Epstein-Barr virus Epstein-Barr virus Shane C. McAllister, MD, PhD Shane C. McAllister, MD, PhD Pediatric Infectious Diseases Fellow Pediatric Infectious Diseases Fellow Stony Brook Long Island Children’s Hospital Stony Brook Long Island Children’s Hospital

Overview History of EBV and mononucleosis EBV structure and gene expression Pathogenesis/Clinical manifestations Epidemiology Diagnosis Treatment Cases

History of EBV and mononucleosis

INTRODUCTION Infectious mononucleosis (IM) was first recognized in1920 however the etiology was unknown The heterophile test was discovered to be diagnostic for Infectious Mononucleosis in 1932 A viral cause of mononucleosis was suspected in the 1960s when a laboratory worker became ill while working with Burkitt lymphoma tissue samples

INTRODUCTION In 1968, Epstein-Barr Virus was actually identified as the cause of Infectious Mononucleosis Since the initial discovery, EBV has been implicated in a wide variety of both benign and malignant diseases

EBV structure and gene expression

Herpesviruses 8 herpesviruses known to infect humans Strict species specificity Closely related rodent and non-human primate strains used as animal models

Herpesviruses Large double-stranded DNA Viruses ≥ 84 different proteins Maintenance/replication of genome in host cell nucleus

Herpesviruses Life-long latency with periodic lytic reactivation Control of infection requires cellular and humoral immunity

Kinetic Classes of EBV Genes Latency  LMP-1, EBNAs Immediate Early: initiate lytic cycle  Zta, Rta Early: condition the host cell environment and synthesize viral DNA  EA-D Late: structural components of capsids/mature virions  gp350

Pathogenesis/Clinical manifestations

DISEASE ASSOCIATIONS Infectious Mononucleosis Chronic Infectious Mononucleosis Burkitt Lymphoma Nasopharyngeal carcinoma Hodgkin Lymphoma Lymphoproliferative disease X-linked Lymphoproliferative disease Oral Leukoplakia (AIDS)

PATHOGENESIS EBV infection is considered immunopathologic rather than tissue destructive EBV initially infects and replicates in the oropharyngeal epithelial cells B-cells are subsequently infected Infected B-cells disseminate throughout the lymphoreticular system

PATHOGENESIS EBV infection triggers an impressive but self-limited immune response Infected B-cells are transformed and secrete a diverse group of antibodies Heterophile antibodies Antibodies to specific EBV antigens Various auto-antibodies EBV induced polyclonal activation of B- cells also leads to an increase in serum immunoglobulins

PATHOGENESIS Infected B-Lymphocytes induce T- Lymphocyte proliferation Manifested as Atypical Lymphocytosis Proliferation of reactive T-cells and infected B-cells leads to Lymphadenopathy Hepatosplenomegaly

CLINICAL MANIFESTATIONS SYMPTOM%RANGE (%) SORE THROAT MALAISE HEADACHE ANOREXIA MYALGIAS CHILLS NAUSEA ABDOMINAL DISCOMFORT COUGH 5 5 VOMITING 5 5 ARTHRALGIAS 2 2

CLINICAL MANIFESTATIONS SIGNS%RANGE (%) LYMPHADENOPATHY PHARYNGITIS FEVER SPLENOMEGALY HEPATOMEGALY PALATAL ENANTHEM JAUNDICE RASH

PHYSICAL FINDINGS EXUDATIVE PHARYNGITIS

PHYSICAL FINDINGS TONSILLAR HYPERTROPHY

EBV – Physical Findings Rash and Antibiotics Rash and Antibiotics Nonspecific maculopapular eruption associated with administration of Ampicillin/Amoxil (50 – 100%) Nonspecific maculopapular eruption associated with administration of Ampicillin/Amoxil (50 – 100%) May be associated with other  -lactam antibiotics (40 – 60%) May be associated with other  -lactam antibiotics (40 – 60%) Usually develops 7 – 10 days after the first dose Usually develops 7 – 10 days after the first dose Does not represent Penicillin allergy Does not represent Penicillin allergy Mechanism is unclear Mechanism is unclear Transient hypersensitivity reaction Transient hypersensitivity reaction Immune complex production Immune complex production

Complications of Primary EBV ORGAN/SYSTEMCOMPLICATIONS LIVER:Abnormal Liver Function Tests (80 – 90%) Clinical jaundice (5%) Fulminant hepatitis (rare) RESPIRATORY: Airway Obstruction (<5%) Interstitial Pneumonitis (Rare) HEMATOLOGIC:Thrombocytopenia (25-50% Mild/Mod) Neutropenia (50 – 80% - Mild/Mod) Pancytopenia (Rare) Hemolytic Anemia (3%) Aplastic Anemia (Rare)

Complications of Primary EBV ORGAN/SYSTEMCOMPLICATIONS SPLEEN:Splenic rupture (0.1 – 0.5%) Splenic Infarction RENAL:Hematuria Interstitial nephritis Glomerulonephritis CARDIAC:Myocarditis Pericarditis Arrhythmias

Complications of Primary EBV ORGAN/SYSTEMCOMPLICATIONS SECONDARY INFECTION:Streptococcal pharyngitis Secondary sepsis due to neutropenia IMMUNOLOGIC:Depressed T-cell immunity NEUROLOGIC:Encephalitis Aseptic Meningitis Guillain-Barre Syndrome Cranial nerve palsies Transverse Myelitis Optic Neuritis Cerebellar Ataxia Brachial Plexus Neuropathy

Neurologic Complications 2 2 May be the first or sole manifestation of EBV mononucleosis 2 2 Occurs in 1 – 5% of cases 2 2 Prognosis is generally good with 85% complete recovery 2 2 Most frequent cause of death related to EBV Infectious Mononucleosis 2 2 Diagnosis is difficult 2 2 Heterophile is negative 2 2 Atypical lymphocytosis is minimal or absent

Neurologic Complications “Because EBV may present atypically and has been associated with a myriad of neurologic diseases, EBV should be considered in all acute neurologic illnesses of unknown etiology in the Pediatric population” Connelly et al., Pediatr Neurol 1994; 10:

INFECTIOUS MONO IN CHILDREN Compared with adolescents, young children more commonly had the following features: URI symptoms Rash Splenomegaly and/or Hepatomegaly Higher peak leukocyte counts with fewer Atypical Lymphocytes More frequent neutropenia

Epidemiology

EPIDEMIOLOGY   Antibodies to EBV found in all populations   Lower socioeconomic groups- higher prevalence   By adulthood, % of most populations have demonstrable antibodies   In the US, EBV seroconversion occurs before age 5 years in 50% of the population   Second wave in the second decade of life   No predilection for male or female

EPIDEMIOLOGY   In the US cases/100,000/year   The incidence is highest in the year-old age group   College and military populations have the highest morbidity   Not a reportable disease   No clear seasonal pattern

Diagnosis

DIFFERENTIAL DIAGNOSIS OF MONO-LIKE SYNDROME Epstein-Barr virus Cytomegalovirus Toxoplasma gondii Adenovirus Human herpesvirus 6/7 Hepatitis A Influenza A and B Rubella virus Diphtheria HIV Malignancies

DIFFERENTIAL DIAGNOSIS OF MONO-LIKE SYNDROME CMV mononucleosis is most frequently confused with EBV: Patients are generally older (Adults) Pharyngitis and lymphadenopathy – less common Fever and malaise are the major manifestations Heterophile negative

Atypical Lymphocytes Activated T cells responding to the EBV-infected B-cells Features of Atypical Lymphs: Larger than mature Lymphocytes Have vacuolated basophilic cytoplasm

DIAGNOSIS ATYPICAL LYMPHOCYTES

Heterophile Antibody Test Heterophile antibodies comprise a broad class of antibody characterized by ability to agglutinate antigens on RBCs from different mammalian species IM heterophile Ab (IgM) does not react with EBV- specific antigens characterized by its ability to react with beef, sheep and horse RBCs The antigen that stimulates this heterophile ab is unknown

Heterophile Antibody Test Replaced by the monospot slide test (Antigen-coated beads on a slide) 15% of patients with IM may be initially heterophile negative and become positive within 2 – 3 weeks High false negative rate in children less than 4 years (>50%) False positive rate - 7% Remains positive for up to 9 months Sensitivity and specificity: 85%/97%

MONOSPOT NEGATIVE POSITIVE

Sumaya et al., Pediatrics 1985; 75:

FALSE – POSITIVE MONOSPOT Collagen Vascular diseases Leukemia/Lymphoma Malaria Pancreatic Carcinoma Viral Hepatitis Other

EBV SEROLOGY The appearance of antibodies induced by EBV specific antigens correlates with the phase of replication IgM antibody to VCA appears at the onset of symptoms and typically disappears within 1 – 3 months IgG antibody to VCA begins to rise shortly after the onset of symptoms Peaks at 2 – 3 months Gradually decreases to a steady-state and persists for life

EBV SEROLOGY Antibodies to EA are not always detectable IgG to EA begins to rise at the onset of symptoms Peak concentration occurs at 3 – 4 weeks Subsequently decreases and disappears Antibodies to EBNA appear during the convalescent period and persist for life along with anti-VCA IgG Past infection: No anti-VCA IgM (Potential for false-positives) No anti-EA IgG

DIAGNOSIS

Interpretation of EBV Serology INFECTION ANTI-VCA-IgMANTI-VCA-IgGANTI-EA ANTI-EBNA NONE NEGATIVENEGATIVENEGATIVE NEGATIVE ACUTE POSITIVEPOSITIVEPOS/NEG NEGATIVE RECENT POS/NEG POSITIVEPOS/NEG POS/NEG PAST NEGATIVEPOSITIVENEGATIVE POSITIVE

EBV SEROLOGY Effective lab diagnosis can be made on single acute phase serum sample Antibody response appears rapidly (onset of symptoms) Acute and Convalescent phase serum will not demonstrate a significant change in antibody titer

EBV SEROLOGY Literature supports the general concern that there is considerable variation in the performance of serological test kits for EBV and other infectious agents VCA-IgM Cross-reactivity occurs Especially with other herpesviruses (CMV)

EBV - DIAGNOSIS The ability of EBV to maintain lifelong latency with low levels of replication and viral shedding results in enduring antigen exposure and continued humoral immune response Variation of EBV antibody titers may be due to reactivation of latent virus due to infection with another virus and development of cross-reactive antibodies

EBV - DIAGNOSIS Important to be aware of nuances of serologic testing as well as viral detection for latent viruses such as EBV Must be cautious with utilization of serologic testing/DNA detection as the sole means for establishing causal relationship between illness and EBV infection

Treatment

TREATMENT   Supportive care   Avoid contact sports   Corticosteroids for selective complications   Airway obstruction   Massive splenomegaly   Myocarditis   Hemolytic Anemia/ITP   Acyclovir – No clearly documented benefit

ANTIVIRAL TREATMENT 5 randomized clinical trials conducted to evaluate treatment of Infectious Mononucleosis with Acyclovir (339 patients) Studies showed no statistically significant benefit or clinical effectiveness Met-analysis also showed no significance No evidence that therapy shortens duration of symptoms or prevents complications

Prognosis and Outcome The majority of cases are uncomplicated resolving in 1 – 2 months Minority of patients experience persistent fatigue for up to 6 months Although EBV remains latent lifelong in a few cells in throat and blood, not thought to be clinically significant Periodically the virus can reactivate (virus isolated in saliva)

Cases

Case 1 5 year old female with acute febrile illness including URI symptoms and sore throat Physical exam significant for exudative tonsillitis and cervical adenopathy Rapid GAS test negative but patient started on Amoxil pending culture. Patient developed diffuse rash on second day of antibiotic treatment. Culture negative

Interpretation of EBV Serology INFECTION ANTI-VCA-IgMANTI-VCA-IgGANTI-EA ANTI-EBNA NONE NEGATIVENEGATIVENEGATIVE NEGATIVE ACUTE POSITIVEPOSITIVEPOS/NEG NEGATIVE RECENT POS/NEG POSITIVEPOS/NEG POS/NEG PAST NEGATIVEPOSITIVENEGATIVE POSITIVE

Case 2 15 year old female with acute febrile illness of 6 days duration including sore throat, malaise, fatigue, and swollen glands Physical exam significant for no tonsillar tissue, positive cervical adenopathy, no HSM Lab work demonstrated: elevated WBC with lymphocyte predominance Heterophile antibody positive, VCA IgM positive; VCA IgG, EAD IgG, and EBNA IgG negative

DIAGNOSIS

Interpretation of EBV Serology INFECTION ANTI-VCA-IgMANTI-VCA-IgGANTI-EA ANTI-EBNA NONE NEGATIVENEGATIVENEGATIVE NEGATIVE ACUTE POSITIVEPOSITIVEPOS/NEG NEGATIVE RECENT POS/NEG POSITIVEPOS/NEG POS/NEG PAST NEGATIVEPOSITIVENEGATIVE POSITIVE

Case 3 17 year old male diagnosed with mono one year ago presents with continued fatigue. He sleeps 7 to 8 hours a night and is a long distance runner Physical exam is normal Lab work demonstrated: Normal CBC Heterophile antibody, VCA IgM, and EAD IgG negative; VCA IgG and EBNA IgG positive

DIAGNOSIS

Interpretation of EBV Serology INFECTION ANTI-VCA-IgMANTI-VCA-IgGANTI-EA ANTI-EBNA NONE NEGATIVENEGATIVENEGATIVE NEGATIVE ACUTE POSITIVEPOSITIVEPOS/NEG NEGATIVE RECENT POS/NEG POSITIVEPOS/NEG POS/NEG PAST NEGATIVEPOSITIVENEGATIVE POSITIVE

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