EPSTEIN-BARR VIRUS: INFECTIOUS MONONUCLEOSIS

Epstein-Barr Virus Family- Herpeviridae, DNA virus Surrounded by a protein capsid, an amorphous tegument, and outer envelope. 180-200 nm in diameter Carries within it a genome that encodes about 80 proteins. 2 classes: a. EBV-A - Found primarily with B cells of immunocompetent hosts. b. EBV-B - Isolated mainly from B cells of immunocompromised patients, suffering from a . Of EBV-related diseases.

PATHOGENECITY One of the most prevalent human viruses. Causative agent of KISSING DISEASE INFECTIOUS MONONUCLEOSIS - Formerly called glandular fever, characterized with fever, lymphadenopathy and prostration. - LYMPHOPROLIFERATIVE DISEASES - Strongly associated with 2 cancers: BURKITTS LYMPHOMA NASOPHARYNGEAL CARCINOMA: Oral Hairy Leukoplakia is indicative of N Carcinoma

1. TRANSMISSION Oral contact with infected saliva. Less frequently through blood transfusion and perinatal route. 2. EBV initially infects the OROPHARYNGEAL EPITHELIAL CELLS where it enters a lytic cycle, characterized by viral replication, lysis of infected cell and release of infectious virions until the acute infection is resolved. 3. The B CELLS in the lymphoid tissue of oropharynx are also infected. The infection cycle begins when the EBV envelope glycoprotein gp350/220 recognizes and binds to the receptor for CD3 (variously known as CR2 or CD21) on the surface of B cells. Uses B cell to manufacture. 4. The virus replicates and disseminates throughout the RES during the 30-to-50 day incubation period of disease. The virus replicates only within the B cells and nasopharyngeal epithelial cells. No overt cytopathic effects are seen. The B cells become immortalize. 5. The EBV transforms (immortalize) B cells, where the viruses can persist in the body indefinitely in small percentage of B cells in which a latent infection is later established. The infected B cells become polyclonally activated, proliferating and secreting number of

Antibodies including: EBV-specific antibodies, heterophile antibodies and autoantibodies. 6. The infected cells produce EBV-antigens associated with different phases of viral infection (and antibodies to these have become important diagnostic tool). a. Early acute phase: EA-R (early antigen restricted) - in the cytoplasm only. EA-D (early antigen diffuse) - diffuse distribution in both cytoplasm and nucleus. b. Late phase (appear during the lytic cycle following viral DNA synthesis): VCA (viral capsid antigen) in the protein capsid. MA (membrane antigen) in the viral envelope. c. Latent phase: EBNA (EBV nuclear antigens) ; EBNA-1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C and EBNA-LP LYDMA (lymphocyte-detected membrane antigens) LMPs (latent membrane proteins) LMP-1, LMP-2A, and LMP-2B.

Clinical manifestations vary: age and immune status. Infants and children generally asymptomatic or show mild symptoms. Adolescents and adults primary infection results into IM (Infectious Mononucleosis). Classic symptoms: fever, lymphadenopathy. Other symptoms include hepatomegaly, splenomegaly, and periorbital edema.

IMMUNE RESPONSE 1. 2. 3. Antibody production Natural Killer cells Specific cytotoxic T cells However, the EBV can persists in small number of B cells, and later establish a latent infection.

LABORATORY FINDINGS 1. Absolute lymphocytosis (>50% of total leukocyte counts, >4500/uL) 2. Atypical lymphocytes (at least 10%), thought to be activated cytotoxic T cells. 3. Serologic findings of heterophile antibodies and antibodies to EBV antigen.

HETEROPHILE ANTIBODIES Definition: antibodies that are capable of reacting with similar antigens from two or more unrelated species. IM heterophile Abs = IgM, produced as a result of polyclonal B cell activation, capable of reacting with horse red cells, sheep red cells, and beef red cells. Produced by 60-70% of patients with IM during the first week of clinical illness, and up to 90% of patients by the 4th week. Antibodies disappear by 3 months after onset of symptoms, or up to 1 year in some patients. Negative heterophile antibody occurs in about 20% of patients with IM, and 50% children under 4 years old. HETEROPHILE AGGLUTININ TEST Rapid slide agglutination test MONOSPOT TEST Principle : Based on the ability of serum absorbed with guinea pig kidney antigen or beef erythrocyte antigen to agglutinate horse red blood cells. HETEROPHILE ANTIBODIES: PAUL-BUNNEL Presumptive Test Principle: Serum dilution of PT serum is added with sheep RBC incubate at 37 oC for 1 hour, or at room temperature overnight centrifuge read agglutination. Titer : 1:56 presumptive evidence of heterophile antibodies; with clinical or cytologic findings suggestive of infectious mononucleosis. DAVIDSOHN Differential Test: To distinguish heterophile sheep red cell agglutinins in human serum due to Forssman antigen, serum sickness and IM. Principles : Some of the antigens that cause agglutination of sheep red cells are carried on ox (beef) erythrocytes but not on kidney cells of the guinea pigs; thus, exposure of the test serum to both GP kidney cells and beef red cells causes absorption of either one or both of these antibodies. The absorbed agglutinins can removed by centrifugation and aspiration of the resultant fluid, is then tested with sheep red cells. Tube 1: PT serum + GP kidney antigen Tube 2: PT serum + Beef erythrocyte antigen Incubate + Sheep RBC centrifugation agglutination? HETEROPHILE ABSORBED SHEEP RBC ABSORBED BY SHEEP RBC ANTIBODY BY GP AGGLUTINATION BEEF AGGLUTINATION KIDNEY Ag ERYTHROCYES YES NO Forssman YES Iinfectious Mono NO YES YES Serum Sickness

Rapid tests that make use of slides or cards: a. Equipan purified antigens from bovine red cells are coated onto latex particles. b. Seradyn Color Slides II Mononucleosis test (Alex on Trend) uses disposable card and color enhanced horse erythrocytes. VCA IgM + VCA IgG ++ + + +++ +/VCA IgA +\EA-D + +/+ EA-R +/+/++ EBNA IgG + + +/Heterophile + +/+/5

No previous exposure Recent (acute) infection Convalescent period

Reactivation of +/Latent Infection Chronic Active Infection Past Infection Post transplant Lymphoproli Ferative DSE Burkitts Lymphoma -

+ ++

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+ +/-

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Nasopharyngeal Carcinoma

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NOTE THAT IN: Convalescent Period: IGG can persist Past Infection: IgG can persist even after the infection was gone EBNA: positive only when VCA IgG is positive EA-D: is positive in Acute Infection EA-R: is positive in Chronic Active Infection VCA-IgG: good marker for Burkitts Lymphoma and Nasopharyngeal Carcinoma

EBV-specific antibodies in IM Anti-EA-D IgG- highly indicative of acute infection, but not detectable in 10-20% of patients with IM. It disappears in about 3 months, however a rise in titer is demonstrated during reactivation of latent EBV infection. Anti-EA-R IgG not usually found in young adults during acute phase. It appears transiently in the later, convalescent phase. Anti-EBNA IgG- appears only when patient has entered the convalescent period. Antibodies are almost always present in serum containing VCA IgG unless patient is in early acute phase. Patients with severe immunologic defects or immunosuppressive disease may not have EBNA antibodies, even if VCA antibodies are present. Antibodies to NA remains at moderate measurable level indefinitely because of the persistent viral carrier state established after primary EBV infection. Most healthy individuals have titers to EBNA ranging from 1:10 to 1:160. Common method: Indirect immunofluorescence Principle: Antigen substrate slide containing EBV-infected B cells + PT serum incubate addition of fluorescent conjugated antihuman IgG or IgM. ELISAA to detect anti-EBNA, using a synthetic peptide antigen to determine relative amounts of IGM and IgG antibodies in PT serum; reported with 99% specificity.