Serologic Evidence that a Herpes-Type Virus is the Etiologic Agent of

Proc Nat Acad Sci USA Vol 68, No 7, pp 1407-1411, July 1971 Serologic Evidence that a Herpes-Type Virus is the Etiologic Agent of Heterophile-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-type virus (HTV) of heterophile-positive infectious mononucleosis sera and "normal" sera from humans was determined with immunoferritin The antiviral activity of the mononucleosis sera was associated with the IgM antibodies, while the antiviral activity of the "normal" human sera was associated with the IgG antibodies It was concluded from these findings that the appearance of antibodies to herpes-type virus in heterophile-positive sera represents a primary immunogenic exposure to this virus, or to a serologically related virus This conclusion, in turn, suggests that herpes-type virus or a serologically related virus is the etiologic agent of heterophile-positive mononucleosis A serologic relationship has been proposed between the herpes-type virus (HTV) associated with human lymphoblastoid cell lines derived from "normal" buffy coats and from Burkitt tumors (1) and the virus of infectious mononucleosis This relationship is based on a correlation between the onset of heterophile-positive mononucleosis with (a) the detection of antibodies to HTV (2-4) and (b) the ability of buffy-coat cells to undergo blastoid transformation and establishment in vitro (2) The available evidence, however, is not sufficient to allow conclusions as to whether the antibodies to HTVt that have been detected in heterophilepositive sera from patients with mononucleosis are caused by a primary immunogenic exposure to this virus Information relating to this question is important in attempts to ascribe an etiologic role for HTV in the onset of heterophile-positive mononucleosis Our studies were based on the assumption that after a primary antigenic stimulation, 19S or IgM antibodies should be detectable before 7S or IgG antibodies (5, 6), provided that the antigen in question (eg, HTV) induces synthesis of both classes of antibody (7) The specificity of human antibodies for antigens of the HTV virion was determined by the immunoferritin technique, which allows visual observation of the reaction between antibody and virus This technique was previously shown effective for determination of the specificity of 7S and 19S antibodies of the rabbit for herpes-simplex virion antigens (8) Abbreviations: HTV, herpes-type virus; MSV, herpes simplex virus t The term herpes-type virus (HTV) refers to both the virus associated with Burkitt tumors and the virus of mononucleosis 1407 MATERIALS AND METHODS Cell lines The HTV-positive HR1K cell line was maintained by cocultivation with a continuous line of African Green monkey (Vero) cells This procedure results in a high percentage of HTV-positive HR1K cells (30-70%) in the medium of cocultivated cultures (9) The HTV-negative Raji cell line served as control The Raji cells showed no evidence of HTV infection by electron microscopy when maintained alone or in cocultivation with Vero cells Antisera Three heterophile-positive mononucleosis sera from humans and three "normal" HTV-positive sera from humans were studied in detail (Table 1) The mononucleosis sera were selected on the basis of high titers of heterophile antibody The "normal" HTV-positive sera were selected on the basis of no recent clinical illness and the absence of heterophile antibodies Since at least 10 ml of each serum was required for the various test procedures, the number of sera studied in detail was necessarily limited by the available volumes However, at least four additional mononucleosis sera and three additional HTV-positive "normal" sera were subjected to one test procedure (blocking reaction); the results were similar to those obtained with sera studied in detail In addition to the above, three rabbit anti-htv sera prepared against HTV capsids (10) were included One HTV-negative serum from a human and three preimmunization sera from rabbits served as 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 in the human sera (Table 1) were determined by the radial immunodiffusion method (Hyland Labs, Inc) Immunoferritin test Human and rabbit serum globulins were precipitated with sodium sulfate (6) The human globulins were filtered on Sephadex G-200 gels to isolate 7S and 19S antibodies (10) The 7S fractions contained IgG and IgA; the 19S fractions contained IgM and IgA, as determined by immunoelectrophoresis The globulins, 7S, and 19S antibodies were conjugated with ferritin (11) The conjugates were absorbed with lyophilized

1408 Microbiology: Hampar et al Proc Nat Acad Sci USA 68 (1971) TABLE 1 Reactivity of human sera with HTVa Mononucleosis Direct Blocking reactions concentration tested heterophile immuno- Serum fraction Sex Age titer ferritin Result mg IgG mg IgM mg IgA Mononucleosis serac MS: Serum M 19 896 NT + 10 007 008 7S - - - :30 00 004 19S 498 + + 00 01 005 SS: Serum F 17 1792 NT + 11 012 008 7S - +d + 30 00 007 19S 896 + + 00 01 004 EE: Serum M 18 896 NT + 25 015 031 7S - - - 30 00 019 19S 896 + + 00 01 009 "Normal" HTV Positivef DS: Serum M 23 - NT + 15 008 025 7S - + + 10 00 005 19S - - 00 03 035 LM: Serum F 42 - NT + 14 003 008 7S - + + 00 00 001 19S - - - 00 03 025 B-76: Serum M 28 - NT + 16 004 024 7S - + + 10 00 006 19S - - - 00 03 06 "Normal" HTV Negativef B-289: Serum M 24 - - 12 02 054 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 concentration 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 immunoferritin assay against HTV-negative Raji cells The methods used for cell fixation and reaction with ferritin conjugates have been described (8, 10) All ferritin conjugates (01 ml) were tested initially by one reaction with HR1K cells Those conjugates found to be negative were retested 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 conjugates (8, 10) Blocking tests with whole sera were performed with volumes of 01 and 03 ml In the case of 7S antibodies, concentrations of 10 and 30 mg of IgG were used, while 19S antibodies were tested at concentrations of 01 and 03 mg of IgM (Table 1) Each blocking reagent was tested for its ability 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 three different HRIK cell preparations Various human antibody fractions were reduced and alkylated by treatment with 01 M 2-mercaptoethanol at room temperature for 2 hr, followed by dialysis against 002 M iodoacetamide for 3 hr (12) The treated fractions were then dialyzed overnight at 40C against 002 M phosphate-buffered saline, ph 72 RESULTS Direct immunoferritin tests Ferritin tagging (Table 1) of HTV (Fig 1) by the "normal" HTV-positive human sera was observed with the 7S conjugates (Fig 2), but not with the 19S conjugates, even though the latter were reacted twice with HR1K cells Ferritin tagging of HTV particles by the mononucleosis sera was noted with the 19S conjugates after one reaction with HR1K cells (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-density core antigens (arrow) X 77400

Viral Etiology of Mononucleosis Proc Nat Acad Sci USA 68 (1971) 47F o t- - 'Nb, Ai¼ All:: - 2 5 1409

1410 Microbiology: Hampar et al Proc Nat Acad Sci USA 68 (1971) * nweaktagging of HTV particles when reacted twice with HR1K cells The three rabbit anti-htv conjugates showed ferritin 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 that were negative for antibodies to herpes simplex virus (HSV) by neutralization 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 ~~~~~~~~and rabbit conjugates was restricted to the outer surface of the virus capsids Ferritin tagging of HTV-enveloped particles was not observed with these reagents Similarly, no ferritin J -Ad lptagging was observed of either low-density (Fig 4) or highdensity (Fig 5) virus-core antigens ;M Ferritin tagging by the human and rabbit conjugates was also noted with fine granular nuclear antigens, some of which were in proximity to capsids (Fig 6) Ferritin tagging of similar structures was reported (8) in HSV-infected cells, where it was suggested that capsids were assembled from the finely granular antigens A--/@ Blocking tests Blocking of the immunoferritin reaction (Table 1) was observed with 01 ml of the "normal" HTV-positive human sera and mononucleosis sera and with the rabbit antisera The 7S antibodies from the "normal" human HTV-positive sera showed blocking activity at a concentration of 10 mg of IgG, but blocking activity was not observed with 19S antibodies at a concentration of 03 mg of IgM The 19S antibodies from the mononucleosis sera showed blocking activity at a concentration of 01 mg of agm, whereas the 7S antibodies from > two mononucleosis sera (sera MS and EE) showed no blocking activity at a concentration of 30 mg of IgG The 7S antibodies from a third mononucleosis serum (serum SS) showed weak blocking activity at a concentration of 30 mg of IgG, but not at a concentration of 10 mg Blocking activity was not observed with 03 ml of the "normal" human HTVnegative serum or with the rabbit preimmune sera Absorption of the mononucleosis sera and 19S antibodies with boiled beef-kidney antigen removed the heterophile,',<e,,-'a antibody activity, but did not eliminate the ability of these 'reagents to block the ferritin tagging of HTV This suggests that the anti-htv activity and the heterophile-antibody activity of the mononucleosis sera were distinct Treatment with 2-mercaptoethanol eliminated the blocking activity of the mononucleosis 19S antibodies, but it did not affect the blocking 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 reacin, 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 from which 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 homogenousantibody coat X 78,300 FIG 8 Blocking of immunoferritin reaction by human 19S ;(gm) antibodies The blocked particles show a "halo of structures resembling loops" X 78,300 "' ",-"' -z, "I m -'m 7, -f-evil i W '% ' 7'

Proc Nat Acad Sci USA 68 (1971) antibody coat (Fig 7), while particles coated with 19S antibodies (Fig 8) showed "a halo of structures resembling loops," suggestive of IgM antibodies (14) Similar differences in 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 mononucleosis sera tested contained 19S HTV antibodies, while the "normal" HTV-positive human sera tested contained 7S HTV antibodies The presence of both 7S and 19S HTV antibodies in one mononucleosis serum (serum SS) was undoubtedly 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 shown immunoelectrophoretically Second, IgA was present in both 7S and 19S fractions, and the HTV-negative fractions usually contained more IgA than did the HTV-positive fractions (Table 1) Third, the HTV blocking activity of the 19S antibodies, but not the 7S antibodies, was eliminated by treatment with 2-mercaptoethanol It was reported (15) that treatment with the sulfhydryl reagent destroyed the neutralizing activity of rabbit 19S antibodies to HSV, even when complement or anti-gamma globulin was added to the reaction mixture Finally, the antibody coating seen with the 7S antibodies in the blocking tests was suggestive of IgG (Fig 7), while the coating seen with the 19S antibodies was suggestive of IgM (Fig 8) Since HTV in humans can induce synthesis of both IgG and IgM antibodies (Table 1), we conclude that the presence of IgM antibodies in heterophile-positive mononucleosis sera reflects a primary immunogenic exposure to HTV This conclusion, in turn, suggests that HTV is the etiologic agent of mononucleosis However, neither our findings nor those reported by others prove conclusively that the virus associated with Burkitt tumors (HTV) and the virus of mononucleosis are 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-associated antigens (Figs 2-6) was of particular interest, since previously reported findings with HSV (8) indicated that the immunoferritin technique could also detect antibodies directed against virus core and envelope antigens Studies by acrylamide gel electrophoresis (16) suggest that there are seven major structural proteins associated with HSV (two in the core, two in the capsid, and three in the envelope), and electrophoretic patterns of HTV proteins indicate close similarities to the proteins found with HSV (17) This suggests that the specificity of our human and rabbit sera for HTV was limited to one, or possibly both, of the major antigens associated with the capsid, and the blocking experiments further suggest that these sera were reactive with the same antigens The inability of our human sera to react by immunoferritin with HTV-enveloped particles agrees with findings reported by others (13, 18) However, this finding was somewhat unexpected, since at least one serum (serum B-76) has been reported capable of neutralizing HTV infectivity (19), which finding suggests envelope activity In contrast, the inability of our rabbit antisera to react with HTV-enveloped particles Viral Etiology of Mononucleosis 1411 was not unexpected, since these sera were prepared against concentrates containing essentially virus capsids (10), and these sera do not neutralize HTV infectivity (19) The apparent discrepancy between the ability of human sera to neutralize HTV infectivity and the reactivity of these sera with HTV by immunoferritin may be due to one or more of the following factors First, infectivity of Herpes viruses may not be limited to enveloped particles (20) Second, neutralization of Herpes viruses may occur at antigenic sites below the surface of the envelope (8) In this case, the reactivity of antibody with complete virions may not be apparent with immunoferritin (8), probably because of the inability of the large antibody-ferritin complex to penetrate the virus envelope The same antibody when unconjugated, however, could still neutralize virus (15) Third, most HTV particles associated with lymphoblastoid cells appear defective by electron microscopy, in that the particles lack either a dense nucleoid or an envelope (unpublished observations) This suggests that HTV infection of lymphoblastoid cells may be abortive (21) or restrictive (22), with unincorporated envelope antigens accumulating in the cells The accumulation of unincorporated envelope antigens may account for the positive immunofluorescent reactions seen with fixed HTV-positive lymphoblastoid cells and human sera, since we have shown (10) that rabbit antisera whose specificity for HTV by immunoferritin is limited to capsid particles (10) do not react by 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 1 Epstein, M A, B G Achong, and Y M Barr, Lancet, i, 702 (1964) 2 Henle, G, W Henle, and V Diehl, Proc Nat Acad Sci USA, 59, 94 (1968) 3 Niederman, J C, R W McCollum, G Henle, and W Henle, J Amer Med Ass, 203, 205 (1968) 4 Gerber, P, D Hamre, R A Moy, and E N Rosenblum, Science, 161, 173 (1968) 5 Uhr, J, Science, 145, 457 (1964) 6 Freeman, M J, and A B Stavitsky, J Immunol, 95, 981 (1965) 7 Pike, R M, Bacteriol Rev, 31, 157 (1967) 8 Miyamoto, K, C Morgan, K C Hsu, and B Hampar, J Nat Cancer Inst, 46, 629 (1971) 9 Hampar, B, L M Martos, and J L Walker, submitted to J Nat Cancer Inst 10 Hampar, B, P Gerber, K C Hsu, L M Martos, J L Walker, R F Sigfienza, and G A Wells, J Nat Cancer Inst, 45, 75 (1970) 11 Hsu, K C, in Methods in Immunology and Immunochemistry, ed Williams, C A and M W 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