CHAPTER 10
Epstein-Barr Virus
Alfred S. Evans and James C. Niederman
1. Introduction 2. Historical Background
Epstein-Barr virus (EBV), a member of the herpes In 1889, Emil Pfeiffer of Wiesbaden, Germany, group of viruses, is the cause of heterophile-positive described a condition called Drusenfieber (glandular infectious mononucleosis, of most heterophile-neg• fever), characterized by fever, adenopathy, mild ative cases, and of occasional cases ot tonsillltIs and sore throat, and in severe cases enlargement of the pharyngItis in childhood. Rarely, it may involve the liver and spleen. (63, 126) Since Pfeiffer's original de• liver or central nervous system as primary mani• scription, as well as Filatov's, (53,54) a Russian, in festations. This virus is strongly implicated as hav• 1892, antedated by some 30-50 years recognition of ing a causal relationship to African Burkitt lym• the hematological changes and the heterophile an• phoma and to nasopharyngeal cancer. High antibody tibody, it is uncertain whether these were true in• titers are also present in 30-40% of cases ot Hod&• fectious mononucleosis. However, his description kin's disease, in some patients with sarcoidosis, and of this febrile syndrome in older children and young in systemic lupus erythematosus. adults seems best to fit this diagnosis. There is little This chapter will deal with the epidemiology of doubt about the classic description of the disease EBV infections and the epidemiology of infectious made by Sprunt and Evans, (139) from Johns Hop• mononucleosis. Infectious mononucleosis can be kins, in 1920. They described the disorder in young defined as an acute febrile illness involving children adults as we now know it, named the disease "in• and young adults characterized clinically by sore fectious mononucleosis," and reported in detail the throat and lymphadenopathy, hematologically by hematological changes. This description was fol• lymphocytOSIs of 50% or more, of which 10% or lowed rapidly by similar reports from other work• more are atypICal, and sel'OIogIcally by an elevated ers. (12,14,22,96,108) A definitive presentation of hema- absorbed heterophile-antibody titer and the devel• tological changes was made by Downey and opment of EBV immunoglobulin M (lgM) and other McKinlay(28) in 1923. The next major development EBV antibodies. This chapter also mentions the re• was the discovery in 1932 of the heterophile anti• lationship of high antibody titers to certain chronic Dod] By lohn K--VauI ai10William W. BunneIFlLi) and malignant diseases, but the major discussion ot Yale University. Their report was based on an of Burkitt lymphoma and of nasopharyngeal cancer accidental observation while studying the occur• will be found in later chapters of this book. rence of heterophile antibodies in rheumatic fever. Alfred S. Evans . WHO Serum Reference Bank, Section This search had been initiated because of the clinical of International Epidemiology, Department of Epidemiol• similarity of rheumatic fever and serum sickness and ogy and Public Health, Yale University School of Medicine, because of the work of Davidsohn(24) describing the New Haven, Connecticut. James c. Niederman . De• partment of Epidemiology and Public Health, Yale Uni• presence of heterophile antibodies in serum sick• versity School of Medicine, New Haven, Connecticut. ness. Among the control subjects for rheumatic 253
A. S. Evans (ed.), Viral Infections of Humans © Plenum Publishing Corporation 1984 254 Chapter 10 • Epstein-Barr Virus
fever patients was one who had infectious mono• fectious agent from the throat or blood of patients nucleosis and was found to have a much higher with infectious mononucleosis using several tissue• heterophile antibody titer than present in any other culture systems, long-term cultures of lymphocytes condition. Paul and Bunnell then continued these on a feeder layer, and fluorescent antibody tech• observations in 3 additional cases of infectious niques to identify an agent were unsuccessful. (37) mononucleosis and utilized 275 controls for com• Epidemiologically, the key events during this time parison. Their paper also describes what they be• were the observations of Hoagland, who suggested lieved to be a false-positive heterophile antibody that the disease might be transmitted by kissing(79) occurring in a patient with aplastic leukemia. A re• and that the incubation period was of the order of view of the details of this case(43) reveals that the 30-49 days. (81) heterophile antibody occurred about 20 days after Early in 1968, evidence first appeared that EBV the administration of several units of blood, and was the cause of infectious mononucleosis. (72, 114) therefore this patient may represent the first case This virus, identified by Epstein et al. (33) in a culture of transfusion infectious mononucleosis. Soon after of African Burkitt tumor tissue, (128) was found to be the discovery of the presence of heterophile anti• a new member of the herpes group of viruses. While bodies, Davidsohn and Walker(23) reported on the working with this agent, a technician in the Henles' use of guinea pig kidney and of beef cells to absorb laboratory in Philadelphia developed infectious serum prior to heterophile testing in order to in• mononucleosis. Her serum, which lacked antibody crease the specificity of the test. Both these proce• several months prior to disease, developed EBV an• dures have withstood the test of time well and still tibody during illness, and her lymphocytes, which constitute one of the major criteria of diagnosis. had previously failed to be cultivated successfully, Regular alterations in various liver-function tests now grew well in tissue culture and were shown to during acute infectious mononucleosis were rec• contain EBV antigen. (72) This serendipitous obser• ognized in several laboratories in the late 1940s and vation was rapidly confirmed and extended at that 1950s, (13,35,86) even though only 5% of patients had time by the Henles in conjunction with Niederman clinical jaundice. This was followed by the discovery and McCollum(114) of Yale and later in several other of alterations in serum glutamic oxalacetic trans• prospective studies carried out by the Yale aminase (SCOT) and other hepatic enzymes during team(50,113,132) and in one English study.(148) Subse- the course of disease. (6,131,158) quent investigations established the presence and Search for the etiological agent of infectious mono• persistence of EBV in the throat during and after nucleosis began in the 1920s, but met with little acute infectious mononucleosis,
3.2. Morbidity Data way, an elevated titer quite accurately reflects the occurrence of infectious mononucleosis even in the Infectious mononucleosis is not a reportable dis• absence of clinical and hematological data and has ease in most states and in most countries. Excep• been utilized as an indicator of infectious mononu• tions are the stataDf Connecticut,. where it has been cleosis in sera sent to hospitals and state labora• reportable since 1948,1"1) and the United States tories. (38) The major limitation of this approach is armed forces, which collect hospitalization data on the extent to which physicians have sent sera from all diseases. (41) Unless strong emphasis is placed on suspected cases to the diagnostic laboratory for the need for fulfilling clinical, hematological, and analysis. The increasing use of simple laboratory serological criteria for diagnosis before reporting, kits for identifying heterophile-antibody elevations the reliability of morbidity data from these sources in the physician's office probably results in much must be seriously questioned. This requirement is less utilization of state and hospital laboratories, so emphasized by the fact that even for the 15-25 age that morbidity data from these sources may greatly group-in which the disease has its highest inci• underestimate the occurrence of the disease. Thus, dence, its most characteristic clinical features, and utilization of the heterophile antibody as an epi• the highest frequency of elevated heterophile anti• demiological tool to identify the acute illness has body tests-only one-third of the serum samples high reliability and specificity but low sensitivity. sent to a state laooratorv for diagnosis of suspected Heterophile-positive cases diagnosed in the Con• cases were heterophile antibody positive. ("38)" necticut State Public Health Laboratory alone rep• To collect mOrbIUlty uata, specIal surveys of se• resented 74.5% of all the reported cases in the state lected populations for infectious mononucleosis in 1972. have been carried out in college infirma• ries, (50,83a,113,132) community medical care groups, (69) Since the discovery of EBV as the cause of infec• tious mononucleosis in 1968, many serological sur• and general practitioners' offices, (83) and by physi• veys in different countries have been made for the cians and laboratories serving defined communi• ties. (29a,36,68,131a) The Center for Disease Control presence of antibody to this virus in sera collected from healthy persons, usually employing the indi• (CDC) also periodically publishes a Surveillance rect immunofluorescence test(70) for viral capsid an• Report on infectious mononucleosis based on data tigen (VCA). This IgG antibody persists for many derived from 19 colleges. (18) years, pernaps tor Ilte. (U !!:'i 14) 1 nese stuctlesyidd The problems of data derived from such surveys prevalence data on prrm·EBV infection, but give no are related to the extent to which the numerator or dIrect mdIcahon of the occurrence of clinical infec• case report reflects the proper diagnosis and whether tious mononucleosis. More recently, the EBV-spe• adequate surveillance has been carried out with re• cific IgM antibody has been included in surveys to spect to the denominator-the population at risk. identify recent infection. (l44a) The most accurate information on the incidence of both EBV infections and clinical infectious mono• 3.3. Serological Surveys nucleosis has come from prospective seroepidemio• Up to 1968, when the causal association of EBV logical studies of defined populations with close clin• with infectious mononucleosis was discovered, the ical surveillance for the occurrence of suspected heterophile antibody constituted the only serologi• cases of infectious mononucleosis and other ill• cal approach to diagnosis and survey work. Because nesses. Sera taken at the start of the observation this is an IgM-type antibody and is transient in na• period are tested to define the number of suscep• ture, it can be used as a serological tool only for tibles, i.e., those lacking EBV antibody; samples incidence data-i.e., during the acute illness-and showing seroconversion at the end of the observa• as an essentIal diagnostic feature. (114) The specificity tion period will identify the total EBV infection rate; of a properly performed quantitative heterophile• those collected during interim illnesses and tested antibody test is high provided that the serum has for EBV and heterophile antibodies will delineate been preabsorbed with guinea pig kidney in the the number and spectrum of clinical illnesses, such sheep and horse red cell tests or that the beef hem• as infectious mononucleosis, associated with EBV olysin test has been used. In a test performed in this infection. 256 Chapter 10 • Epstein-Barr Virus
3.4. Laboratory Methods mononucleosis, However, it has not proved very useful as a diagnostic test for infectious mono• 3.4.1. EBV Isolation. The virus cannot be grown nucleosis because antibody is usually present by inJhe~usual tissue cultures employed for other her• the time the patient seeks medical care and rises in pesviruses. The currently available isolation tech• titer are detectable in only 15-20% of cases. Second, nique is tedious, difficult, and usually confined to antibody to "early antigen" is also identified by im• research laboratories. It is based on the ability of munofluorescence techniques, (74,76) and its presence EBV to transform uninfected human leukocytes into is indicative of recent or active infection. Unfortu• continuous cell lines and the identification of this nately, from a diagnostic standpoint, it is demon• effect as due to EBV.(19,103) Leukocytes derived from strable only in about 75% of patients with infectious the cords of newborn infants or from persons lack• mononucleosis(77); it also occurs in the sera from ing EBV antibody are employed to ensure absence patients with Burkitt lymphoma and nasopharyn• of EBV antigen in the lymphocytes. Throat washings geal cancer. Third, antibodies to EBNA are detect• or other materials to be tested are usually filtered able by an immunofluorescence technique based on to remove debris and bacteria, then added to the CF; these usually arise only 1 month or more after leukocytes, and placed on a placental fibroblast onset of infectious mononucleosis and after primary feeder layer. If EBV is present, evidence of trans• infections and probably persist for life. (73) Their late formation is indicated by an abrupt increase in the appearance impairs their usefulness in routine di• total number of cells, the production of acid, growth agnosis. Fourth, there is an indirect immunoflu• of cells in clumps, and the development of the ca• orescence test for EBV-specific IgM antibody, and pacity to be subcultured indefinitely. Usually, trans• this is the most useful procedure for the diagnosis formation occurs 30-90 days after addition of the of heterophile-negative infectious mononucleo• throat washing. SiS(29,114b,133); however, in its present form, this test The presence of EBV-associated nuclear antigen is technically difficult to perform, and it is not cur• (EBNA) can be demonstrated in acetone-fixed smears rently available in most diagnostic laboratories. Fi• of transformed cord cells using an indirect comple• nally, a membrane fluorescence test has been de• ment-fixation (CF) fluorescence technique, (129) but veloped by Klein et al. (88-90) Other antibody tests VCA cannot be demonstrated by ordinary immu• include CF using either the virus(l) or soluble anti• nofluorescence methods, presumably because the gens, (56,57) neutralization tests based on contact in• virus does not mature sufficiently in such cells. EBV• hibition, (78, 105) and immunodiffusion tests. (116) transformed leukocytes may also be grown further 3.4.3. Heterophile Antibody Tests. Three gen• in culture to prepare a CF antigen as a means of eral methods are employed: (1) the classic identification; however, this is a laborious method. Paul-Bunnell test(121) using sheep or horse red cells More sensitive methods of antigen detection, chem• after absorption of the serum with guinea pig kidney ical means of enhancing the rapidity of viral mul• as developed by Davidsohn and Walker(23); (2) the tiplication to shorten the long observation time, or beef-cell hemolysin test of Bailey and Raffel(2) adopted discovery of a more sensitive cell line will be needed for diagnostic use by Mason, (98) which does not re• before viral isolation will be practical in the diag• quire absorption of sera with guinea pig kidney be• nostic laboratory. Robinson and Miller(129a) have cause beef-cell he moly sins are absent or at very low demonstrated that DNA stimulation occurs early in titers in normal sera; and (3) the enzyme test of EBV-infected cord cells and can be detected by in• Wollner, (157) in which red-cell receptors for hetero• creased uptake of radio-labeled thymidine. phile antibody are specifically removed by treatment 3.4.2. EBV Antibody. A wide variety of tech• with papain or a similar enzyme. Recent evidence niques to measure EBV antibody have been devel• suggests that heterophile-antibody titers may reach oped. Five antibody methods based on immunoflu• diagnostic levels even after mild or asymptomatic orescence have been used. (73a) First, for epidemio• EBV infections provided serial specimens are tested logical purposes, the indirect immunofluorescence over a month or so by the horse-cell differential test of the Henles(70) for VCA has been widely test. (52) The test is also useful in childhood EBV in• employed in serological surveys as a reliable indi• fections, which are often mild. Most state and large cator of susceptibility and immunity to infectious diagnostic laboratories employ either the Davidsohn Chapter 10 • Epstein-Barr Virus 257 differential absorption test or the beef-cell hemo• by DNA hybridization or EBNA tests, but only 1-3% lysin test; the former, using horse cells, is more sen• have demonstrable VCA(1l5,1l7,129,160); cell clones sitive, and heterophile antibody to this antigen often grown from such cultures show a similar low per• persists at diagnostic levels for as long as a year or centage of complete virus. (99,159) One line of cells so. (52,93) The beef hemolysin test is more specific but from Burkitt lymphoma, the P3J line of Pulvertaft, (128) less sensitive, and disappears in 3 months or less; and its cloned derivative, the HR1K, produce more it is perhaps the most reliable test to diagnose in• extracellular virus than other lines but fail to induce fectious mononucleosis during the acute illness. A transformation. (106) Another line, B95-8, derived new immunoadherence test of high sensitivity is from EBV-infected marmoset cells by Miller and also promising. (94c) Lipman, (102) releases about 1000 times more trans• A number of commercial testing kits for the di• forming virus but about the same number of viral agnosis of infectious mononucleosis in the physi• particles as HR1K. It has been useful in viral char• cian's office are now available. Most are slide ag• acterization. (68c,d) glutination tests, usually performed at a single Some of the biological properties of EBV are im• dilution and commonly based on the agglutination portant epidemiologically. The capacity for persist• of formalinized horse cells. Some of these employ ence of a lytic infection in the throat provides a guinea pig kidney to remove nonspecific agglutinins source of potential transmission; the low yield of from the serum prior to testing. Other tests employ extracellular virus may bear on the need for inti• papain-treated red cells in the agglutination test. A mate, oral contact tor transmission in young spot test using horse red cells and absorption pro• adults. (ll'!O) The reasons for the higher efficiency ot cedures is recommended. Such tests are useful if transmission of EBV infection in young children carried out by trained personnel. than in adults are unknown, but might include the production of more infectious virus in the pharynx of children, more intense exposure, or indirect 4. Biological Characteristics of the Agent spread by saliva in settings with poor hygiene. The capacity for persistence and latency of EBV 4.1. The Virus in a nonproductive form sets the stage for later reac• tivation under conditions of immunosuppreSSIOn EBV is a distinctive member of the herpes group (e.g., malana, therapeutic immunosuppression in of viruses. On electron microscopy, EBV appears renal transplants). African Burkitt lymphoma and similar to other herpes-group viruses. (33) Currently, nasopharyngeal cancer may be expressions of this. two laboratory strains of EBV have been identi• reactivation. The long-:!erm persistence of EBV in fied, (106) but the sophisticated techniques needed to lymphocytes is of importance epidemiologically in differentiate strain differences as small as exist be• the transmission of infection during blood transfu• tween herpes types I and II are not yet available. sions to susceptible reCipients. Of great importance Definitive evidence of differences in EBV isolates is the capacity of EBV to transtorm unintected pri• from nature have not yet been found, although mate lymphocytes, inducing in them the potential minor variations exist. (59a) The virus has been cul• tor unllmited proliferation; this property was termed tivated only in suspension cultures of primate lym• "immortalization" bvMiller, (IOU) and the lympho• phocytes, and most cultures yield only small amounts cytes that result are termed "I lymphocytes." The of extracellular virus. These limitations have made EBV-transformed and -infected cells are B-type lym• characterization of the physical and chemical prop• phocytes. (118,119) Viral induction of new antigens (or erties of EBV very difficult. EBV is a lymphotropic unmasking of preexisting ones) such as the mem• virus and infects B lymphocytes, which have EBV brane antigen of Klein et al.(88) may have immuno• receptors on their surfacp(85a} near to or idenffi:al logical consequences in the development of new an• with the C3 receptors(I54a}; new membrane antigens tibodies, in a graft-vs.-host response, and in the are induced to which T cells respond. Alllympho• induction of cytotoxic T lymphocytes.(83b,131C) cytes in continuous cultures established from infec• A better understanding of the dynamics and ef• tious mononucleosis blood or Burkitt lymphoma fects of EBV activity at molecular and cellular levels biopsies contain the EBV genome as demonstrated ("molecular epidemiology") and of the responses 258 Chapter 10 • Epstein-Barr Virus
of the host of them under varied conditions of age, thus indicating that this is a primary response to concomitant infection, immune status, and genetic EBV infection.(4,29,66,133) Both the IgG and IgM EBV• constitution will be needed before the full spectrum specific antibodies of infectious mononucleosis are of clinical response is known. distinct from the heterophile antibody. The virological evidence consists of the appear• ance of EBV in the oropharynx and in the circulating 4.2. Proof of Causation of Infectious lymphocytes of patients with acute infectious mono• Mononucleosis nucleosis. The agent has been regularly demon• The causation of heterophile-positive infectious strated in the pharynx of over 80% of patients during mononucleosis by EBV has been established beyond the acute illness(19,59,103,125) and may be multiplying 'any reasonable doubt. The proof is based on sero• in_epithelial cells. (94a) In addition, EBV antigens have epidemiological and virological evidence and also been found in tonsillar lymphocytes. (149a,b) They on partial success in the experimental transmission persist for many months and in several cases have of infection to monkeys and man. persisted for as long as a year or so. A chronic carrier Seroepidemiological investigations have repeat• state may exist, as suggested by the presence of edly shown that antibody to EBV of the IgG type has virus in the oropharynx of 15-20% of healthy been consistently absent in sera taken prior to the adults. (20,59,143) onset of infectious mononucleosis, regularly ap• EBV has been regularly demonstrated in lympho• pears during illness, and persists for years there• cyte cultures from patients with acute infectious after. (50,65,114,132,148) The presence of this antibody mononucleosis, where it may remain in a latent indicates immunity to clinical infectious mononu• form for years and may be a source of transfusion cleosis, and its absence indicates susceptibility to mononucleosis. (26,58,75) EBV has now been the disease. Table 1 summarizes 11 prospective demonstrated in fresh B lymphocytes from patients studies involving over 5000 children and young with acute infectious mononucleosis(90a,130a): up to adults in support of this relationship. No other virus 0.5% of circulating mononuclear leukocytes are has been found that induces a similar antibody, and EBV -infected during the acute illness. (130a) The ap• no other viral antibody has been demonstrated dur• pearance and persistence of EBV in the oropharynx ing heterophile-positive infectious mononucleosis. following mild or asymptomatic infections provides The occurrence of some heterophile-negative cases a large pool of healthy carriers capable of transmit• of infectious mononucleosis due to EBV has also ting infection through appropriate exposure. been noted in prospective studies. (50,65) Other mono• Efforts to transmit infectious mononucleosis to like syndromes are due to cytomegalovirus and volunteers using blood, throat washings, or stools other agents. (44a,92) from acutely ill patients were made prior to the dis• EBV-specific antibody of the IgM class has been covery of EBV in 1968; the results were largely in• demonstrat2d during acute infectious mononucleo• conclusive or unsuccessful, probably because of the sis and found to disappear during convalesct nce, presence of prior immunity in tnose inoculated.
Table 1. Summary of 11 Prospective StudIes of Epstein-Barr Virus Infection in Children and Young Adultsa
Subsequent rate/IOO per year
EBV antibody status at Clinical infectious start Number Percentage EBV infection rnononuc1eosisb
With antibody 3733 70.7 o o Without antibody 1547 29.3 16.4 7.1
TOTALS: 5280 100 4.6 2.0
, From ten studies carried out by Yale investigators(SO,132,148) and one by an English team, (148) b Clinical infectious mononucleosis was recognized in 47% of those infected with EBV, Chapter 10 • Epstein-Barr Virus 259
However, there are a few exceptions. Wising(156) 5. Descriptive Epidemiology successfully transmitted the full-blown disease to a female volunteer by transfusion. Evans(37) and Tay• 5.1. Prevalence and Incidence lor(145) reported suggestive evidence of successful transmission by inoculation of pooled sera from pa• The prevalence of antibody to the VeA of EBV has tients with acute infectious mononucleosis into pa• been determined in many countries and in many tients with acute leukemia as a therapeutic effort to age groups. (42) Figure 1 indicates the percentage of induce a remission; the young age of this group children in several areas of the world with EBV an• probably meant that some were susceptible because tibody. In developing and tropical areas, most chil• they had not been previously exposed. About 50 dren have been infected by age 6 years. Because other experiments in humans were equivocal or un• infections with EBV are often mild and asympto• successfuly,34,36,112) Similarly, earlier efforts to in• matic in young children, infectious mononucleosis duce infectious mononucleosis in monkeys were not may not be commonly recognized as a clinical entity rewarding. (49,13 7) in such countries. However, more intensive clinical Recent studies with this virus in humans have and serological studies, especially employing newer been very limited because of concern for the onco• diagnostic techniques, have permitted identification genicity of EBV. Grace et al. (61) repeatedly inoculated of both heterophile-positive and heterophile-nega• partially purified EBV into a terminal cancer patient tive cases of infectious mononucleosis in children who lacked prior antibody; both EBV and hetero• in such settings as Singapore(12a) and Brazil. (11 7a) The phile antibodies developed. Inoculation of EBV-in• prevalence of EBV antibody in young adults living fected lymphocytes into gibbons has resulted in an in different parts of the world is depicted in Fig. 2. exudative tonsillitis and the appearance of EBV an• A similar socioeconomic pattern exists. It is only tibodyy54) Shope and Miller(135) have induced tran• when a significant percentage of the population sient EBV and heterophile antibody in squirrel mon• reaches ages 15-25 before exposure to and infection keys inoculated with virus-transformed leukocytes. with EBV that infectious mononucleosis emerges as The current evidence of successful transmission of an important clinical entity. This delay in exposure infectious mononucleosis to monkeys must be re• is largely limited to nations with high economic and garded as incomplete at this time. hygienic levels and to middle and upper socioeco• In summary, the results of seroepidemiological, nomic classes in any country. The most susceptible virological, and transmission studies in man and college group tested thus far were entering fresh• monkeys indicate that EBV is the cause of all cases man students at Yale University in the period of heterophile-positive mononucleosis and most 1958-1963, when nearly 75% were at risk to infec• heterophile-negative cases. (40-42,44a) tious mononucleosis because they lacked antibody;
PERCENT POS ITIVE 10 20 30 40 50 60 70 80 90 100
BARBADOS
UGANDA
INDONESIA
MEXICO
HAWA II
BRAZIL
FRANCE
SWEDEN
Fig. 1. EBV antibody prevalence at age ENGLAND 4-6 years in different populations. CONN. Adapted from Evans. (42) 260 Chapter 10 • Epstein-Barr Virus
NO PERCENT WITH ANTIBODY TESTED 1-""10=--720~3::'0~4:-:0~!lC::0"';"':"6:-::0~7~0~80';'-:-:::-90:;:--,"10""'0~
MILITARY RECRUITS COLOMBIA 303 100~ ARGENTINA 120 ~~~~~~~~~~~~~9~8~1 ~ UN ITED STATES 484
COLLEGE FRESHMEN
THAILANDII I 45 "':::::':':"::':'::W~:::"::;~::',,:::'''W::::::::@::::: :'W,;':::::::@; ;::W;:;: 100; ;:;::
PHILI PPINES 122 ,:,::,,:::·:'::::;:;(~::;:;;:;:ml::;:;;;W!(;:::::;m~;::';: ;::·::::;:;:;:::::i;:;~:;:; ;:;:;:: 82
U.S. ' WEST PO INT 140 t :.. '''~.'X:.:... .:::::::::::: ::::::::::::::: ::::::::::::: :::::::::::::::~::~:::::l 63
U.S.' YALE GIRLS ('70) I 39 :;:;:: ;:;:::: :.:.:.:.:.:.:.:.:.:.:.:.:::,.:.:.:.:.::::; :::::::::::':::::~':'I 60
U.S.' YALE BOYS ('68) 35 5 ::,:.: :::::::t<::::::::~"":::::::::~m::::':·:·:::::::"" 51
NEW .ZEALAND ('69) 5 0 ...::.:.:.:.,.:.:.:.:.:.:.:.:.:.:.: ::::::;::":::::::;::";':::;1 48
U.S. ' SMITH GIRLS ('SO) 8 7 ~ffl::::::::&;:::::::::::::::::; ::::::::::::: 38
U.S.' YALE BOYS <'58-'63) 424 :"'::.:'::::::' :.:.:::::::,,;:,: 26
Fig. 2. EBV antibody prevalence in young adults in different populations. (x) Student nurses. Adapted fro m Evans. (42)
coincident with programs that broadened admission were recorded in 1972. (21) In 1979, a simplified report to include students with widely differing socioeco• form and an aggressive surveillance system were nomic backgrounds, among them many minority initiated for all reportable diseases, resulting in a groups, the susceptibles decreased to 40-50%. In rate for infectious mononucleosis of 137.7 per contrast, under 20% of undergraduate students at 100,000. (21a) This rate of infectious mononucleosis the University of Philippines lacked EBV antibody, is surpassed only by chicken pox and gonorrhea and all of 145 freshman student nurses in Thailand among reportable diseases in Connecticut for 1979 had antibody. and greatly exceeds mumps, rubella, measles, and Hospitalization rates for infectious mononucleosis hepatitis (Table 2). As comparison, the United States in the armed forces range from 148 to 250 per rates for these communicable diseases per 100,000 100,000. (41) In the Navy and Marine Corps, for which in 1978 were as follows: gonorrhea, 464.7; chicken comparative data are available, it ranks as the fifth pox, 80.4; hepatitis A, 13.5; measles, 12.3; primary most common infectious disease and the fourth and secondary syphilis, 10.0; and rubella, 8.4. most common cause of days lost. Among college students, the rates of infectious The incidence of clinical infectious mononucleosis mononucleosis are very high, averaging 840 per is not well documented, since reporting is not oblig• 100,000 students in 19 colleges in 1971-1972.(18) atory in most states in the United States, and the The most accurate measure of EBV infection and available data have usually been derived from spe• of disease has been obtained in prospective sero• cial surveys such as the community survey in At• logical and clinical studies, where the number of sus• lanta, Georgia, where a rate of 45 per 100,000 of ceptibles, the infection (seroconversior) rate, and heterophile-positive cases was found, and in Olm• the clinical attack rate can be critically defiled. Com• stead County, which includes the Mayo Clinic, parative data are available from three prospective where resident rates were 200 per 100,000. In Den• investigations of freshman students: at Yale Uni• mark, the rate of notified cases is 60 per 100,000. (131 a) versity, (132) at five English colleges and universi• The state of Connecticut requires reporting of in• ties,(148) and at the U.S. Military Academy at West fectious mononucleosis, and rates of 48 per 100,000 Point, New York. (65) As depicted in Table 3, the Chapter 10 • Epstein-Barr Virus 261
Table 2. Reported Cases and Cases per 100,000 idence on the influence of psychological factors is Population for Infectious Mononucleosis and emerging(88a) (see Section 5.11). Other Reportable Diseases in Connecticut for 1979 a S.2. Epidemic Behavior Reported Disease cases Cases/1 00, ()()(t True epidemics of infectious mononucleosis that fu'lliII appropriate dia~ostic criteria have not oc• Chicken pox 11,525 363.0 curred in modern times.138,82) Earlier, many pur• Gonorrhea 9,627 303.2 ported epidemics were described, of which the most 4,371 Infectious mononucleosis 137.7 impressive are those described by West(155) in the Rubella 258 8.1 United States in 1896, by Moir(lQ7) in the Falkland Mumps 231 7.3 Hepatitis B 213 6.7 Islands in 1930, and by Carlson et al. (15) in Wisconsin Hepatitis A 185 5.8 in 1926. More recent and suggestive outbreaks have Measles 4 0.1 been described from an Emergency Medical Hos• pital(64) and from Oxford, England, reported by , Derived from data provided by the Connecticut State Department Hobson et al. (83) A small outbreak involving 9 of 29 of Health. b Based on a population of 3,174,784. staff members in an outpatient clinic was recently reported by Ginsburg et al. (59c); however, the source of the outbreak and means of spread were not iden• incidence rate of EBV infection was strikingly similar tified. in all three settings: 12-13% of susceptible students The high incidence in military camps during were infected with EBV during the freshman year, World War II probably reflects the rapid turnover and of those with known EBV infection, 27.7-74.0% of large numbers of men. (46,149,153) Some reported developed clinical infectious mononucleosis. At the hospital "outbreaks" are suggestive of a true out• U.S. Military Academy, where a prospective inves• break, (64,83) but in general do not fully meet diag• tigation was carried on in a single cohort of fresh• nostic criteria, On a hypothetical basis, the early men over 4 years, the EBV infection rate in suscep• acquisition of immunity to infectious mononucleosis tible cadets was 12.4% in the first year, 24.4% in the by mild and inapparent infections with EBVin child• second year, 15.1 % in the third year, and 30.8% in hood and the probable route of transmission via the fourth college year. (65) Over the 4-year period, intimate oral contact in young adults weigh heavily 45.9% of susceptible cadets were infected with EBV, against the occurrence of "epidemic infectious mono• and 26.4% of these were known to have clinical nucleosis," infectious mononucleosis; others may have been ill The high prevalence rates of EBV antibody in chil• but not have reported to the clinic for treatment. dren in developing countries, (42,48) in nurseries, (124) The reasons for the varying rates of clinical expres• and in orphanages(147) suggest that EBV spreads sion among EBV-infected young adults in similar effectively in young children under circumstances settings are not known. The variation may be related of crowding and poor hygiene to reach almost all to the intensity of clinical surveillance, the students' susceptibles. However, the contagiousness of infec• attitude toward the health service, the average time tious mononucleosis has been notoriously low in of hospitalization, or various host factors. Some ev- young adult populations; secondary cases have
Table 3. Epstein-Barr Virus Infection Rates during Freshman Year and Percentage Clinically Expressed in Different Colleges
Infection rate in Clinical infectious Place Number in study Susceptible (%) susceptibles (%) mononucleosis (%)
U.s. Military Academy(6S) 1401 36 12.3 27.7 Five English schools(148) 1487 43 12.0 59.1 Yale University(132) 355 49 13.1 74.0 262 Chapter 10 • Epstein-Barr Virus
been rare in roommates of index cases, (37,65,79) in revealed a very high level of prior immunity. The college dormitories,
a~ ~ 100 0-' _. _ . .o-.-.-.-o-.-.~ j: z ",,- c ~ 80 .",' '" ILl 0'" ...oII: ILl 60 > j: iii NO. TESTED ~ 40 _. Conn. 1701
...Z A-~ Hawaii (Oahu) 304 ILl o c>.-o Barbado. 248 ffi 20 IL
Fig. 3. Acquisition of EBV antibody by age in three different areas. Data derived in part from Evans(42) 4 8 12 16 20 24 and Jennings. (84) AGE (YEARS)
5.5. Age France, (138) and Denmark, (146) on heterophile-posi• tive cases identified in state public-health labora• The acquisition of EBV antibody by age is shown tories, (25,38,108a,109,131a) and on recent community sur- in Fig. 3 for three different geographic areas. An• veys in which the population at risk can be tibody occurs early in life in economically under• defined. (68,69) In results from the Atlanta community developed countries, often reaching close to 100% survey based on 575 heterophile-positive cases, the immunity by age 10. In a prospective study of EBV highest rate, 345.2 per 100,000, occurred in the infections in newborns living in Accra, Ghana, 81 % 15-19 age group and the next highest, 122.8, in the had acquired antibody by age 21 months, (lOa) but 20-24 age group; 27 heterophile-positive cases oc• none showed evidence of clinical infectious mono• curred in the 5-9 age group, and 4 in the 0-4 age nucleosis. (lOb) In contrast, clinical infectious mono• group. Figure 4 shows the distribution of cases in nucleosis is clearly a disease of older children and this study. A similar age distribution was observed young adults in economically developed countries, in the Wisconsin State laboratory data based on el• with its highest incidence in the 15- to 25-year-old evated antibody titers in sera from suspected cases age group. This has been true of data based on hos• sent in for heterophile testing. (38) The peak fre• pitalized cases in the United States, (55,68,69,95,110) quency was in the 20-24 age group, in which 29.6%
50 _MALE --- FEMALE ,Ii 40 , \ I I ) ( en 30 I W I en I « I u 20 ,I I I J 10 / /
Fig. 4. Cases of infectious mononucleosis, by age and 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36+ sex, metropolitan Atlanta, Georgia, 1968. AGE (YEARS) 264 Chapter 10 • Epstein-Barr Virus
of the sera were positive. Some cases occurred at 5.S. Occupation the extremes of age: 11.9% of the sera from sus• pected cases were positive in the 5-9 age group and Infectious mononucleosis is most often a disease 5.8% in the 65-69 age group. Use of more sensitive of the college student and of the white-collar heterophile tests and of the EBV IgM tests now per• worker. (55,110) It is these persons who are likely to mits an increasing number of cases to be identified escape infection until young adult life because of in childhood. (54a,59b,117a) The mean age of mononu- higher socioeconomic level or hygienic standards or cleosis cases was 18.4 years in Wisconsin(38) and 19.3 both. It is also common in the military.(30,4I) years in Norway. (I08a) The age pattern described is that of developed 5.9. Occurrence in Different Settings countries. In developing countries, the age distri• The Original cases of Pfeiffer(I26) suggested that bution will be shifted downward because of the the disease had a familial pattern, and this has been small number of children who escape infection until partially borne out by subsequent studies. Analyses an age when the host response is that of recogniz• of sera from the Cleveland family study(27,71) pro• able infectious mononucleosis. In a recent study in duced evidence of several cases in three of seven Sao Paulo, Brazil, the average age of 15 heterophile• families. There appeared to be a paucity of EBV negative patients was 4.7 years and that of 31 het• infections in the 6-12 year age group in this setting, erophile-positive cases was 13.2 years. (ll7a) The old• with higher rates observed in children under 6 and est case was age 15; 5 cases occurred in the 0-2 age over 12. Among 75 Canadian families, Joncas and group. In this setting, over 90% of the population Mitnyan(85) identified 67 persons lacking EBV anti• has EBV antibody by age 10.(17) body; in follow-up over approximately 2 years, only 10.5% of these susceptible persons developed EBV 5.6. Sex antibody. In Sweden, Wahren et al. (151) found EBV antibody increases in 7 of 21 members exposed to No difference in EBV-antibody prevalence rates an index case; 6 of the 21 contacts lacked EBV an• by sex have been noted in population surveys. tibody initially, and 3 of these seroconverted. Infectious mononucleosis occurs equally in both The high rates of infectious mononucleosis in col• sexes, although girls appear to develop the disease lege and military settings have already been noted. earlier than boys, (68,69) with a peak occurring in girls However, during the early recruit-training period at age 16 and boys at age 18 (see Fig. 4). in the armed forces, infectious mononucleosis is not a common disease, unlike adenovirus, Mycoplasma pneumoniae, and other respiratory infections. This 5.7. Race is probably due both to a high level of preexisting EBV infection occurs in all ethnic groups; no evi• immunity among recruits and to the long incubation dence of differential susceptibility has been found. period of about 4-7 weeks, so that cases usually Infectious mononucleosis in developed countries develop after the end· of the usual training pe• has been rare in blacks, but this probably reflects riod and after dispersal of recruits to other mili• socioeconomic levels and earlier acquisition of in• tary assignments. The former point is supported by fection rather than any difference in susceptibility. the finding of an antibody prevalence rate of 85% The incidence of the disease in whites in Atlanta, in entering Marine recruits at Parris Island(94) and Georgia, was 30 times higher than in blacks. (68) An• of 93% in Army recruits at Fort Jackson, South Car• tibody prevalence to EBV among entering black ca• olina.(5I) Among the Marine recruits at Parris Island dets at the U.S. Military Academy was 85% as com• whose sera lacked antibody, the infection rate was pared with 65% among whites. (65) In an analysis of 18.5 per 1000 over the 16-week training period; in prevalence rates among different ethnic groups in those returning from a 13-month overseas assign• Hawaii, (84) higher rates were observed in Hawaiians ment, the EBV infection rate was estimated at 23.8 and Filipinos than in Caucasians of the same age. per 1000. Among the 34 Fort Jackson recruits who However, socioeconomic levels, hygienic habits, lacked EBV antibody, 3 recruits developed EBV an• and varying cultural practices in the home cannot tibody during the 16 weeks of basic and advanced be separated from the ethnic backgrounds. training/51 ) a rate of 88 per 1000 recruits. Chapter 10 • Epstein-Barr Virus 265
5.10. Socioeconomic Factors mononucleosis in cadets infected with EBV(8Sa): (1) having fathers who were "overachievers"; (2) hav• Socioeconomic settings influence the incidence of ing a strong commitment to a military career; (3) both EBV infection and infectious mononucleosis, ascribing strong values to various aspects of the but in opposite directions. Low socioeconomic training and military career; (4) scoring poorly on gouP~ have bi&1"' Tatps of EBV infection paTh' in life, indices of relative academic performance; and (5) but little clinical infectious mononucleosis; high so• having strong motivation and doing relatively poorly cioeconomic groups have low levels of EBV infection academically. The same factors seemed to influence earlvln life, but a high rate of clinieal disease that development of heterophile antibody during mild occurs in the 15- to 25-year-old group. Two exam• or inapparent EBV infections, a result free of bias ples illustrate this eUect on infection rates. At the toward seeking medical care. U.S. Military Academy at West Point, the EBV-an• tibody prevalence rate was 77.1 % in cadets coming from families earning under $6000 and only 58.6% 6. Mechanism and Route of Transmission among those from families with incomes of over $30,000. (65) In New Haven, the antibody prevalence among first-graders in three schools serving a low The major route of transmission of infectious socioeconomic group was 84.8%, and in three mononucleosis in young adults is probably through schools serving a high socioeconomic group, it was intimate oral contact in kissing with the exchange 37.8%. (136) A second serum sample collected from ot saliva. as first suggested by Hoagland(19jTn1955. these same children 4-5 years later revealed an EBV This concept is supported by three types of circum• seroconversion rate of 50% among susceptible chil• stantial evidence: First, close personal contact with• dren from lower socioeconomic areas and only 2.4% out kissing, as in roommates of infected patients(132) or in such confining environments as a destroyer(122) in susceptible children in the higher socioeconomic group. or a Polaris submarine, rarely leads to secondary cases(142); this has been true even when the exposed roommate is known to lack EBV antibody and is 5.11. Other Factors followed closely over 2 months for the appearance of antibody or of clinical symptoms. (65) Second, a Little is known of the role of nutritional and ge• history of intimate oral contact within the appro• netic factors in relation either to EBV infections or priate incubation period is common in young adults to infectious mononucleosis. However, it is recog• who develop infectious mononucleosis(79) and oc• nized that ABO blood groups are not correlated with curs statistically more frequently than in healthy susceptibility to infection or to clinical disease. (65,132) controls or patients with acute respiratory infec• The relationship to HLA antigens has not been tions. (37) Third, the presence of IiBV has been dem• clearly established. (132b) It seems likely that genetic onstrated in the pharynx during acute illness and control of the immune response plays a role in the during convalescence for periods of many months severity of clinical illness, in the persistence of virus, (Table 4). (19,59;nIT.TPm] In addition, cross-sectional and in possible oncogenic sequelae as suggested in studies of presumably healthy adults have also recent studies of EBV infections in the X-linked lym• shown EBV pharyngeal excretion in 15-20% of phoproliferative syndrome. (128a,b) young adults. (59.143) One investigation found a leu• Psychological and behavioral factors may influ• kocyte-transforming factor, presumably EBV, in the ence the frequency with which clinical infectious throats of 18% of 368 patients attending an outpa• mononucleosis develops after EBV infection. By cor• tient clinic. (20) Transmission of EBV infection may relation of psychological scores taken on entry into also occur via blood transfusions, usually without the school and subsequent academic achievement illness. (58,75,150) with the prospective serological and clinical data This prolonged carrier state after clinical infec• collected in cadets at the United States Military tious mononucleosis and tollowinginaprarent EBV Academy at West Point,<65) the following factors intection may serve as the principal source of ex• were found to be significantly associated with an posure in young adults. The long duration ot ViruS increased risk of development of clinical infectious excretion may explain the difficulties in tracing 266 Chapter 10 • Epstein-Barr Virus
Table 4. Recovery of Epstein-Barr Virus from mate is based on well-defined, often single contacts Oropharyngeal Excretions of 32 Patients with between an index case and a member of the opposite Infectious Mononucleosis sex involving intimate oral contact. Throat washings Studies of the recovery of EBV from oropharyn• geal secretions of patients with infectious mono• Positive nucleosis have revealed that virus shedding occurs Number during acute illness and from several weeks to Days after onset tested Number % months after onset, but the type of cells involved 0-6 5 20.0 in viral maturation and release is unknown. Trans• 7-14 20 15 75.0 formation of umbilical-cord leukocytes into contin• 15-21 8 5 62.5 uous cell lines has been the assay system used for 22-28 10 6 60.0 demonstration of the virus, and this transformation 29-60 13 8 61.5 has been neutralized by sera containing EBV anti• 61-150 12 11 91.7 body, but not affected by. sera lacking this anti• >150 19 6 31.6 body. (103) In addition, transformed leukocytes ac• quire EBV genome, demonstrated by nucleic acid TOTALS: 87 52 59.7 hybridization, and express EBV-associated anti• gens. The virus has been detected in throat wash• ings for prolonged periods and as indicated in Table transmission of disease from case to case. Virus ex• 4 is regularly recovered several months after clinical cretion occurs in the presence of circulating anti• illness. In 6 of 19 patients, the agent was still present body, which suggests that humoral antibody does over 5 months after disease had occurred and in 1 not have a major role in the regulation of orophar• case was detected 24 months after onset. No special yngeal shedding. Clearly, identification of the spe• clinical characteristics have as yet been identified in cific oropharyngeal cells that produce infectious those cases associated with prolonged oropharyn• virus remains an important area for further inves• geal virus shedding. tigation. EBV was not found in the urine of 10 acute Prior to the onset of definite symptoms in young cases or in the cervix of 175 pregnant or postpartum adults, there is frequently a history of ill-defined women. (149c) complaints, such as malaise and easy fatigue. It has The mechanism of transmission accounting for been suggested that an early, abortive infection of the rapid and high rate of acquisition of EBV anti• this type may occur in children without subsequent body in nurseries and in young children in low so• development of classic infectious mononucleosis. (37) cioeconomic circumstances(59a,124,147) is not defi- In an analysis of 100 presumed heterophile-negative nitely known. Presumably, transfer of infected cases of infectious mononucleosis involving the 0-9 saliva on fingers, toys, and other inanimate objects year age group studied in England, (83) the incuba• in settings of poor hygiene can account for much of tion period was shorter than for adults and esti• the spread of infection. Perhaps more cell-free virus mated at 4-10 days. is released in childhood infections. A recent report The pathogenesis of infectious mononucleosis is of hepatitis due to EBV in a hemodialYSis unit raises intriguing not only because the self-limited clinical the possibility of airborne spread. (21b) disease is manifested primarily in young adults but From a practical standpoint, the low contagious• also because of the suspected oncogenicity of the ness of the disease in young adults eliminates the virus and its relationship to African Burkitt lym• need for strict isolation procedures. phoma and to nasopharyngeal carcinoma. (32a) An understanding of what immunological mechanisms turn infectious mononucleosis on and what mech• anisms turn it off is important in this context. 7. Pathogenesis and Immunity Transient depression of delayed hypersensitivity has been described during acute infectious mon• The incubation period of infectious mononucleo• onucleosis,<1O·62) and depressed T-cell stimulation(l34) sis is 4-7 weeks(37,81) in college students. This esti- by phytohemagglutinin has been recorded. Re- Chapter 10 • Epstein-Barr Virus 267 cently, profound alterations in cell-mediated im• A schematic diagram of the possible pathogenesis munity were demonstrated by intradermal skin of infectious mononucleosis (Fig. 5) summarizes a tests, in vitro lymphocyte stimulation, and enumer• hypothetical concept. EBV enters the oropharynx ation of absolute numbers of peripheral-blood T and through salivary transfer, probably by kissing in B cells. (97) Lymphocyte responsiveness to a variety young adults or saliva-contaminated objects in of mitogens and antigens was found to be depressed young children. It multiplies locally, presumably in during the first weeks of illness. Serial studies of epithelial cells,(94a) possibly cells of the salivary the interaction of T- and B-cell populations during gland. (94a,107b) A sore throat accompanies this, often acute disease indicated that peripheral-blood B cells with exudative pharyngotonsillitis. A persistent, in• increase during the first week of illness and return termittent oropharyngeal excretion over many years to normal levels several weeks later. In contrast, T occurs in 15-20% following either apparent or in• cells reach peak values during the second week of apparent EBV infection, (20, 114b) which may be greatly disease and remain elevated for approximately 5 increased during immunosuppression. (143) The local weeks. (97) lymphatics are probably invaded, resulting in cerv• Recent investigations indicate that both T andB icallymphadenopathy. The virus enters the blood• cells marbe transformed into atyPicallymphocvt~s stream from one of these sources, involves B lym• characteristic of~ this disease. (31,f>U,~,I7,12UJ These phocytes, and spreads hematologically to the liver, observanons suggest that B cells may hp ~ t= producing hepatitis, and to the spleen, causing sple• formed by infection with EBV and T cells trans• nomegaly; involvement of the brain, lung, or other formed as an immunological response to. the viral organs occurs rarely. antigen itseU or to altered antigens on the surtace EBV has at least three functional effects on B lym~ of the B celli. phocytes, although it is not known whether sepa-
PERSISTENT LYTIC INFECTION IN 15- 20% 1 ./ BLOOD ...... - UVER / 1 ~ SPLEEN -I SPLENOMEGALY I L-____-' B LYMPHOCYTE INTERACTIONS
~:E;T6~~~~~S~:M:J6~~T~:D ::f~'FERATION f----+ C3 RECEPTOR ON SURFACE (EBNA+B LYMPHS) '----.-----' (EARLY ATYPICAL LYMPHOCYTOSIS)
PROLIFERATION OF T CELLS LACKING FC RECEPTOR FOR 19G AND 19M (LATER ATYPICAL LYMPHOCYTOSIS)
ANTI• HETEROPHILE AUTO NON~ ACTIVATION EBNA ANTIBODIES ANTIBODIES EBV OF SUPPRESSOR EA ANTIBODIES T CELLS AND CLINICAL INFECTIOUS MONONUCLEOSIS 1 I MA MACROPHAGES
Fig. 5. Hypothetical pathogenesis of EBV infection. 268 Chapter 10 • Epstein-Barr Virus
rate B-cell populations are involved. One is due to number survive, however, to initiate long-term in the action of virus on a subset of specific antibody• vitro cultures years later. (32) The third functional ef• producing cells, the second results from infection fect of EBV on B cells is similar to that of pokeweed and transformation, and the third is a consequence mitogen, but- is probably due to infection of B cells, of polyclonal B-cell activation. The first leads to the rather than a surface effect, and results in a poly• production of EBV antibody. The second is a com• clonal B-cell proliferation; an increase in immuno• plex event related to the existence of EBV receptors globulin-secreting cells then occurs, synthesizing IgA on the surface of B cells near receptors for C3(85b); early, with IgD, then later IgM and IgG. Multiclonal the virus enters and multiplies in the B cell, pro• antibodies are stimulated via other B cells such as ducing EBNA-positive cells and a variety of antigens sheep-cell antibodies of the Forssman, and possibly such as early antigen, VeA, and EBNA, against of the Paul-Bunnell, type, plus other antibodies which specific antibodies are produced via other B unrelated to EBV. (87a,147a) These antibodies are prob• cells. The EBNA-positive B cell transforms and pro• ably not produced directly by EBNA-positive B cells. liferates, and this constitutes most of the early atyp• In the wake of these B-T cell interactions, there ical lymphocytosis of infectious mononucleosis. (97) appear suppressor T cells, and perhaps macro• In vitro, the transformed B cell is "immortalized"• phages, which inhibit the proliferation of B i.e., made capable of continual multiplication. (lOOa) cells(68a,147a) and probably underlie the severe anergy EBV induces neoantigens on the surface of some that occurs in infectious mononucleosis. To date, infected B cells, more from adults than children and EBV -specific suppressor T cells have not been dem• not in cord cells. (lOOa) These antigens include lym• onstrated, but they may exist. phocyte-determined membrane antigen (LDMA) While the appearance of transformed and prolif• and possibly heterophile antigen of the Paul-Bunnell erating B cells and of cytotoxic and suppressor T type, antibodies to which are formed by other B cells has been documented, their origin, specificity, cells. The neoantigens stimulate a B-T cell interac• interrelationship, and relationship to EBV await tion similar to a graft-vs.-host response, which may clarification. The proliferation of B cells, and the account for some of the clinical symptoms of the acute disease itself, are limited by cytotoxic T cells, disease. The mechanism of production of the clinical suppressor cells, and humoral antibody. The de• features of infectious mononucleosis is not well velopment of specific cell-mediated immunity to understood, but might result from these B-T cell EBV during infectious mononucleosis has been interactions, from immune complex deposition or demonstrated. (114b) A failure of these immune mech• other immunopathological events, from direct ef• anisms, either on a genetic basis, as in the X-linked fects of EBV on cells, or from some combination of lymphoproliferative syndrome of males, (128a,b) or these mechanisms. The rarity of classic infectious due to acquired immunodeficiencies, as possible in mononucleosis in early childhood might be related a fatal case in a 4-year-old girl, investigation. For example, EBV-infected fetallym• pharyngitis, and cervicallvmphadenopathy,_ accom• phocytes do not have demonstrable VCA, but do pamea oy splenomegaly in 50% and hepatomegaly contain EBNA by the EBNA test(lOU29) and CF an• in 10%. The pharyngitis is often associated with a tigen. (10U04) In lymphocytes from adults and from whitish or a gray-green exudate having an offensive marmosets, EBV may mature more fully, resulting odor. The eyelids may be swollen, and petechiae in release of EBV antigens to other lymphocytes. occur on the hard palate in 25% of cases. Heterophile antibody may occur in response to Abnormalities of liver-function tests are a regular membrane-induced antigens of EBV expressing feature of infectious mononucleosis, and clinically themselves more fully in lymphocytes of young recognizable jaundice occurs in 5% of cases. Rare adults than in those of younger children or in fetal manifestations include a variety of central nervous lymphocytes. There is preliminary evidence of het• .. ystem syndromes (encephalitis, meningoence• erophile-antibody production by lymphocytes cul• phalitis, Guillain-Barre syndrome), pneumonitis tured from acute cases of infectious mononucleo• and pneumonia, thrombocytopenic purpura, my• sis, (107a) and the various possible mechanisms of its carditis, and nephritis. (39,82) Hepatitis and central appearance have been reviewed by Kano and Mil• nervous system involvement may occur in the ab• grom.(87a) sence of other features of infectious mononucleo• While the presence of antibody to the VCA of sis. (29a,61a) The major complications include sple.l1.ic EBV has been shown to indicate protection against rupture and airway obstruction from exudative infectious mononucleosis and its absence indicates pharyngotonsillitis. About 50 deaths have been re• susceptibility,<44,50,65,113,132) the actual antibody that ported due mostly to central respiratory failure; re• provides immunity is probably the neutralizing an• cently, cell-associated EBV was detected in the cer• tibody for which tests have recently been devel• ebrospinal fluid of a case with complicating oped. (78,105) One attack of infectious mononucleosis meningoencephalitis. (132a) An immunological defi• confers a high degree of durable immunity to sub• cit, especially in cell-mediated immunity, may in• sequent attacks of clinical infectious mononucleo• fluence the severity of the clinical response, as sug• sis. (16,50,105) Presumably, subclinical or inapparent gested by the report of two deaths in a family(5) and EBV infections also confer lasting immunity. One a recent severe illness in a women with a T-cell fairly well-documented case of clinical recurrence defect. (136a) An X-linked recessive lymphoprolifer• has been reported, (9) and a resurgent anamnestic ative syndrome in which EBV has been implicated heterophile response after infectious mononucleosis has also been described by Purtilo et al. (128a,b) Both has also been noted in patients who subsequently lytic and proliferative manifestations have appeared develop a respiratory infection. (8,80) Reinfection with in one kindred. These include fatal infectious mono• or without clinical illness has not yet been fully doc• nucleosis, malignant lymphoma, and agamma• umented by appropriate heterophile and EBV an• globulinemia. tibody tests, but asymptomatic endogenous reacti• The frequency with which EBV infections are ex• vation was found in 32% of renal-transplant pressed as clinicaTillness in young adults has varied recipients. (Ia) . m aurerem popUlanons. In a sniay or a conort of U.S. Military Acaaemy caaets over a 4-year period, only 26.4% of 201 infected with this agent developed 8. Patterns of Host Response heterophile-positive clinical infectious mononucleo• sis. (65) The apparent: inapparent EBV infection ratio 8.1. Clinical Features in different years ranged from 1: 1 to 1: 2.6 in this population. Comparison of the frequency of clini• When infection with EBV occurs in childhood, a cally expressed infectious mononucleosis in fresh• mild, nonspecific illness or an inapparent infection man students in three different settings is presented may develop, both of which are associated with the in Table 3 (Section 5.1). As mentioned, the reasons appearance and persistence of antibody to EBV. If for the differences are not known, by may relate to exposure and primary infection are delayed until the motivation to seek medical care, phYSical fitness, adolescence or young adulthood, the characteristic psychological factors, or concern about the effect of clinical picture usually occurs. This conSIsts ot fever, hospitalization on academic and school activities. 270 Chapter 10 • Epstein-Barr Virus The relationships between clinical features and fever, sore throat, and cervical lymphadenopathy, antibody levels in a typical heterophile-positive case the occurrence of at least 50% lymphocytosis with in an 18-year-old student are shown in Fig. 6. Fol• at least 10% atypical lymphocytes, and the appear• lowing a prodromal period associated with fatigue, ance of heterophil antibodies. The presence of IgM fever, and headache over several days, the onset of or early antigen antibodies to EBV, or both, is an sore throat, cervical adenopathy, and recurrent absolute requirement in doubtful or heterophile• fever developed during the second week. Charac• negative cases. Antibody to VCA is usually present teristic blood changes were present on the 3rd day at the time the physician first sees the patient, and after onset, and on culture the patient's lympho• only 15-20% of patients will show a subsequent rise cytes contained EBNA CF antigens in a nuclear lo• in titer; antibodies to early antigen appear later, but cation. The heterophile-antibody titer was negative are present in only 75% of typical cases. (77) Anti• on the first day of symptoms, rose to 1: 14 after bodies to EBNA usually arise 1 month or more after guinea pig absorption 2 days later, and then in• illness and probably persist for life. (73) EBV -specific creased to 1 :896 on the 15th day. In contrast, Ep• IgM antibodies are demonstrable in 85% of cases stein-Barr VCA antibodies of IgG type, undetecta• during acute illness. Figure 7 depicts the course of ble on the 3rd day of illness, were present on the EBV -specific IgG and IgM antibodies during the 8th day and rose to a level of 1 : 320 by the 2nd week. course of illness. IgG antibodies persist for years, EBV -specific IgM antibodies were demonstrable on perhaps for life; IgM antibodies usually disappear the 8th day at a titer of 1 :2.5, which then increased in 3-6 months. At present, the EBV-VCA-IgM is the to 1: 10 by the 15th day. best antibody test for diagnosis in cases that are No direct correlation has been found between the heterophile-antibody negative(29,52,59b,114C); the pres- levels of Epstein-Barr VCA and heterophile anti• ence of false positives due to rheumatoid factor must bodies or between VCA, early antigen, and EBNA be excluded. antibody levels and the severity of clinical symp• The main reliance in diagnosis must be placed on toms and hematological changes. (73,77,114) Neither the heterophile antibodies, which are of the 115M does their persistence correlate with the duration of type. MetfiOds in most common use are the-sheep• clinical illness. and horse-cell agglutination tests after absorption of the serum with guinea pig kidney to remove 8.2. Diagnosis Forssman antibody, and the beef-cell hemolysin The diagnosis of infectious mononucleosis is test, which does not require absorption. The ap• based on a typical clinical picture with the triad of pearance and persistence of these tests during acute SORE THROAT TO OF FATIGUE EDEMA OF INFIRMARY 104 _ADENO. EYELIDS I 103 CLINICAL 102 ONSET 101 6/Y172 BETTER __ SCHOOL 100 99~~~~~-L~~~~~L-~~~~~~~~~~~~ WBC 26.5 12.2 LYMPHS + M 28 38 45 15 ATYPICAL 27 27 30 38 HETEROPHILE NEG 1-14 Mono Spol + 1-896 EBY - IGG <1-5 <1-5 1,80 1-320 80 EBY - IGM 0 0 1-2,5 1'10 EBY IN THROAT 0 + + LYMPHOCYTE Fig. 6. Relationship between clin• + (SHOWN TO CONTAIN EBY) GROWTH ical and laboratory features of in• 5 10 15 20 fectious mononucleosis in an 18- DAY OF DISEASE year-old male. Chapter 10 • Epstein-Barr Virus 271 100 90 ~II I I I 80 10 1\1- 70 i I I I&J > 60 I I I ~ Ul 50 0 I I I Fig. 7. Appearance and duration of IgG and IgM antibod• 0.. ies specific for EBV during infectious mononucleosis. From 40 I- I I I Evans et al. (52) z I&J 0 30 II:: I I I&J 0.. 20 I I 10 Gl 0 0 PRE 2 3 4·6 7-9 10-12 13-18 ~19 NUMBER OF MONTHS AFTER ONSET OF ILLNESS and convalescent infectious mononucleosis are shown pearance and persistence of antibody to horse cells in Fig. 8. The beef hemolysin test is the most specific have been found to follow mild and subclinical ep• but has a short duration; the horse-cell test is the isodes of infectious mononucleosis provided that most sensitive and most persistent, with positive sera are collected over a long enough time. This test tests present for a year or more in 75%. (52) The ap- is useful in childhood infections with EBV, which 100 I II:: I&J I I J: 90 HORSE RBe ~ J: 80 I I c z « 70 I or0 I - 60 !;i 50 Fig. 8. Persistence of heterophile antibodies during infec- I&J tious mononucleosis. (RBC) Red blood cells. From Evans > 40 et a/(52) t: Ul 0 30 0.. « II:: 20 I&J (/) 0 10 ~ 0 0 #- 0 PRE 2 3 4-6 7-9 10-1213-18 ~19 NUMBER OF MONTHS AFTER ONSET OF ILLNESS 272 Chapter 10 • Epstein-Barr Virus are often heterophile-negative by other tests or ilar humoral, cell-mediated, and local immunity when inadequately followed. A new immune ad• might be highly effective. An attenuated live vaccine hera nee heterophile test may also be very useful in administered orally would be the most desirable. childhood infections. (S4a,94c) However, the apparent limitation of viral multipli• Development of EBV antibody has also been cation in vitro to primate lymphocyte suspension shown in cases where the clinical and hematological cultures and the low yield of infectious virus re• characteristics are those of infectious mononucleosis leased are formidable technical obstacles at present. but the heterophile antibody remains persistently Work on EBV-membrane antIgens is under way to negative. (SO,114b) These heterovhile-nee:ative. EBV• circumvent some of these problems. (123,I28c) antibody-positive cases appear tooecommon m m• If an effective vaccine were available, it might best fantsa:nd children.: but are rare-In adults.llUa.b,~~a) be given on entrance to high school to permit natural lnrection with cytomegalovirus (LMV)may produce infection with little clinical illness to occur before a clinical picture of heterophile-negative mononu• that and then to prevent clinical illness in the young cleosis that is hard to distinguish from classic infec• adult. It would be useful only in developed coun• tious mononucleosis; however, it usuallv occurs at tries with a high incidence of infectious mononu• a later agt:. and adenopathy and exudatIve phar• cleosis. yngitis are rare. (91) EBV may possibly be reactivated The oncogenic and transformin~ potential of FRV during the course of other herpesvirus infections, . poses a senous question of risk for use of a vaCCine. especially CMV. (94b) Infection with EBV can result The numerous problems associated with long-term in development of a false-positive CMV-IgM-anti• surveillance for possible complications would be body response; however, the reverse does not considerable, but these risks in developed countries occur. Toxoplasma gondii, adenovirus, rubella, and may not be great. The association between EBV and hepatitis A infections may also resemble hetero• cancer is evident primarily in Burkitt lymphoma in phile-negative infectious mononucleosis, but the Africa and in nasopharyngeal carcinoma (NPC) pa• lymphocytosis is relative and not absolute. The in• tients of Chinese descent in the Far East. In these fectious-mononucleosis-like syndromes have re• settings, clinical infectious mononucleosis is too rare cently been reviewed. (44a) a disease to merit vaccination. Furthermore, tumor development is associated with malaria in African Burkitt lymphoma and immunogenetic susceptibil• 9. Control and Prevention ity to NPC in Chinese. In the absence of malaria and genetic susceptibility, as in the United States, both Attempts to control infectious mononucleosis and these tumors are very rare. Such considerations sug• EBV infections by interrupting the presumed chain gest that if the technical problems of viral attenua• of transmission in young adults seem neither real• tion and vaccine production can be overcome, on• istic nor perhaps desirable in light of our current cogenicity would be a hazard of negligible magnitude knowledge. If salivary exchange represents the in the United States and other countries where clin• major route of spread, there seems little likelihood ical infectious mononucleosis is a common and dis• of interdicting this practice. If poor hygienic con• abling disease. ditions promote the spread of EBV infections in young children, then improvement in hygienic and socioeconomic circumstances might reduce their in• cidence. Unfortunately, control of spread at this 10. Unsolved Problems time when infection is largely mild and asympto• matic might simply delay exposure to later child• The problems that remained to be solved con• hood and young adult life when the majority of EBV cerning the nature of EBV infections are summa• infections are expressed as clinical infectious mono• rized in Table 5. While EBV is well established as nucleosis. the cause of heterophile-positive infectious mono• The high degree of protection against infectious nucleosis and "turns on" the immunological events mononucleosis provided by natural infection with that are involved in the pathogenesis of the clinical EBV suggests that a vaccine capable of evoking sim- disease, we need more information as to what Chapter 10 • Epstein-Barr Virus 273 Table 5. Unsolved Questions Concerning Epstein-Barr Virus 1. What turns infectious mononucleosis off and makes it a self-limited disease? 2. What cell supports pharyngeal multiplication of EBV? 3. What is the route of transmission in young children? 4. Where does heterophile antibody originate? 5. Can the EBV-IgM-antibody test be simplified for routine diagnostic use? 6. What causes different responses to EBV at different ages? 7. Is an infectious mononucleosis vaccine possible? 8. Does EBV cause cancer? 9. Why are EBV antibody titers high in some chronic diseases? 10. What accounts for the geographic distribution of Burkitt lymphoma and nasopharyngeal cancer? "turns off' the lymphoproliferative cY121e and makes brane antigens. The possibility of a vaccine has al• the disease a benign, self-limited one. ready been discussed, and the relationship of EBV The persistence of EBV in circulating lymphocytes to cancer is explored in other chapters of this book. following infection and the presence of a small num• Infectious mononucleosis continues to be an im• ber of "atypical lymphocytes" in healthy persons portant model for studying the immunological and suggest that the proliferative proct:ss is not fully virological events involved in a persistent and pos• "turned off" but held under careful control and im• sibly neoplastic infection. munological surveillance; the continued presence of anti-VCA, anti-EBNA, CF, and other viral antibod• 11. References ies supports the concept of continued antigenic stimulation. Viral excretion in the pharynx certainly 1. ARMSTRONG, D., HENLE, G., AND HENLE, W., Com• continues long after infection, perhaps intermit• plement-fixation tests with cell lines derived from tently for life, but the cells supporting this multi• Burkitt's lymphoma and acute leukemias, J. Bacterial. plication are at present unknown. The source of 91:1257-1262 (1966). heterophile antibody and of the variety of other non• 1a. ARMSTRONG, J. A., EVANS, A. 5., RAo, N., AND Ho, viral antibodies that appear during the course of M., Viral infections in renal transplant recipients, infectious mononucleosis remains a mystery; whether Infect. Immun. 14:970-975 (1976). these antibodies appear in milder EBV infections in 2. BAILEY, G. H., AND RAFFEL, 5., Hemolytic antibodies smaller amounts and at a later time is not known, for sheep and ox erythrocytes in infectious mono• but recent evidence suggests this may be so. A sim• nucleosis, J. Clin. Invest. 14:228-244 (1935). 3. BANATVALA, J. E., BEST, J. M., AND WALLER, D. K., ple EBV-IgM antibody test is needed in the diag• Epstein-Barr virus-specific IgM in infectious mon• nostic laboratory to confirm presumably hetero• onucleosis, Burkitt lymphoma, and nasopharyngeal phile-negative cases. An explanation is lacking for carcinoma, Lancet 1:1205-1208 (1972). the observation that a more severe clinical response 4. BANG, J., Forsoeg paa at overfoere mononucleosis to EBV infection occurs when exposure is delayed infectiosa til mennesket, Ugeshkr. Laeg. 105:499-504 until later childhood and young adult life, similar (1943). to the more frequent occurrence of jaundice in hep• 5. BAR, R. 5., DELoR, J., CLAUSEN, K. P., HURTUBISE, P., atitis and paralysis in poliomyelitis infections in HENLE, W., AND HEWETSON, J. F., Fatal infectious adults. If the clinical syndrome of infectious mono• mononucleosis in a family, N. Engl. J. Med. nucleosis is primarily an immunological response 290:363-367 (1974). of T cells to EBV-induced neoantigens on the B-cell 6. BARONDESS, J. A., AND ERLE, H., Serum alkaline phos• phatase activity in hepatitis of infectious mononu• membrane, then one can speCUlate that induction cleosis, Am. J. Med. 29:43-54 (1960). of membrane antigens occurs more commonly in 7. BAUSCHER, J. C, AND SMITH, R. T., Studies of Ep• lymphocytes from mature persons in whom more stein-Barr virus-host-relationship: Autochthonous complete virus is formed than in lymphocytes from and allogeneiC lymphocyte stimulation by lymphob• young children; this idea is supported by the failure last cell lines in mixed cell culture, Clin. Immunal. of EBV-infected cord cells to produce VCA or mem- Immunapathal. 1:270-281 (1973). 274 Chapter 10 • Epstein-Barr Virus 8. BENDER, C. E., Interpretation of hematologic and a human population, N. Engl. J. Med. 289:1328-1329 serologic findings in the diagnosis of infectious mon• (1973). onucleosis, Ann. Intern. Med. 49:852-865 (1958). 21. CHRISTINE, B. W., Infectious mononucleosis, Conn. 9. 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