Proc. Nat. Acad. Sci. USAVol. 68, No. 7, pp. 1407-1411, July 1971

Serologic Evidence that a Herpes-Type Virus is the Etiologic Agent ofHeterophile-Positive Infectious Mononucleosis

(human/rabbit/purified antibodies/antisera/blocking/electron microscopy)

BERGE HAMPAR, KONRAD C. HSU*, LIDIA M. MARTOSt, AND JOHN L. WALKERt

Viral Carcinogenesis Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014;* Department of Microbiology, Columbia University, New York; and t Flow Laboratories, Inc., Rockville, Maryland 20852

Communicated by Robert J. Huebner, April 22, 1971

ABSTRACT The antibody activity against herpes-typevirus (HTV) of heterophile-positive infectious mono-nucleosis sera and "normal" sera from humans wasdetermined with immunoferritin. The antiviral activityof the mononucleosis sera was associated with the IgMantibodies, while the antiviral activity of the "normal"human sera was associated with the IgG antibodies. It wasconcluded from these findings that the appearance ofantibodies to herpes-type virus in heterophile-positivesera represents a primary immunogenic exposure to thisvirus, or to a serologically related virus. This conclusion,in turn, suggests that herpes-type virus or a serologicallyrelated virus is the etiologic agent of heterophile-positivemononucleosis.

A serologic relationship has been proposed between theherpes-type virus (HTV) associated with human lympho-blastoid cell lines derived from "normal" buffy coats and fromBurkitt tumors (1) and the virus of infectious mono-

nucleosis. This relationship is based on a correlationbetween the onset of heterophile-positive mononucleosis with(a) the detection of antibodies to HTV (2-4) and (b) theability of buffy-coat cells to undergo blastoid transformationand establishment in vitro (2). The available evidence, how-ever, is not sufficient to allow conclusions as to whether theantibodies to HTVt that have been detected in heterophile-positive sera from patients with mononucleosis are caused bya primary immunogenic exposure to this virus. Informationrelating to this question is important in attempts to ascribean etiologic role for HTV in the onset of heterophile-positivemononucleosis.Our studies were based on the assumption that after a

primary antigenic stimulation, 19S or IgM antibodies shouldbe detectable before 7S or IgG antibodies (5, 6), provided thatthe antigen in question (e.g., HTV) induces synthesis of bothclasses of antibody (7). The specificity of human antibodiesfor antigens of the HTV virion was determined by theimmunoferritin technique, which allows visual observation ofthe reaction between antibody and virus. This technique was

previously shown effective for determination of the specificityof 7S and 19S antibodies of the rabbit for herpes-simplexvirion antigens (8).

Abbreviations: HTV, herpes-type virus; MSV, herpes simplexvirus.

t The term herpes-type virus (HTV) refers to both the virusassociated with Burkitt tumors and the virus of mononucleosis.

1407

MATERIALS AND METHODS

Cell lines

The HTV-positive HR1K cell line was maintained by co-cultivation with a continuous line of African Green monkey(Vero) cells. This procedure results in a high percentage ofHTV-positive HR1K cells (30-70%) in the medium of co-cultivated cultures (9). The HTV-negative Raji cell line servedas control. The Raji cells showed no evidence of HTV infec-tion by electron microscopy when maintained alone or incocultivation with Vero cells.

Antisera

Three heterophile-positive mononucleosis sera from humansand three "normal" HTV-positive sera from humans werestudied in detail (Table 1). The mononucleosis sera wereselected on the basis of high titers of heterophile antibody. The"normal" HTV-positive sera were selected on the basis of norecent clinical illness and the absence of heterophile anti-bodies. Since at least 10 ml of each serum was required for thevarious test procedures, the number of sera studied in detailwas necessarily limited by the available volumes. However,at least four additional mononucleosis sera and three addi-tional HTV-positive "normal" sera were subjected to one testprocedure (blocking reaction); the results were similar tothose obtained with sera studied in detail. In addition to theabove, three rabbit anti-HTV sera prepared against HTVcapsids (10) were included. One HTV-negative serum from ahuman and three preimmunization sera from rabbits servedas controls.

Heterophile-antibody titers were determined by the Paul-Bunnell test and the Davidson differential test (Table 1).Concentrations of IgG, IgM and IgA immunoglobulins inthe human sera (Table 1) were determined by the radialimmunodiffusion method (Hyland Labs, Inc.).

Immunoferritin test

Human and rabbit serum globulins were precipitated withsodium sulfate (6). The human globulins were filtered onSephadex G-200 gels to isolate 7S and 19S antibodies (10). The7S fractions contained IgG and IgA; the 19S fractions con-tained IgM and IgA, as determined by immunoelectro-phoresis.The globulins, 7S, and 19S antibodies were conjugated with

ferritin (11). The conjugates were absorbed with lyophilized

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1408 Microbiology: Hampar et al.

TABLE 1. Reactivity of human sera with HTVa

Mononucleosis Direct Blocking reactions concentration testedheterophile immuno-Serum fraction Sex Age titer ferritin Result mg IgG mg IgM mg IgA

Mononucleosis seracMS: Serum M 19 896 NT + 1.0 0.07 0.08

7S - - - :3.0 0.0 0.0419S 498 + + 0.0 0.1 0.05

SS: Serum F 17 1792 NT + 1.1 0.12 0.087S - +d + 3.0 0.0 0.0719S 896 + + 0.0 0.1 0.04

EE: Serum M 18 896 NT + 2.5 0.15 0.317S - - - 3.0 0.0 0.1919S 896 + + 0.0 0.1 0.09

"Normal" HTV PositivefDS: Serum M 23 - NT + 1.5 0.08 0.25

7S - + + 1.0 0.0 0.0519S - - 0.0 0.3 0.35

LM: Serum F 42 - NT + 1.4 0.03 0.087S - + + 0.0 0.0 0.0119S - - - 0.0 0.3 0.25

B-76: Serum M 28 - NT + 1.6 0.04 0.247S - + + 1.0 0.0 0.0619S - - - 0.0 0.3 0.6

"Normal" HTV NegativefB-289: Serum M 24 - - 1.2 0.2 0.54

a NT = not tested; (-) negative; (+) positive.bAbility to block ferritin tagging of HTV by human 7S, human 19S, and rabbit globulin conjugates.c Sera MS and SS were from students at the Vanderbilt University Clinic and were obtained from Dr. J. M. Flexner through Blood

Research, Inc. Serum EE was from a student at the University of Michigan Clinic and was obtained from Dr. J. D. Zarafonetis.d Ferritin tagging of HTV required two reactions with HR1K cells.a A weak blocking reaction was observed at a concentration of 3 mg of IgG immunoglobulins, but no blocking was observed at a concen-

tration of 1 mg of IgG immunoglobulins.f Sera B-76 and B-289 were obtained from Dr. P. Gerber.

HeLa and Raji cells and were negative by immunoferritinassay against HTV-negative Raji cells.The methods used for cell fixation and reaction with

ferritin conjugates have been described (8, 10). All ferritinconjugates (0.1 ml) were tested initially by one reaction withHR1K cells. Those conjugates found to be negative wereretested by a second reaction with HR1K cells (8).Blocking tests were performed by reaction of the cells for

2 hr at room temperature with unconjugated antibody,followed by washing and reaction for 1 hr with various con-jugates (8, 10). Blocking tests with whole sera were performedwith volumes of 0.1 and 0.3 ml. In the case of 7S antibodies,concentrations of 1.0 and 3.0 mg of IgG were used, while 19Santibodies were tested at concentrations of 0.1 and 0.3 mg ofIgM (Table 1). Each blocking reagent was tested for itsability to block the ferritin tagging of HTV by human 7S,19S, and rabbit conjugates. All conjugated and unconjugated

reagents were tested at least one time with each of threedifferent HRIK cell preparations.

Various human antibody fractions were reduced and alkyl-ated by treatment with 0.1 M 2-mercaptoethanol at roomtemperature for 2 hr, followed by dialysis against 0.02 Miodoacetamide for 3 hr (12). The treated fractions were thendialyzed overnight at 40C against 0.02 M phosphate-bufferedsaline, pH 7.2.

RESULTSDirect immunoferritin testsFerritin tagging (Table 1) of HTV (Fig. 1) by the "normal"HTV-positive human sera was observed with the 7S con-jugates (Fig. 2), but not with the 19S conjugates, even thoughthe latter were reacted twice with HR1K cells. Ferritintagging of HTV particles by the mononucleosis sera wasnoted with the 19S conjugates after one reaction with HR1Kcells (Fig. 3), but only one 7S conjugate (serum SS) showed

FIG. 1. HTV capsids. X 77,400.FIG. 2. HTV capsids ferritin-tagged with human 7S (IgG) antibodies. X 77,400.FIG. 3. HTV capsids ferritin-tagged with human 19S (1gM) antibodies. X 77,400.FIG. 4. HTV capsids ferritin-tagged with human 7S (JgG) antibodies. No tagging is seen with high-density core antigens (arrow).

X 77,400.-FIG. 5. HTVcapsids ferritin-taggedwith human 7S (IgG) antibodies. No tagging is seen with low-densitycore antigens (arrow). X 77.400.

Proc. Nat. Acad. Sci. USA 68 (1971)

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Proc. Nat. Acad. Sci. USA 68 (1971) Viral Etiology of Mononucleosis 1409

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1410 Microbiology: Hampar et al. Proc. Nat. Acad. Sci. USA 68 (1971)

*. n.weaktagging of HTV particles when reacted twice withHR1K cells. The three rabbit anti-HTV conjugates showedferritin tagging of HTV particles, as reported previously (10).At least one 7S conjugate from a "normal" HTV-positive~~~~~ ~ ~ ~ ~ serum and one 195 conjugate from a mononucleosis serum thatwere negative for antibodies to herpes simplex virus (HSV) byneutralization were tested against HSV-infected Raji or vero

`;;fcells. No tagging of HSV capsids or enveloped particles was

Ferritin tagging of HTV by the various human (Table 1)4 ~~~~~~~~andrabbit conjugates was restricted to the outer surface of

the virus capsids. Ferritin tagging of HTV-enveloped particleswas not observed with these reagents. Similarly, no ferritin

A--/@J|-Ad.... . . . ..... .lPtagging was observed of either low-density (Fig. 4) or high-density (Fig. 5) virus-core antigens.

;M Ferritin tagging by the human and rabbit conjugates wasalso noted with fine granular nuclear antigens, some of whichwere in proximity to capsids (Fig. 6). Ferritin tagging ofsimilar structures was reported (8) in HSV-infected cells,where it was suggested that capsids were assembled from thefinely granular antigens.

Blocking tests

Blocking of the immunoferritin reaction (Table 1) was ob-served with 0.1 ml of the "normal" HTV-positive humansera and mononucleosis sera and with the rabbit antisera. The7S antibodies from the "normal" human HTV-positive serashowed blocking activity at a concentration of 1.0 mg of IgG,but blocking activity was not observed with 19S antibodiesat a concentration of 0.3 mg of IgM. The 19S antibodies fromthe mononucleosis sera showed blocking activity at a con-centration of 0.1 mg of agM, whereas the 7S antibodies from

.> two mononucleosis sera (sera MS and EE) showed no blockingactivity at a concentration of 3.0 mg of IgG. The 7S anti-bodies from a third mononucleosis serum (serum SS) showedweak blocking activity at a concentration of 3.0 mg of IgG,but not at a concentration of 1.0 mg. Blocking activity wasnot observed with 0.3 ml of the "normal" human HTV-negative serum or with the rabbit preimmune sera.

Absorption of the mononucleosis sera and 19S antibodieswith boiled beef-kidney antigen removed the heterophileantibody activity, but did not eliminate the ability of these

,',<E,,,-'.a'reagents to block the ferritin tagging of HTV. This suggeststhat the anti-HTV activity and the heterophile-antibodyactivity of the mononucleosis sera were distinct. Treatmentwith 2-mercaptoethanol eliminated the blocking activity ofthe mononucleosis 19S antibodies, but it did not affect theblocking activity of the 7S antibodies from the "normal"HTV-positive human sera.The appearance by electron microscopy of antibody-

~~~~~~~~~~coated HTV particles (13), as observed in the blocking re-acin, differed for 7S and 19S antibodies. The virus particles

coated with 7S antibodies showed a relatively homogenous

FIG. 6. HTV capsids ferritin-tagged with human. 78 (IgG)antibodies. Tagging of finely granular nuclear antigens fromwhich capsid is presumably formed, is also seen. X 78,300.

FIG. 7. Blocking of immunoferritin reaction by human 78.(IgQ) antibodies. The blocked particles show a relatively homo-genousantibody coat. X 78,300.

FIG. 8. Blocking of immunoferritin reaction by human 19S;(gM) antibodies. The blocked particles show a "halo of struc-

tures resembling loops." X 78,300."'",-"' -z, "I ..m -'m .7., -f-evili '%. '..W .7'.

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Viral Etiology of Mononucleosis 1411

antibody coat (Fig. 7), while particles coated with 19S anti-bodies (Fig. 8) showed "a halo of structures resemblingloops," suggestive of IgM antibodies (14). Similar differencesin morphology of antibody-coated particles was reported(8) for rabbit 7S and 19S antibodies reacted with HSV.

DISCUSSION

The results indicate that the heterophile-positive mono-

nucleosis sera tested contained 19S HTV antibodies, whilethe "normal" HTV-positive human sera tested contained 7SHTV antibodies. The presence of both 7S and 19S HTVantibodies in one mononucleosis serum (serum SS) was un-

doubtedly related to the time the serum was collected.The following findings suggest that the human 7S and 19S

antibodies to HTV represented IgG and IgM antibodies,respectively. First, the absence of IgM in the 7S fractions,and the absence of IgG in the 19S fractions, was shownimmunoelectrophoretically. Second, IgA was present in both7S and 19S fractions, and the HTV-negative fractionsusually contained more IgA than did the HTV-positivefractions (Table 1). Third, the HTV blocking activity of the19S antibodies, but not the 7S antibodies, was eliminated bytreatment with 2-mercaptoethanol. It was reported (15) thattreatment with the sulfhydryl reagent destroyed the neu-

tralizing activity of rabbit 19S antibodies to HSV, even whencomplement or anti-gamma globulin was added to the reactionmixture. Finally, the antibody coating seen with the 7S anti-bodies in the blocking tests was suggestive of IgG (Fig. 7),while the coating seen with the 19S antibodies was suggestiveof IgM (Fig. 8).

Since HTV in humans can induce synthesis of both IgG andIgM antibodies (Table 1), we conclude that the presence ofIgM antibodies in heterophile-positive mononucleosis sera

reflects a primary immunogenic exposure to HTV. This con-

clusion, in turn, suggests that HTV is the etiologic agent ofmononucleosis. However, neither our findings nor thosereported by others prove conclusively that the virus associatedwith Burkitt tumors (HTV) and the virus of mononucleosisare truly identical and not merely serologically related.The finding that the specificity of the human and rabbit

antibodies tested for HTV was limited to capsid-associatedantigens (Figs. 2-6) was of particular interest, since previouslyreported findings with HSV (8) indicated that the immuno-ferritin technique could also detect antibodies directed againstvirus core and envelope antigens. Studies by acrylamide gelelectrophoresis (16) suggest that there are seven majorstructural proteins associated with HSV (two in the core, twoin the capsid, and three in the envelope), and electrophoreticpatterns of HTV proteins indicate close similarities to theproteins found with HSV (17). This suggests that the speci-ficity of our human and rabbit sera for HTV was limited toone, or possibly both, of the major antigens associated withthe capsid, and the blocking experiments further suggest thatthese sera were reactive with the same antigens.The inability of our human sera to react by immunoferritin

with HTV-enveloped particles agrees with findings reportedby others (13, 18). However, this finding was somewhatunexpected, since at least one serum (serum B-76) has beenreported capable of neutralizing HTV infectivity (19), whichfinding suggests envelope activity. In contrast, the inabilityof our rabbit antisera to react with HTV-enveloped particles

was not unexpected, since these sera were prepared againstconcentrates containing essentially virus capsids (10), andthese sera do not neutralize HTV infectivity (19).The apparent discrepancy between the ability of human

sera to neutralize HTV infectivity and the reactivity of thesesera with HTV by immunoferritin may be due to one or moreof the following factors. First, infectivity of Herpes virusesmay not be limited to enveloped particles (20). Second,neutralization of Herpes viruses may occur at antigenic sitesbelow the surface of the envelope (8). In this case, the re-activity of antibody with complete virions may not be appar-ent with immunoferritin (8), probably because of the inabilityof the large antibody-ferritin complex to penetrate the virusenvelope. The same antibody when unconjugated, however,could still neutralize virus (15). Third, most HTV particlesassociated with lymphoblastoid cells appear defective byelectron microscopy, in that the particles lack either a densenucleoid or an envelope (unpublished observations). Thissuggests that HTV infection of lymphoblastoid cells may beabortive (21) or restrictive (22), with unincorporated envelopeantigens accumulating in the cells. The accumulation of un-incorporated envelope antigens may account for the positiveimmunofluorescent reactions seen with fixed HTV-positivelymphoblastoid cells and human sera, since we have shown(10) that rabbit antisera whose specificity for HTV byimmunoferritin is limited to capsid particles (10) do not reactby immunofluorescence with fixed, HTV-infected cells.

The technical assistance of Mrs. M. Tagametz, Mr. M.Chakrabarty, Mrs. M. A. Burroughs, Mr. D. Simms, Miss C.Owen, and Mrs. D. Krell is gratefully acknowledged.

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5. Uhr, J., Science, 145, 457 (1964).6. Freeman, M. J., and A. B. Stavitsky, J. Immunol., 95, 981

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Nat. Cancer Inst., 46, 629 (1971).9. Hampar, B., L. M. Martos, and J. L. Walker, submitted to

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Buscheck, Cancer Res., 27, 2020 (1967).14. Svehag, S-E., and B. Bloth, Virology, 31, 676 (1967).15. Hampar, B., A. L. Notkins, M. Mage, and M. A. Keehn,

J. Immunol., 100, 586 (1968).16. Olshevsky, U., and Y. Becker, Virology, 40, 948 (1970).17. Weinberg, A., and Y. Becker, Virology, 39, 312 (1969).18. Sugawara, K., and T. Osato, Abstr. 10th Int. Cancer Con-

gress, Houston, Texas, 1970.19. Gerber, P., and G. Goldstein, J. Immunol., 105, 793 (1970).20. Spring, S. B., and B. Roizman, J. Virol., 2, 979 (1968).21. Aurelian, L., and B. Roizman, Virology, 22, 452 (1964).22. Hampar, B., and M. A. K. Burroughs, J. Nat. Cancer Inst.,

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