Simultaneous infection with multiple herpesviruses

Simultaneous Infection with Multiple Herpesviruses

STANLEY M. LEMON, M.D.* LINDSEY M. HUTT, Ph.D. YUNG-TSUN HUANG, M.S. JAY BLUM. M.D.’ JOSEPH S. PAGANO, M.D. Chapel Hill, North Carolina

From the Division of Infectious Diseases, Department of Medicine, and the Cancer Research Center, School of Medicine, University of North Carolina, Chapel Hill, North Carolina. This study was supported in part by Grants POlCAl6086 and lPOlCA19014 awarded by the National Cancer Institute. Requkts for reprints should be addressed to Dr. Stanley M. Lemon. Manuscript accepted July 13.1978. * Recipient of National Research Service Award l-F32-AI05125. Present address: Department of Virus Diseases, Walter Reed Army Institute of Research, Walter Reed Army Medical Center, Washington, DC. 20612. + Present address: Pulmonary Unit, Massachusetts General Hospital, Boston, Massachusetts 02114.

270

Active dual infection with Epstein-Barr virus (EBV) and cytomegalovirus (CMV) was observed in four otherwise healthy persons with mononucleosis syndromes. A secondary serologic response to EBV occurred in three patients as determined by the presence of antibodies to EBV-induced nuclear antigen (EBNA) early in the illness. All four patients lacked heterophil antibodies; in the one case tested, immunoglobulin M (IgM) antibodies specific for EBV viral capsid antigen (VCA) were absent as well. The Guillain-Barr6 syndrome occurred in one patient, who also had active infection with herpes simplex virus 1 (HSV-1). In a fifth patient, herpes zoster developed complicating heterophil-positive infectious mononucleosis due to primary infection with EBV. These five cases demonstrate that mononucleosis syndromes may occur in association with dual or multiple herpesvirus infections and that reactivation of EBV may be common during heterophil-negative mononucleosis. Reactivation of latent virus is most likely related to depressed cellular immunity due to a primary infection with another herpesvirus. An alternate hypothesis is that viral DNA polymerase induced by infection with one herpesvirus might simultaneously permit the productive replication of a second herpesvirus previously latent within the same cell. Thus, reactivation may result from molecular interactions between viruses at the cellular level. The possibility of multiple infections must be considered whenever determining the specific viral etiology of heterophil-negative mononucleosis. Heterophil-negative infectious mononucleosis is frequently related to infection with cytomegalovirus (CMV) but less often occurs as a response to primary infect& with Epstein-Barr virus (EBV) [l-3]. Virus-specific diagnostic tests are required to determine which of these two herpesviruses is responsible for any individual case, although cervical lymphadenopathy,’ pharyngitis and hepatitis are less pronounced in cases due to CMV [3,4]. Other agents which have been incriminated as occasional causes of heterophil-negative mononucleosis-like illness include toxoplasma gondii, adenovirus and rubella [3,5,6]. During heterophil-negative mononucleosis associated with infection with CMV, various investigators have noted apparent serologic responses to EBV [z-4~1. Rising or elevated titers of antibody to EBV viral capsid antigen (VCA) [2-4,7], as well as high titers of antibody to the D or R components of EBV early antigen (EA) [3] have been observed. The significance of these EBV antibody titers is unclear. It is unlikely that they reflect cross reactions with CMV antigens, however, and more

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likely that they are indicative of dual infections with both viruses. CMV and EBV share neither homologous nucleic acid sequences [8] nor common antigens, although they are both considered herpesviruses and are similar in morphology and other biologic characteristics. Both viruses have a well recognized proclivity for latent infection [9,10], and reactivation is characteristic of CMV in man [lo]. Reactivation of EBV is not well established, although it probably occurs frequently in immunocompromised and perhaps older subjects [11-K?]. The recent demonstration of EBV-induced nuclear antigen (EBNA) and anti-EBNA antibody has enhanced the interpretation of EBV serology [14]. Following a primary infection with EBV, antibodies to different EBV antigens develop in a characteristic temporal fashion [15]. At first presentation, immunoglobulin G (IgG) antibodies to EBV VCA usually are present at high titers and subsequently persist for life [16]. Immunoglobulin M (IgM] antibodies to VCA are found in nearly all patients with EBV mononucleosis but persist only briefly during the acute illness [17-191.Antibodies to EA reach a maximum titer shortly after the onset of symptoms but also rarely persist for more than a few months [16]. A titer >l:2O is considered strong evidence for current infection. Antibodies to EBNA, however, follow a quite distinct pattern as they usually do not appear until one or more months after the onset of symptoms [14]. Antibodies to EBNA, as those to VCA, persist for life. Therefore, the finding of VCA antibodies in a patient lacking antibody activity against EBNA indicates primary EBV infection, whereas increasing titers of antibody to VCA and EA, in the presence of activity against EBNA, would strongly suggest a secondary serologic response. We have recently observed four cases of heterophilnegative mononucleosis in which active dual infection with both CMV and EBV was present. Serologic results suggest that infection with EBV was not primary, but probably secondary reactivation of latent virus. We also present a fifth case in which primary infection with EBV was complicated by reactivation of varicella-zoster virus (VZV). These cases demonstrate that simultaneous active infections with more than one herpesvirus may not be uncommon in patients with mononucleosis. Multiple infections must be considered when determining a specific viral diagnosis in heterophil-negative mononucleosis. Reactivation of endogenous EBV may occur in this setting in otherwise healthy young adults. Reactivation may be related to functional immunosuppression during a primary herpesvirus infection or to hypothetical molecular interactions at the cellular level. MATERIALS AND METHODS Virus Isolations. Urine, throat and cervical washings were inoculated onto monolayers of WI38 human fibroblasts maintained in Eagle’s minimal essential medium (Gibco) supplemented with 3 per cent fetal calf serum, penicillin, 100

U/ml: and streptomycin, 100 fig/ml. Peripheral blood leukocytes, separated either in z per cent dextran (Gentran 75, Travenol) or on Ficoll-Hypaquee gradients (LSM solution, Bionetics Laboratory Products) by a modification of the method of Boyum [20], were cocultivated with WI38 fibroblasts. Viruses were identified by cytopathic effect and immunofluorescent staining of infected monolayers with specific antiserums [21]. Continuous lymphoid cell lines were established by culturing separated blood mononuclear cells in RPM1 1640 media (Gibco) supplemented with 20 per cent fetal calf serum. Transformed cell cultures displayed typical morphologic changes [15]and contained EBNA as determined by the method of Reedman and Klein [22]. For detection of EBV, throat washings were passed through a 0.45 p Millipore filter and added to freshly separated mononuclear cells from human umbilical cord blood [23].Leukocyte-transforming activity was assessed by tritiated [3H)-thymidine incorporation after six weeks of culture [24], presence of EBNA and typical morphologic changes of transformation. Serology. The “Monospot” slide test (Ortho-Diagnostics, Inc.] was used for detection of heterophil antibodies. Antibodies to EBV VCA, to D or R components of EA and to EBNA were determined by immunofluorescence [14,16]. The specificity of anti-EBNA staining was confirmed by retesting positive serums against two EBV-negative lymphoid cell lines (U-698 and Bjab). Serum from one patient [Case 1) was tested for IgM antibodies to VCA by Dr. J. Nikoskelainen [18]. Antibodies to CMV were determined by indirect immunofluorescence (FA) [25]. Additionally, complement-fixation tests for CMV, herpes simplex virus (HSV) and VZV were performed by the Division of Health Services, North Carolina Department of Human Resources, Raleigh, North Carolina. Indirect hemagglutination titers to CMV and IgM-specific IHA titers to CMV and HSV were determined by the Center for Disease Control, Atlanta, Georgia.

RESULTS Over a four-year period, five cases of dual herpesvirus infection were observed in association with mononucleosis syndromes. No systematic effort was made to detect such cases, although three patients were encountered during an investigation of other aspects of infectious mononucleosis. Two cases came to attention because of unusual clinical findings (Guillain-Barr6 syndrome in one and the appearance of herpes zoster in the other). Infection was considered active if either a fourfold or greater change in virus-specific IgG antibody titer occurred, or if the herpesvirus was isolated from a patient with high or changing titers of antibody. CASE REPORTS Case 1. This 23year old nurse (NCMH 38-50-05)had enjoyed excellent health until the onset of myalgias, chills and fever with temperatures to 39%. Examination disclosed minimally enlarged anterior cervical and submental lymph nodes, and a hyperemic pharynx. The spleen was not enlarged, and there was no hepatic tenderness. The total leukocyte count was 7,000/mm3, with 46 per cent polymorphonuclear leukocytes,

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TABLE I

ET AL.

Viral Antibodies In Case 1 l

Days After onset 01 Illness 11 20 27 42 139

INFECTION WITH MULTIPLE HERPESVIRUSES-LEMON

CF <1:8

. *. ‘i:32 1:64

Cytomsgalovlrust FA

IHA

1:40

1:32

..

. .

“’ 1:160

;;4096

...

. .

Epstein-Barr Virus* EA EBNA

VCA 1:640 1:640 1:320 1:320 1:320

I:80 1:160 1:160

I:20 1:lO i:bb

iI80

. .

IaM VCA
.. . .

NOTE: In this and subsequent tables VCA = viral capsid antigen; EA = early antigen; EBNA = Epstein-Barr nuclear antigen, l There were no changes in antibody titers to herpe$ simplex virus, rubella or to toxoplasma. + Antibody titers determined by complement fixation (CF); immunofluorescence (FA) and indirect hemagglutination (IHA). t All antibody titers determined by immunofluorescence.

21 per cent band forms, 26 per cent lymphocytes, 1 per cent atypical lymphocytes and 6 per cent monocytes. Urinalysis and a roentgenogram of the chest disclosed no abnormalities. Fever, malaise and intermittent headaches persisted over the ensuing two weeks. The total leukocyte count was then 11,800/mm3 with 36 per cent polymorphonuclear leukocytes, 15 per cent band forms, 23 per cent lymphocytes, 19 per cent atypical lymphocytes and 6 per cent monocytes. The serum glutamic oxaloacetic transaminase @GOT) level rose to 110 SF U (normal 8 to 60 SF LJ),glutamic pyruvate transaminase (SGPT] 66 SF U (normal 8 to 60 SF U), the alkaline phosphatase 7.2 NP units (normal 2 to 6 NP U] and lactic dehydrogenase [LDH) 811 U/ml (normal 159 to 350 U/ml). The serum haptoglobin was 16 mg/dl [normal 40 to 290 mg/dl), suggesting mild hemolysis, but direct and indirect Coombs’ tests were negative, and the hematocrit value remained stable. The heterophil test was negative and remained negative several weeks later. Radioimmunoassay for hepatitis B surface antigen (HBsAg) was negative. The next two weeks were marked by general improvement with resolution of the liver function abnormalities. Peripheral blood lymphocytes, taken during the third week of illness, underwent spontaneous proliferation to form a continuous lymphoid cell line. Throat washings induced transformation of cord lymphocytes. However, CMV was not recovered from urine, throat, cervix or blood leukocytes. Serologic studies are shown in Table I.

The illness experienced by this patient is typical of heterophil-negative mononucleosis. The minimal degree of pharyngitis and lymphadenopathy are characteristic of CMV mononucleosis [34]. Although this virus was not isolated from any body site, high and rising antibody titers to CMV were documented by complement fixation, indirect immunofluorescence and indirect hemagglutination methods. At the same time, very high antibody titers to VCA and EA, the presence of transforming EBV in throat washings and spontaneous outgrowth of peripheral blood lymphocytes indicated an active infection with EBV. The presence of antibodies to EBNA early in the illness and the lack of IgM antibodies to VCA suggest that this was a secondary serologic response. Thus, this patient apparently experienced reactivation of latent EBV in concert with active CMV infection and heterophil-negative mononucleosis. Comment:

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Case 2. A 22 year old white salesman (NCMH 35-23-82) was admitted to the hospital with a two day history of progressive ascending weakness. One week earlier he had experienced a mild upper respiratory tract infection with rhinorrhea and a nonproductive cough but with neither sore throat nor fever; he had not received any recent immunizations. On admission, he was afebrile, the pharynx appeared normal, and there was no lymphadenopathy. The liver \hras neither enlarged nor tender, but the spleen tip was 2 cm below the left costal margin. There was generalized weakness, and deep tendon reflexes were absent in all four extremities. Position sensation was decreased in the right lower extremity. The total leukocyte count was 8,100/mm3 with 41 per cent polymorphonuclear leukocytes, 6 per cent band forms, 27 per cent lymphocytes, 16 per cent atypical lymphocytes and 10 per cent monocytes. Heterophil antibodies were not detected initially or subsequently. The cerebrospinal fluid was under normal pressure and contained 2 lymphocytes/mm3, 27 mg/lOO ml protein and 80 mg/lOO ml glucose. Bacteriologic studies were negative. Over the first five days of hospitalization, the patient’s weakness progressed to a complete flaccid quadriplegia, and a right facial nerve palsy developed. Increasing respiratory insufficiency necessitated tracheotomy and positive pressure ventilation. Episodic hypertension and tachycardia were treated effectively with apresoline and propranolol. No corticosteroids were given. Liver function abnormalities developed by the 12th hospital day. The SCOT level rose to 107 SF U, the SGPT to 95 SF U, alkaline phosphatase to 7.3 NP U and LDH to 532 U/ml. The patient required mechanical ventilatory assistance for 42 days, but he eventually regained normal neurologic function and was discharged from the hospital. On the day of admission, herpes simplex type 1 virus (HSV-1) was recovered from throat washings. One month later, CMV was isolated from a urine specimen. Although lymphocytes separated from the peripheral blood at the same time did not yield CMV, they spontaneously proliferated to form a continuous cell line. Three months after the onset of the illness, studies of peripheral lymphocytes, urine and throat washings disclosed no abnormalities. Serologic results are shown in Table II. Comment: In this patient the Guillain-Barr6 syndrome developed in association with heterophil-negative mononucleosis. Three separate herpesvirus infections were documented. HSV-1 was isolated from the orop’harynx at the onset of illness and a late rising comVolume 66

SIMULTANEOUS

TABLE II

WITH

MULTIPLE

HERPESVIRUSES-LEMON

ET AL.

Viral Antibodies in Case 2

Days After Onset of Illness 2 15 30 98 164

INFECTION

Herpes simplex Virus IgM IHAf CF’
Complement fixation. t Indirect hemagglutination. t All antibodies to EBV were determined

CF

Cytomegalovirus fgM IHA

iIS .. .

1:lO 1:40 1:160


iIS

i/l60

<;18

VCA

Epstein-Ban Viru$ EA

1:40 1:160 1:160 L1:320 1:160

EBNA

1:20 1:80 1:160 11:320 1:80

1:20 ::: . .. . ..

l

by immunofluorescence.

plement-fixing antibody titer to this virus, preceded by an IgM response, suggests that this may have been a primary infection. IgM antibodies to HSV are occasionally present during clinical reactivation, however [26]. An active infection with EBV is indicated by the marked increases in antibody titers to EA and VCA as well as the spontaneous growth of peripheral blood lymphocytes in vitro. The presence of antibodies to EBNA at the onset of the patient’s illness, when titers to both VCA and EA were relatively low, suggests that this was a secondary serologic response. CMV was recovered from the urine one month into the patient’s illness. The Guillain-Bar& syndrome is frequently associated with primary EBV infections [27,28] but also occurs in association with active CMV infection [28-301. Wahren and Link [28] found evidence of dual infection with both viruses in a single patient. The pathogenetic relationship, however, of herpesvirus infections to the Guillain-Barr6 syndrome remains unclear. Case 3.

This 22 year old construction worker (NCMH 32referred by his local physician after two weeks of malaise, fever and night sweats with arthralgias, photophobia and conjunctivitis. Temperature on admission was 38’C. There was moderate conjunctival injection, palatal petechiae and small, nontender inguinal lymph nodes. The liver was not enlarged, but the spleen tip was 2 cm below the left costal margin. The hematocrit value was 46, and the total leukocyte count was 9,300/mm3 with 22 per cent polymorphonuclear leukocytes, 8 per cent band forms, 2 per cent eosinophils, 31 per cent lymphocytes, 24 per cent atypical lymphocytes and 13 per cent monocytes. The SGOT was 82 SF U, the SGPT 58 SF U, alkaline phosphatase 5.4 NP U and LDH 806 U/ml. The cold-agglutinin titer was 1:128.Heterophil antibodies were not present, and radioimmunoassay for HBsAg was negative. One month later the patient continued to complain of fatigue and malaise, and his liver enzyme levels remained elevated: SGOT 219 SF U, SGPT 222 SF U and LDH 384 U/ml. Two months later, however, he was asymptomatic, and serum chemistries were normal. CMV was isolated from urine collected at the time of admission. Throat washings and peripheral blood leukocytes taken at the same time yielded no virus, and peripheral blood lymphocytes did not develop into a continuous lymphoid cell line. The results of serologic studies are in Table III. 45-09)was

CMV Comment: This patient had heterophil-negative mononucleosis, documented by high titers of complement-fixing antibodies to CMV and isolation of the virus from urine. The greater than fourfold increase in antibody titers to VCA and EBNA indicates an active EBV infection, and the presence of EJ3NA early in the illness again suggests that this infection was secondary. Case 4. A 22 year old white male undergraduate [SHS ll95.-08) came to the Student Health Service with a one week history of fever, sore throat and fatigue. Examination revealed marked anterior cervical lymphadenopathy, a hyperemic pharynx and tonsillar enlargement without exudate. The spleen tip was palpable below the left costal margin. The total leukocyte count was 10,500/mm3 with 29 per cent polymorphonuclear leukocytes, 3 per cent band forms, 66 per cent lymphocytes (many atypical] and 2 per cent monocytes. The heterophil test remained negative throughout the illness. A throat culture did not grow any pathogenic streptococci. The patient remained symptomatic for the ensuing three weeks with intermittent fever, fatigue and sore throat. In early March, after a period of general improvement, the pharyngitis worsened, and a 3 cm by 3 cm left tonsillar node developed. Bacterial studies remained negative, however, and recovery was complete. CMV was recovered from throat washings during the exacerbation of the pharyngitis. Peripheral blood leukocytes, cultured at the same time, neither yielded virus nor developed into a continuous lymphoid cell line. Serologic studies are shown in Table IV. Antibodies to EBNA could not be determined in the first specimen (day 7) because of the presence of nonspecific antinuclear antibodies. This student had heterophil-negative moComment: nonucleosis in association with infection with both CMV and EBV. CMV was isolated from the oropharynx, and a high titer of antibody to CMV was present.

TABLE III Days After on@ of lflness

Cytomegafovlrus’

14 104

1:1280 1:1280

Determined t Determined l

February 1979

Viral Antibodies

in Case 3 Epstein Barr Vlrust VCA EA ‘EBNA 1:80 11:320

1:20 1~40

1:20 1:320

by complement fixation. by immunofluorescence.

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Viral Antlbodles in Case f’

TABLE IV

Ep@ln-M Vkus

DaysAfter owtoflktess

CMnsasbvha

7 38 88

lYl60 1:40

VCA

EA

E8NA

1:320 1:320 1:320

1:80 1:180 1:20

ID’ 1:180 1:80

antibodies

were

Determined by immunofluwescence. t Indeterminate, nonspecific antinuclear present. l

abnormalities were confined to an SGPT of 182 U/liter (normal 7 to 40 U/liter), as the SGOT, alkaline phosphatase and LDH remained within normal limits. The subsequent courSe was one of uneventful recovery with resolution of both rashes. Virus studies were carried out on the fifth hospital day. Peripheral blood lymphocytesspontaneouslyproliferated to form a continuous, EBNA-positive cell line. VZV was recovered from a fresh vesicular lesion.,Serologic studies are shown in Table V. Comment:

A high and declining antibody titer to EA indicated an active infection with EBV, but the presence of nonspecific antinuclear antibodies in the serum specimen obtained during the acute phase did not permit classification of the EBV infection as secondary on the basis of determinations of antibody to EBNA. Case 9. This 23 year old male student (SHS 13-47-14) presented to the Student Health Service with a four-day history of sore throat and fever. The “Monospot” test (Ortho-Diagnostics. Inc.) was positive, and a blood smear revealed 63 per cent lymphocytes [many atypical] with a total leukocyte count of 5,79tl/mms. Three days later he returned with marked pharyngeal discomfort and a temperature of 40°C. and he was admitted. Anterior and posterior cervical lymph nodes were greatly enlarged, the posterior pharynx was hyperemic and the tonsils were moderately enlarged without exudate. The spleen was not palpable, and there was no hepatic tenderness. Several vesicular lesions with erythematous bases were present over the left flank. The total leukocyte count was 11.299/mm3 with 44 per cent polytnorphonuclear leukocytes, 9 per cent band forms, 38 per cent lymphocytes and 9 per cent atypical lymphocytes. The following day, prednisone therapy, 20mg twice a day, was instituted. Over the next two days, fever subsided, but the vesicular eruption over the left flank worsened dramatically and became intensely pruritic. Four days after admission, fever with a temperature of 39% recurred, and shallow ulcers with exudate were present over both tonsils. The administration of penicillin, 250 mg orally four times a day, was started. Two days later, florid vesicular lesions of zoster were present in the 11th and 12th thoracic dermatome on the left. Prednisone therapy was discontinued. Eight days after admission, a generalized erythematous maculopapular rash developed and became confluent over the thorax and abdomen; penicillin therapy was discontinued. Liver function TABLE V

Viral Antlbodles In Case 5’

Day8sftr OMstsfuhmss 7 months prior 7 13 40

Epdm VW+ VCA EA EEHX
j:bb ... ...


V@csHa zost*Vlr& ;I;8 1:8

...

’ Complement fixing antibodies to cytomegalovirus and herpes simplex virus remalned <1:8 dwing tjw illness; antibodies to toxoplasma determined by immunofluorescence rema!ned
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Febwary 1979 The American Journal of Medicine

This patient related a history of chickenpox at age four. Serum had been obtained seven months prior to his illness for research purposes and had shown no antibodies to EBV. Heterophil-positive mononucleosis occurred, and high titers of antibodies to EBV antigens were present. The unusual feature of this case is the simultaneous reactivation of WV, which was cultured from a fresh vesicle. Although prednisone may well have contributed to the severity of the zoster, the earliest lesions were present at the time of admission, prior to any corticosteroid therapy.

COMMENTS Ahhough morphologically similar, EBV and CMV do not share common antigenic specificities. There are no reports of cross reactions among IgC antibodies to EBV and CMV, although there is evidence that antibodies produced during primary EBV infections may occasionally produce false-positive reactions in the IgMCMV test [3,31]. The reverse is not true however, as antibodies reactive in the IgM-VCA test are not found during CMV mononucleosis [3]. Dual infections with EBV and CMV are highly probable in Cases 1 through 4, as determined by significant changes in IgC antibody titers or isolation of each virus. Our finding of four such cases over a relatively short time period indicates that dual infection with CMV and EBV is not uncommon and probably occurs more often than coincidence alone would allow. In Cases 1 through 3, the presence of anti-EBNA in serum obtained during the acute phase strongly suggested a secondary serologic response which could be due to either EBV reinfection or reactivation [l4]. However, no clear antigenic differences exist among the various strains of EBV, and there is no evidence that reinfection with EBV can occur. It seems more reasonable to propose that in our cases reactivation of latent EBV was triggered by a primary infection with CMV or, as in Case 2, possibly HSV-I. Existing evidence for reactivation of EBV is limited, despite the well known proclivity of the virus for lifelong latency 191.Patients receiving immunosuppressive drugs have an increased likelihood of oropharyngeal shedding of virus [ll), and antibody titers to VCA may rise following renal transplantation [12). Both of these findings suggest reactivation of virus in immunocompromised subjects. In addition, a recent serologic survey demonstrated a high prevalence of IgM-VCA antibodies and

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elevated anti-EA titers among asymptomatic older adults [13]. As all of these subjects possessed anti-EBNA antibodies, they may have experienced a reactivation of endogenous EBV. Reactivation of EBV during active CMV infections may be related to alterations in cellular immunity known to accompany active infection with CMV [7b]. Lymphocyte responses to concanavalin A and pokeweed mitogen, but not to phytohemagglutinin, are transiently depressed during CMV mononucleosis [32]. This may in part be due to quantitative changes in lymphocyte subpopulations as decreases in thymusderived lymphocytes, relative to other lymphocyte subpopulations, have been observed in both infants and adults with active CMV infection [7b,33,34]. In contrast to the first four cases, one patient (Case 5) experienced a primary infection with EBV complicated by herpes zoster, a clinical presentation associated with reactivation of latent VZV. Although to our knowledge this complication has not previously been reported, it is reasonable to propose that VZV reactivation was due to the recognized effects of primary infection with EBV on cellular immunity. Primary infections with EBV are associated with transiently diminished cutaneous responses to intradermal tuberculin and other antigens [35,36]. In vitro transformation of lymphocytes by phytohemagglutinin or pokeweed mitogen is also decreased, as are lymphocyte responses in the mixed lymphocyte reaction [36,37]. A reversible defect in monocyte migration to chemotactic stimuli also exists [38]. Although little clinical significance has been attached to these defects in cellular immune functions, it is reasonable to propose that such impairments may have allowed the reactivation of VZV in Case 5. Other mechanisms, however, may be responsible for the reactivation of EBV in patients with active CMV infection. EBV has classically been associated with infection of lymphocytes [9] whereas CMV has been primarily associated with epithelial cell infections [lo]. However, during heterophil-negative mononucleosis, CMV can be isolated from highly purified mononuclear cell preparations [32,39] as well as polymorphonuclear leukocytes. At least limited replication of CMV DNA occurs in infected lymphoid cells in vitro [40]. Also, recent evidence suggests that EBV infects epithelial cells in vivo [4l], as does CMV. Thus, both viruses are able to infect similar cell types, and occasional single cells may become infected with both.

ET AL.

All herpesviruses induce specific DNA polymerases that are required for productive replication of the viral DNA. The DNA polymerase enzymes induced by the different herpesviruses are similar in that their activity is enhanced by high salt concentrations and inhibited by phosphonoacetic acid, in contrast to the DNA polymerases of mammalian cells [42,43]. The EBV genome exists during latency in an episomal form [44] and is replicated by cellular DNA polymerases (451.Viral DNA polymerase induced by infection with CMV might initiate productive replication of a latent EBV genome resident in the same cell, leading to the production and release of virus particles. In this fashion, a primary infection with one herpesvirus could lead to reactivation of another. However, despite the similarities between CMV and EBV DNA polymerase, evidence has yet &_I be presented that either enzyme can replicate the DNA of a different herpesvirus. IgM antibodies to VCA were absent in Case 1, even though they were present in 95 per cent of apparent secondary serologic responses to EBV among normal older subjects [13]. IgM anti-VCA was not found in six other subjects with CMV-mononucleosis and high anti-EA titers [3]. The role of IgM in the secondary serologic response to EBV thus remains to be defined. Heterophil antibodies were notably absent in Cases 1 through 4, as they have been in most other cases of possible EBV-reactivation [3,12], further supporting the concept that heterophil antibodies, which are also of the IgM class, are restricted to primary infections with EBV. The possibility that infection with one herpesvirus might initiate reactivation of another can only serve to confuse the specific viral diagnosis in any single case. Dual infections need to be considered in heterophilnegative mononucleosis, and viral-specific studies for both EBV and CMV should be performed. The clinical consequences of EBV reactivation, however, remain uncertain. ACKNOWLEDGMENT

We should like to thank Dr. W. Eakins for permission to report Case 2, Dr. H. Dascomb who participated in the study of Cases 3 and 4,and Dr. J. McCutcheon for referral of Case 5. We thank D. Crews and C. Gilbert for excellent technical assistance. In addition, we are grateful to Dr. j. Nikoskelainen for determination of IgM anti-VCA titers in Case 1.

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Evans AS: Infectious mononucleosis and other mono-like syndromes. N Engl J Med 286: 836,1972. 6. Hallee TJ, Evans AS, Niederman JC, et al.: Infectious mononucleosis at the United States Military Academy. A prospective study of a single class over four years. Yale J Biol Med 47: 182.1974. 7. (a) Wahren B, Epsmark A, Wallden G: Serological studies on cytomegalovirus infection in relation to infectious mononucleosis and similar conditions. Stand J Infect Dis 1: 145,

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(b] Oil1 PA, Fiala M. Schofferman J, et al.: Cytomegalovirus mononucleosis in a healthv adult. Association with heuatitis, secondary Epstein-Barr virus antibody responseand immunosuppression. Am 1Med 62: 413.1977. 8. Huang E-S, Pagan0 JS: Human cytomegalovirus. II. Lack of relatedness to DNA of herues simulex I and II. EosteinBarr virus, and nonhuman strains of cytomegalo&usI J Virol13: 642,1974. 9. Pagan0 JS: Diseases and mechanisms of persistent DNA virus infection: latency and cellular transformation. J Infect Dis 132: 209,1975. 10. Weller TH: The cytomegaloviruses: Ubiquitous agents with protean clinical manifestations. N Engl J Med 285: 203,267, 1971. 11. Straugh B, Andrews N, Siegal N, et al.: Oropharyngeal excretion of Epstein-Barr virus by renal transplant recipients and other patients treated with immunosuppressive drugs. Lancet 1: 234,1974. 12. Spencer ES, Andersen HK: Antibodies to the Epstein-Barr virus in kidney transplant recipients. Acta Med Stand 191: 107,1972. 13. Sumaya CV: Endogenous reactivation of EBV Infections. J Infect Dis 135: 374,1977. 14. Henle G, Henle W. Horwitz CA: Antibodies to Epstein-Barr virus-associated nuclear antigen in infectious mononucleosis. J Infect Dis 130: 231,1%74. 15. Henle W. Henle G. Horwitz CA: Eustein-Barr virus snecific diagnostic tests in infectious mononucleosis. Hum&&l 5: 551.1974. 16. Henle W, Henle G, Niederman JC, et al.: Antibodies to early antigens induced by Epstein-Barr virus in infectious mononucleosis. J Infect Dis 124: 58, 1971. 17. Schmitz H, Scherer M: IgM antibodies to Epstein-Barr virus in infectious mononucleosis. Arch Gesamte Virusforsch 37: 332, 1972. 18. Nikoskelainen J, Hanninen P: Antibody response to Epstein-Barr virus in infectious mononucleosis. Infect Immun 11: 42. 1975. 19. Evans AS, Niederman JC, Cenabre LC, et al.: A prospective evaluation of heterophile and Epstein-Barr virus-specific IgM antibody tests in clinical and subclinical infectious mononucleosis. Specificity and sensitivity of the tests and persistence of antibody. J Infect Dis 132: 546,1975. 20. Boyum A: Isolation of mononuclear cells and granulocytes from human blood. Stand J Clin Lab Invest 21 [suppl97): 77, 1968. 21. Huang Y-Y, Huang E-S, Pagan0 JS: Antisera to human cytomegalovirus prepared in the guinea pig: specific immunofluorescence and complement fixation tests. J Immunol 112: 528,1974. 22. Reedman BM, Klein G: Cellular localization of Epstein-Barr virus-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int J Cancer 11: 499, 1973. 23. Chang RS, Golden HD: Transformation of human leukocytes by throat washing from infectious mononucleosis patients. Nature (Lond) 234: 359,197l.

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28. 29. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40.

ET AL.

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