Chapter VI.23. Epstein-Barr Virus Infection
Erica Y. Shin
October 2022

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The editors and current author would like to thank and acknowledge the significant contribution of the previous author of this chapter from the 2004 first edition, Dr. Jhoanna D. Mabutas. This current third edition chapter is a revision and update of the original author’s work.

An 18-year-old freshman college student presents to the health center complaining of sore throat and fever for 3 days. She also states that she has been feeling tired for the past week. On physical exam, she is tired and subdued but not toxic in appearance with a temperature of 38 degrees C. Her tonsils are enlarged and erythematous. She also has enlarged posterior cervical lymph nodes bilaterally, which are mildly tender to palpation. She has no supraclavicular, axillary, or inguinal lymphadenopathy. Her spleen tip is palpable below the left costal margin. A throat swab is sent for a group A streptococci NAAT (nucleic acid amplification test), which is negative. Laboratory testing reveals a mild leukocytosis with the presence of atypical lymphocytes. A Monospot test is positive. Her human immunodeficiency virus (HIV) testing is negative. Her symptoms improve in a week.

Epstein-Barr virus (EBV) causes a broad spectrum of disease in humans with several clinical syndromes. Perhaps the best known is the one illustrated in the case above, the syndrome of infectious mononucleosis. This is an acute illness that results from primary infection with EBV. It is characterized by at least two of the following presentations: sore throat, fever, fatigue and cervical lymphadenopathy (1,2).

EBV was first described in 1964 (3) and is currently recognized as one of the most ubiquitous viruses, infecting more than 90% of the world's population (4). The name infectious mononucleosis is derived from the mononuclear lymphocytosis with atypical appearing lymphocytes that accompany the illness. Its clinical manifestations depend on the age at which the infection is first acquired. Most infections occur during infancy or early childhood. These are often asymptomatic or indistinguishable from other childhood illnesses. The prevalence/incidence of EBV infection among children is variable, depending on age and risk of exposure to the virus, which varies by race/ethnicity, socioeconomic status and geographic location. In developed countries, primary infection may be delayed until adolescence or young adulthood. This is when the classic syndrome of infectious mononucleosis often manifests. Most adults over age forty have been infected with EBV and show serologic evidence of prior infection (1).

EBV is a member of the herpes virus family, a gamma-1 herpesvirus and is also known as human herpes virus-4 or HHV-4 (1,2). EBV has a double-stranded DNA genome with an icosahedral protein nucleocapsid and, similar to other herpesviruses, it establishes a lifelong latent infection (5). EBV is transmitted in oral secretions and is acquired from close contact such as kissing or exchange of saliva between children. It initially infects epithelial cells in the oropharynx, where viral replication occurs and lysis of the epithelial cells results in release of new virions into the circulation (5). The virus then infects B lymphocytes in the peripheral blood and the reticuloendothelial system, including the liver, spleen, and lymph nodes. It is in these cells where the virus establishes latency, via formation of a viral episome. The host mounts a cell-mediated immune response to control the number of proliferating infected B lymphocytes. The characteristic atypical lymphocytes seen in infectious mononucleosis are activated CD8+ T-cells with suppressor and cytotoxic functions directed towards the infected B cells. As a result, infection is controlled but not abolished; reactivation may occur intermittently with viral shedding in oral secretions of affected individuals (1).

The incubation period of infectious mononucleosis is 30 to 50 days (2). The onset of symptoms is often insidious, with a prodrome of malaise, headache, fatigue, fever, sore throat, anorexia, and myalgia (6). Patients normally come to medical attention due to worsening sore throat and fever. On physical exam, the most common finding is lymphadenopathy, which is present in 90% of cases; it often occurs in the cervical region, particularly the posterior cervical chain, but may also be generalized with involvement of submandibular, epitrochlear, axillary, and inguinal lymph nodes. The lymph nodes are enlarged, firm and mildly tender to palpation. Fever and pharyngitis also occur in most patients. Pharyngitis is one of the most common complaints which may be moderate to severe with tonsillar enlargement and exudate. Abdominal exam may reveal splenomegaly in 50% to 75% and hepatomegaly in 30% (6). Patients with EBV erroneously treated with ampicillin or amoxicillin for presumed bacterial pharyngitis, characteristically develop a maculopapular rash, which may be useful in diagnosis, but sometimes is misdiagnosed as penicillin allergy (1,2).

The diagnosis of infectious mononucleosis may be made by clinical history, physical exam, and typical laboratory findings. Approximately 70% of patients demonstrate a relative and absolute mononuclear lymphocytosis with total white blood cell counts ranging from 12,000 to 18,000 cells/mm3, and atypical lymphocytes accounting for 20% to 40% of the total number. Compared to typical lymphocytes, atypical lymphocytes appear larger, with eccentrically placed nuclei and a larger amount of cytoplasm (6). A lymphocyte count less than 4,000 mm3 holds a 99% negative predictive value for infectious mononucleosis (6). Mild elevation of liver enzymes (e.g., aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase), occur in a majority (>80%) of cases; however jaundice is not typically present (6). EBV-associated infectious mononucleosis is associated with the transient production of heterophile antibodies. These are IgM antibodies from the patient's serum that cause agglutination of sheep or horse red cells. The most widely used heterophile antibody test is the Monospot, a qualitative rapid slide test which detects horse red cell agglutination (i.e., the modern equivalent of the heterophile antibody). The test has a specificity of 95% to 100% and sensitivity of 70% to 90% for adults and adolescents, but sensitivity for children ranges from 27% to 76% (2). Children, especially those under the age of 4 years, with symptomatic primary EBV infection are often heterophile negative. Overall, 10% of EBV-associated infectious mononucleosis may be heterophile negative. Certain organisms may cause an infectious mononucleosis-like syndrome but are not associated with formation of heterophile antibodies, such as cytomegalovirus (CMV), Toxoplasma gondii, adenovirus, viral hepatitis, human immunodeficiency virus (HIV), and rubella (1,2).

Resolution of infectious mononucleosis varies depending on the presenting symptoms. Fever and pharyngitis typically resolve after 10 to 14 days, adenopathy and splenomegaly can persist for several weeks, and fever and malaise may persist for weeks to months (6). Treatment is primarily supportive, with rest recommended during the acute stage of illness, as well as symptomatic care. Contact sports should be avoided while splenomegaly is present due to the risk of splenic rupture, although the incidence of this complication is low at less than 0.5% (1, 7). Treatment with antiviral agents such as acyclovir, ganciclovir, and foscarnet, or corticosteroids have not proven to be of benefit in uncomplicated cases (4). Corticosteroids may be considered in the treatment of select severe complications of EBV infection, which are rare, such as marked tonsillar inflammation (with impending airway obstruction), massive splenomegaly (with possible splenic rupture), myocarditis, autoimmune hemolytic anemia, aplastic anemia, thrombocytopenia, neutropenia, hemophagocytic syndrome, meningitis, and encephalitis (2).

The host also produces antibodies specific to the EBV. These are more useful for EBV related syndromes other than infectious mononucleosis since these additional antibody tests are not required for the diagnosis of infectious mononucleosis when the Monospot test is positive. These may be useful to clarify the diagnosis of heterophile-negative cases, or for cases of atypical EBV infections when the Monospot test is often negative. Multiple EBV-specific antibody tests are available, including tests for viral capsid antigen (VCA, IgG and IgM), early antigen (EA), and EBV nuclear antigen (EBNA). These antibody titers have a 97% sensitivity and 94% specificity for diagnosis of EBV infections whether they be infectious mononucleosis-like or not (1,2). The presence of IgM antibodies against viral capsid antigen signifies acute infection, while the presence of IgG antibodies signifies recent or past infection. Interpretation of the results of antibody testing for EBV is noted in Table 1 below (2):

Table 1 – Epstein Barr virus infection serologies
InterpretationEarly antigen (EA)Viral capsid antigen (VCA) IgMViral capsid antigen (VCA) IgGEBV nuclear antigen (EBNA)
No present or previous infection
Acute infection
Recent infection
Past infection

EBV has been implicated in benign and malignant proliferative disorders, particularly seen in patients with immunodeficiencies such as those caused by HIV infection, organ transplantation pharmacologic immunosuppression, severe combined immune deficiency, or Wiskott-Aldrich syndrome. The absence of an intact cell-mediated immunity in these patients allows uncontrolled proliferation of EBV-infected B lymphocytes. Examples of benign disorders associated with EBV include oral hairy leukoplakia (which occurs primarily in adults with HIV infection and presents with raised, white lesions on the tongue), and lymphoid interstitial pneumonitis (which occurs primarily in children with HIV infection and is characterized by the presence of diffuse interstitial pulmonary infiltrates). Examples of malignant disorders that have been associated with EBV include nasopharyngeal carcinoma (the most prevalent cancer among adult males in southern China), and endemic Burkitt lymphoma (the most common childhood cancer in equatorial east Africa). The geographic predominance of these diseases may be partially due to genetic and environmental factors. EBV has also been associated with gastric carcinoma, Hodgkin lymphoma, and post-transplant lymphoproliferative diseases (1,2). Additionally, EBV is known to contribute as an environmental risk factor for multiple sclerosis (8). A possible syndrome of congenital infection has been suggested but not confirmed (1).

1. A 16 year old male presents with sore throat, fever, and cervical lymphadenopathy. A throat culture is done which is positive for group A streptococcus. Treatment is initiated with penicillin. He returns two days later with worsened symptoms, despite taking the medicine. Which of the following is the most appropriate step to do next?
   a. Switch to azithromycin
   b. Obtain a CBC and Monospot
   c. Check anti-VCA, anti-EA, and anti-EBNA titers against EBV
   d. Assume the patient has infectious mononucleosis and start acyclovir and prednisone

2. Which of the following is false regarding EBV infection in young children?
   a. Primary infection is usually asymptomatic
   b. Heterophil antibodies are usually positive
   c. Immunocompromised patients are at risk for lymphocytic interstitial pneumonitis
   d. Complications are less common than in adults

3. Which syndrome has NOT been found to be associated with EBV?
   a. Nasopharyngeal carcinoma
   b. Oral hairy leukoplakia
   c. Aplastic anemia
   d. Kaposi's sarcoma

4. An 18-year-old female presents with malaise, fever, sore throat, and lymphadenopathy. Her CBC reveals atypical lymphocytosis, but her Monospot test is negative. Which of the following statements is correct?
   a. The Monospot test has higher sensitivity in children compared to adults
   b. Her symptoms may be due to primary infection by cytomegalovirus (CMV)
   c. There is no role for EBV-specific antibodies in making the diagnosis
   d. The atypical lymphocytes represent circulating infected B lymphocytes

5. Which of the following statements about EBV infection is correct?
   a. The syndrome of infectious mononucleosis results from primary infection with the virus
   b. Infection usually occurs via contact with the blood of an affected person
   c. About 25% of older adults show serologic evidence of prior infection
   d. Splenic rupture is a frequent complication in EBV-associated infectious mononucleosis

1. Katz BZ, Muller WJ. Chapter 208. Epstein-Barr Virus (Mononucleosis and Lymphoproliferative Disorders). In: Long SS, Prober CG, Fischer M, Kimberlin DW (eds). Principles and Practice of Pediatric Infectious Diseases, 6th edition, 2023. Elsevier, Philadelphia. pp:1107-1113.
2. Epstein-Barr Virus Infections (Infectious Mononucleosis). In: Committee on Infectious Diseases, Kimberlin DW, Barnett ED, Lynfield R, Sawyer MH (eds). Red Book: 2021-2024 Report of the Committee on Infectious Diseases, 32nd edition. American Academy of Pediatrics, Itasca, IL. pp:318-322.
3. Epstein MA, Achong BG and Barr YM. Virus Particles in Cultured Lymphoblasts from Burkitt’s Lymphoma. Lancet 1964;1(7335):702-703. doi: 10.1016/s0140-6736(64)91524-7
4. Dunmire SK, Verghese PS, Balfour HH Jr. Primary Epstein-Barr virus infection. J Clin Virol. 2018;102:84-92. doi: 10.1016/j.jcv.2018.03.001
5. Smatti MK, Al-Sadeq DW, Ali NH, et al. Epstein–Barr Virus Epidemiology, Serology, and Genetic Variability of LMP-1 Oncogene Among Healthy Population: An Update. Front Oncol 2018;8:211 doi: 10.3389/fonc.2018.00211
6. Olson D, Levin MJ, Asturias EJ. Chapter 40. Infections: Viral & Rickettsial. In: Hay Jr. WW, Levin MJ, Abzug MJ, Bunik M (eds). Current Diagnosis & Treatment: Pediatrics, 25th edition. 2020, McGraw Hill. Pp:1-64.
7. Lee S, Lin AC, Baerg J, Wu E. Spontaneous splenic rupture secondary to Epstein-Barr Virus-induced infectious mononucleosis. J Pediatr Surg Case Rep 2020;63.
8. Bjornevik K, Cortese M, Healy BC, et al. Longitudinal Analysis Reveals High Prevalence of Epstein-Barr Virus Associated with Multiple Sclerosis. Science 2022;375(6578):296-301. doi: 10.1126/science.abj8222

Answers to questions
1.b. In this case, the group A streptococcus probably represents colonization rather than the etiology of the patient's symptoms. Infectious mononucleosis may have a similar presentation to streptococcal pharyngitis, and must be considered if a patient is not responding clinically to treatment with antibiotics. The diagnosis can be made with a Monospot test as well as the presence of atypical lymphocytes on CBC. EBV titers are not usually needed for diagnosis, but may be considered if the Monospot is negative and EBV infection needs to be ruled out. Treatment with acyclovir or corticosteroids has not been proven to be of clinical benefit in uncomplicated cases of infectious mononucleosis.
2.b. Primary EBV infection occurs more commonly in childhood and is often asymptomatic. In children who do develop symptomatic EBV infection, heterophil antibodies are often negative. Immunocompromised patients, especially those with HIV infection, may present lymphocytic interstitial pneumonitis. Complications occur less commonly in children than in adults.
3.d. The first three have all been found to be associated with EBV infection. Kaposi's sarcoma is associated with a different human herpes virus, referred to as human herpes virus-8 or HHV-8.
4.b. The Monospot test has lower sensitivity (<50%) in children compared to adults/adolescents (>60%), although 10% of EBV-associated infectious mononucleosis can be Monospot negative. There are also a number of organisms that may cause an infectious mononucleosis-like syndrome but are not associated with formation of heterophil antibodies. The most common cause of a heterophil-negative infectious mononucleosis-like syndrome is CMV, which this patient likely has. Obtaining antibody titers specific against EBV and CMV can clarify the diagnosis. The atypical lymphocytes that can be seen with either EBV or CMV infection represent activated T lymphocytes, which proliferate in response to infected B lymphocytes.
5.a. The syndrome of infectious mononucleosis results from primary infection with EBV, particularly when it is delayed until adolescence or young adulthood. It is usually transmitted through close contact with oral secretions of an infected individual. The virus is ubiquitous, and almost all adults over age 40 show serologic evidence of prior infection. Splenic rupture is a rare complication of EBV-associated infectious mononucleosis.

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