo
• Furthermore a few EBV-immortalized B-cells can be
demonstrated in the circulation which are continually cleared by
immune surveillance mechanisms.
• EBV is associated with several very different diseases where it
may act directly or one of several co-factors.
DR.T.V.RAO MD 11

12. DISEASE ASSOCIATION
1. Infectious Mononucleosis
2. Burkitt's lymphoma
3. Nasopharyngeal carcinoma
4. Lymphoproliferative disease and lymphoma in the
immunosuppressed.
5. X-linked lymphoproliferative syndrome
6. Chronic infectious mononucleosis
7. Oral leukoplakia in AIDS patients
8. Chronic interstitial pneumonitis in AIDS patients.
DR.T.V.RAO MD 12

13. INFECTIOUS
MONONUCLEOSIS
• Primary EBV infection is usually subclinical in childhood. However in
adolescents and adults, there is a 50% chance that the syndrome of
infectious mononucleosis (IM) will develop.
• IM is usually a self-limited disease which consists of
fever, lymphadenopathy and splenomegaly. In some patients jaundice may
be seen which is due to hepatitis. Atypical lymphocytes are present in the
blood.
• Complications occur rarely but may be serious e.g. splenic
rupture, meningoencephalitis, and pharyngeal obstruction.
• In some patients, chronic IM may occur where eventually the patient dies of
lymphoproliferative disease or lymphoma.
• Diagnosis of IM is usually made by the heterophil antibody test and/or
detection of EBV IgM.
• There is no specific treatment.
DR.T.V.RAO MD 13

14. DR.T.V.RAO MD 14

15. INFECTIOUS MONONUCLEOSIS
Exudative pharyngotonsillitis
DR.T.V.RAO MD 15

16. INFECTIOUS MONONUCLEOSIS
Hepatosplenomegaly
Cervical
lymphadenopathy
DR.T.V.RAO MD 16

17. INFECTIOUS MONONUCLEOSIS
IM with rash after treatment with amoxicillin or ampicillin
NEJM;343:481-492.
DR.T.V.RAO MD 17

18. INFECTIOUS MONONUCLEOSIS
• Acute infectious mononucleosis
• fatigue and malaise 1-2 wks
• sore throat, pharyngitis
• retro-orbital headache
• fever
• myalgia
• nausea
• abdominal pain
• generalized lymphadenopathy
• Hepatosplenomegaly
DR.T.V.RAO MD 18

19. INFECTIOUS MONONUCLEOSIS
atypical lymphocytes : Downey types
DR.T.V.RAO MD 19

20. BURKITT'S LYMPHOMA
• Burkitt's lymphoma (BL) occurs endemically in parts of Africa
(where it is the commonest childhood tumor) and Papua New
Guinea. It usually occurs in children aged 3-14 years. It
respond favorably to chemotherapy.
• It is restricted to areas with holoendemic malaria. Therefore it
appears that malaria infection is a cofactor.
• Multiple copies of EBV genome and some EBV antigens can be
found in BL cells and patients with BL have high titres of
antibodies against various EBV antigens.
DR.T.V.RAO MD 20

21. EBV+: 90% of cases in developing countries – jaw tumors
20% cases in US – children with abdominal tumors
AIDS patients – tumors in lymph nodes
EBV may be one “hit” but all tumors have c-myc
translocations
Dysregulation of c-myc oncogene
Only EBV EBNA-1 expressed
Therapy: Chemotherapy
Burkitt Lymphoma
DR.T.V.RAO MD 21

22. BURKITT’S LYMPHOMA
• BL cells show a reciprocal translocation between the long arm of
chromosome 8 and chromosomes 14, 2 or 22.
• This translocation result in the c-myc oncogene being transferred
to the Immunoglobulin gene regions. This results in the
deregulation of the c-myc gene. It is thought that this
translocation is probably already present by the time of EBV
infection and is not caused by EBV.
• Sporadic cases of BL occur, especially in AIDS patients which
may or may not be associated with EBV.
• In theory BL can be controlled by the eradication of malaria (as
has happened in Papua New Guinea) or vaccination against EBV.
DR.T.V.RAO MD 22

23. B-CELL LYMPHOMA
• In most individuals infected with EBV, the virus is
present in the B-cells, which are normally
controlled by T-lymphocytes
• When T-cell deficiency exists, one clone of EBV-
infected B-lymphocytes escapes immune
surveillance to become autonomously proliferating.
• EBV induced B cell lymphomas are most
prevalent in immunocompromised patients.
DR.T.V.RAO MD 23

24. EBV+: 60-70% of cases in
developing countries
35-50% cases in US
EBV in Reed-Sternberg cells
Therapy: Chemotherapy,
radiation Anti-EBV CTLs
effective in some cases
Hodgkin Disease
LMP-1 expression
DR.T.V.RAO MD 24

25. NASOPHARYNGEAL CARCINOMA
• Nasopharyngeal carcinoma (NPC) is a malignant tumor of the squamous
epithelium of the nasopharynx. It is very prevalent in S. China, where it is
the commonest tumor in men and the second commonest in women.
• The tumor is rare in most parts of the world, though pockets occur in N.
and C. Africa, Malaysia, Alaska, and Iceland.
• Multiple copies of EBV genome and EBV EBNA-1 antigen can be found
in cells of undifferentiated NPC. Patients with NPC have high titers of
antibodies against various EBV antigens.
• Besides EBV there appears to be a number of environmental and genetic
cofactors in NPC.
• NPC usually presents late and thus the prognosis is poor.
• In theory NPC can be prevented by vaccination.
DR.T.V.RAO MD 25

26. IMMUNOCOMPROMISED PATIENTS
• After primary infection, EBV maintains a steady low grade latent
infection in the body. Should the person become
immunocompromised, the virus will reactivate. In a few
cases, lymphoproliferative lesions and lymphoma may develop. These
lesions tend to be extranodal and in unusual sites such as the GI tract
or the CNS.
• Transplant recipients e.g. renal - EBV is associated with the
development of lymphoproliferative disease and lymphoma.
• AIDS patients - EBV is associated with oral leukoplakia and with
various Non-Hodgkin's lymphoma.
• Duncan X-linked lymphoproliferative syndrome - this condition occurs
exclusively in males who had inherited a defective gene in the X-
chromosome . This condition accounts for half of the fatal cases of IM.
DR.T.V.RAO MD 26

27. MOLECULAR BIOLOGY : REPLICATION
• To infect cells, EBV uses a cell surface receptor
(CR2,CD21) found primarily on B lymphocytes
and nasopharyngeal epithelial cells.
• MHC class II protein functions as a cofactor for
this virus-receptor interaction.
• After infection of epithelial cells, active
replication occurs and leads to lysis and death
of the cell.
DR.T.V.RAO MD 27

28. MOLECULAR CONFIGURATION OF
EPSTEIN BARR VIRUS
DR.T.V.RAO MD 28

29. MOLECULAR BIOLOGY : REPLICATION
• Viral capsid antigens (VCAs) are the primary
structure proteins in viral capsids and are found in
replicating cells.
• EBV early antigens (EAs) consist of >15 proteins
codes by genes distributed throughout the
genome.
• EBV nuclear antigen (EBNA) corresponds to six
virally encoded proteins found in the nucleus of an
EBV-infected cell.
DR.T.V.RAO MD 29

30. MOLECULAR BIOLOGY : LATENCY
• Latently infected B cells are the primary
reservoir of EBV in the body.
• >100 gene products may be expressed during
active viral replication, only 11 are expressed
during viral latency.
• In this way, the virus limits cytotoxic T-cell
recognition of EBV-infected cells.
DR.T.V.RAO MD 30

31. DIAGNOSIS
• Acute EBV infection is usually made by the heterophil antibody test
and/or detection of anti-EBV VCA IgM.
• Cases of Burkitt’s lymphoma should be diagnosed by histology. The
tumour can be stained with antibodies to lambda light chains which
should reveal a monoclonal tumour of B-cell origin. In over 90% of
cases, the cells express IgM at the cell surface.
• Cases of NPC should be diagnosed by histology.
• The determination of the titre of anti-EBV VCA IgA in
screening for early lesions of NPC and also for
monitoring treatment.
• A patient with with non-specific ENT symptoms who have elevated
titers of EBV IgA should be given a thorough examination.
DR.T.V.RAO MD 31

32. INFECTIOUS MONONUCLEOSIS :
LAB
• Heterophile antibodies
• 50% in first week of illness
• 60-90% in the second or third weeks
• begins to decline during the fourth or fifth week and
often is less than 1:40 by 2-3 months after
symptom onset
• 20% of patients have positive titers 1-2 years after
acquisition
• children < 2 years : 10-30%
• children 2-4 years : 50-75%

33. PRIMARY EBV INFECTION :
SEROPREVALENCE
• In developing countries -80-100% of children becoming
infected by 3-6 yrs of age
-clinically silent or mild disease.
• In developed countries
-occurs later in life, 10-30 years of age induce
clinically mononucleosis syndrome (U.S.college students
: 50-75% associated with primary EBV infection)
Hickey SM et al. Pediatr Clin North Am. Dec 1997;44(6):1541-56.
DR.T.V.RAO MD 33

34. INFECTIOUS MONONUCLEOSIS : LAB
• Time course of antibody production
• EA is rising at symptom onset : rise for 3-4 weeks, then
quickly decline to undetectable levels by 3-4
months, although low levels may be detected intermittently
for years
• VCA-IgM usually is measurable at symptom onset, peaks at
2-3 weeks, then declines and unmeasurable by 3-4 months
• VCA-IgG rises shortly after symptom onset, peaks at 2-3
months, then drops slightly but persists for life
• EBNA : convalescence and remain present for life

35. SERUM EPSTEIN-BARR VIRUS (EBV)
ANTIBODIES IN EBV INFECTION
Infection VCA
IgG
VCA
IgM
EA(D) EBNA
No previous
infection
- - - -
Acute
infection
Recent
infection
+
+
+
+/-
+/-
+/-
-
+/-
Past
infection
+ - +/- +
AAP. Red book2006;286-288.

36. DIAGNOSTIC APPROACH
• Examination of the blood usually shows an increase in the
white blood cells, due to the appearance of many atypical
lymphocytes in the blood.
• Blood serum in IM often contains an antibody known as
heterophile antibody that agglutinates, or clumps, the red blood
cells of sheep.
• Heterophile antibodies are antibodies that are stimulated by
one antigen and react with an entirely unrelated surface
antigen present on cells from different mammalian species.
DR.T.V.RAO MD 36

37. DIAGNOSIS
• Heterophile antibody titers rise during the first two or three weeks with
half or more developing a significant titer during the first week of illness.
• The level of antibody gradually declines and usually disappears in eight
to twelve weeks following the onset.
• Elevated titers sometimes linger for four to six months up to a year or
more.
• Heterophile antibody most commonly used in the serological diagnosis
of IM is an IgM antibody which agglutinates sheep red blood cells.
DR.T.V.RAO MD 37

38. DIAGNOSIS
• The original Paul-Bunnell test was a simple titration of sheep cell
agglutinins but this procedure was subsequently modified in order to
distinguish between sheep cell agglutinins formed in IM and the
Forssmann-type antibodies found in normal serum, serum sickness and
in certain other conditions.
• Tissues rich in Forssmann antigen (guinea pig kidney) absorb
Forssmann antibodies but do not affect the heterophile antibodies in IM.
• Heterophile antibodies are absorbed by beef cells,
• Forssmann Hapten is a glycolipid usually associated with a protein, the
determinant being largely carbohydrate and therefore heat stable.
DR.T.V.RAO MD 38

39. PAUL BUNNELL TEST
• The original Paul-Bunnell test was a simple titration of sheep cell
agglutinins but this procedure was subsequently modified in order
to distinguish between sheep cell agglutinins formed in IM and the
Forssmann-type antibodies found in normal serum, serum sickness
and in certain other conditions.
• Tissues rich in Forssmann antigen (guinea pig kidney) absorb
Forssmann antibodies but do not affect the heterophile antibodies
in IM.
• Heterophile antibodies are absorbed by beef cells,
• Forssmann Hapten is a glycolipid usually associated with a
protein, the determinant being largely carbohydrate and therefore
heat stable.
DR.T.V.RAO MD 39

40. DAVIDSOHN DIFFERENTIAL
• The principle behind the Paul-Bunnell-Davidsohn test is that the two
types of sheep agglutinins are distinguished by titrating them before
and after absorption with guinea pig kidney and ox cells.
• Patients serum containing antibodies due to IM is added to guinea pig
kidney cells. These antibodies are not absorbed by the kidney cells.
These antibodies then react with Beef (Ox) red blood cells which
causes agglutination and is a positive test for IM.
• Patients serum containing Forssmann antibodies are added to guinea
pig kidney cells. Antibodies are absorbed by the kidney cells. These
antibodies are then allowed to react with Beef red blood cells which
does not cause agglutination. This is a positive test for Forssmann
antigens.
DR.T.V.RAO MD 40

41. DAVIDSOHN DIFFERENTIAL
* TO BE CONSIDERED ABSORBED THERE MUST BE GREATER THAN A THREE TUBE
DIFFERENCE BETWEEN THE PRESUMPTIVE TITER AND THE DIFFERENTIAL TITER.
Heterophil Antibody
------------------------
Infectious Mono
Kidney Extract
------------------
Not Absorbed
Beef Erythrocyte
---------------------
Absorbed
Forssman Absorbed Not Absorbed
Serum Sickness Absorbed Absorbed
DR.T.V.RAO MD 41

42. DAVIDSOHN DIFFERENTIAL
Advantages
• When properly performed, this test is
specific for Infectious Mononucleosis
and false-positive results are rare.
Disadvantages
• Davidsohn Differential test is very
time consuming and burdensome.
DR.T.V.RAO MD 42

43. • Laboratory detection of EBV is
accomplished in several ways
, and recent progress has
focused on the molecular
analysis of viral DNA and RNA.
In situ hybridization has long
been considered the gold
standard for detecting tumour-
associated viral infection, and
EBV viral load assays are now
being adopted for clinical
evaluation of tumour burden in
affected patients.
RECENT ADVANCES IN DIAGNOSIS
DR.T.V.RAO MD 43

44. IM TREATMENT
Medical Care
• self-limited illness : not require specific therapy
• Inpatient therapy of medical and surgical complications
may be required
• Acyclovir (10 mg/kg/dose IV q8h for 7-10 d)
• inhibit viral shedding from the oropharynx
• clinical course is not significantly
• IVIG (400 mg/kg/d IV for 2-5 d)
• immune thrombocytopenia associated with
Andersson J et al. J Infect Dis. Feb 1986;153(2):283-90.
Cyran EM et al. Am J Hematol. Oct 1991;38(2):124-9.
DR.T.V.RAO MD 44

45. EPIDEMIOLOGY
• Two epidemiological patterns are seen with EBV.
• In developed countries, 2 peaks of infection are
seen : the first in very young preschool children
aged 1 - 6 and the second in adolescents and young
adults aged 14 - 20 Eventually 80-90% of adults are
infected.
• In developing countries, infection occurs at a much
earlier age so that by the age of two, 90% of
children are seropositive.
• The virus is transmitted by contact with saliva, in
particularly through kissing.
DR.T.V.RAO MD 45

46. EPSTEIN BARR VIRUS INFECTION
CONTINUES TO BE IMPORTANT
• In addition to clinical signs and symptoms, laboratory testing is necessary to establish
or confirm the diagnosis of IM. This can provide important information for both the
diagnosis and management of EBV-associated disease.
• If the classic signs and symptoms of IM are absent, a diagnosis of IM is more difficult
to make. A definite diagnosis of IM can be established by serologic antibody testing.
The antibodies present in IM are heterophile and EBV antibodies.
• EBV is widely disseminated. It is estimated that 95% of world’s population is exposed
to the virus, which makes it the most ubiquitous virus known to man.
• EBV is only a minor problem for immunocompetent persons, but it can become a
major one for immunologically compromised patients
• After primary exposure a person is considered to be immune and generally no longer
susceptible to overt reinfection.
DR.T.V.RAO MD 46

47. VISIT FOR MORE ARTICLES OF INTEREST ON
INFECTIOUS DISEASES
DR.T.V.RAO MD 47

48. DR.T.V.RAO MD 48
• Programme Created By Dr.T.V.Rao MD
for Medical and Paramedical Students in
the Developing World
• Email
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