EPSTEIN-BARR VIRUS

Dr.T.V.Rao MD

DR.T.V.RAO MD

EPSTEIN-BARR VIRUS : HISTORY

In 1889, German physician Pfeiffer
fever lymphadenopathy malaise Hepatosplenomegaly abdominal discomfort in adolescents and young adults

In England, Drsenfieber, or glandular fever In the early 1900s

numerous case descriptions of illnesses epidemiologically and clinically compatible with IM.
DR.T.V.RAO MD

FEIGIN et al. Textbook of Pediatric Infectious Diseases5th ed;2004:1952-1957.

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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.
DR.T.V.RAO MD

FEIGIN et al. Textbook of Pediatric Infectious Diseases5th ed;2004:1952-1957.

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IMPORTANCE OF EPSTEIN BARR VIRUS

Epstein-Barr virus (EBV) is a human -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 Burkitts lymphoma (BL) and Hodgkins disease, as well as several AIDS-associated cancers of which the most prevalent is diffuse large B-cell lymphoma (DLBCL).
DR.T.V.RAO MD

Diseases Associated with EBV

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 DR.T.V.RAO MD

Diseases Driven by Epstein-Barr Virus

Infectious mononucleosis Chronic Active EBV X-linked lymphoproliferative disease Lymphoproliferative disease Oral hairy leukoplakia
Hodgkin disease Nasopharyngeal carcinoma T cell lymphoma Burkitt lymphoma DR.T.V.RAO MD EBV Gene Expression EBV-Driven Cell Proliferation
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EPSTEIN BARR VIRUS INFECTION ALSO KNOWN AS

THE KISSING DISEASE"

DR.T.V.RAO MD

DR.T.V.RAO MD

BURKITTS LYMPHOMA

DR.T.V.RAO MD

NASOPHARYNGEAL CARCINOMA

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

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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

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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

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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

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DR.T.V.RAO MD

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INFECTIOUS MONONUCLEOSIS

Exudative pharyngotonsillitis
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INFECTIOUS MONONUCLEOSIS

Cervical lymphadenopathy DR.T.V.RAO MD

Hepatosplenomegaly
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INFECTIOUS MONONUCLEOSIS

IM with rash after treatment with amoxicillin or ampicillin

DR.T.V.RAO MD

NEJM;343:481-492.

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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

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INFECTIOUS MONONUCLEOSIS

atypical lymphocytes : Downey types

DR.T.V.RAO MD

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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

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Burkitt Lymphoma
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

DR.T.V.RAO MD

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BURKITTS 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

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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 EBVinfected 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

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Hodgkin Disease
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
LMP-1 expression
DR.T.V.RAO MD

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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

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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 Xchromosome . This condition accounts for half of the fatal cases of IM.
DR.T.V.RAO MD

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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

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MOLECULAR CONFIGURATION OF EPSTEIN BARR VIRUS

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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

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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

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DIAGNOSIS
Acute EBV infection is usually made by the heterophil antibody test and/or detection of anti-EBV VCA IgM.

Cases of Burkitts 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

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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%

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

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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.

Infection

SERUM EPSTEIN-BARR VIRUS (EBV) ANTIBODIES IN EBV INFECTION

VCA IgG VCA IgM EA(D) EBNA

No previous infection
Acute infection

+/-

Recent infection
Past infection

+
+

+/-

+/+/-

+/+

AAP. Red book2006;286-288.

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

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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

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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

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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

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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.
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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
DR.T.V.RAO MD

Absorbed
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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

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RECENT ADVANCES IN DIAGNOSIS

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 tumourassociated viral infection, and EBV viral load assays are now being adopted for clinical evaluation of tumour burden in affected patients.

DR.T.V.RAO MD

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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.
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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

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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 worlds 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

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

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