Anomalous Antibody Responses in Viral Infection: Specific Stimulation or Polyclonal Activation?

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Sept. 1984, p /84/ $02.00/0 Copyright D 1984, American Society for Microbiology Vol. 20, No. 3 Anomalous Antibody Responses in Viral Infection: Specific Stimulation or Polyclonal Activation? NATALIE E. CREMER,* VERONICA L. DEVLIN, JOHN L. RIGGS, AND SHIRLEY J. HAGENS Viral and Rickettsial Disease Laboratory, California Department of Health Services, Berkeley, California Received 1 March 1984/Accepted 6 June 1984 Eighteen paired serum samples submitted for serodiagnosis of current infection showed anomalous antibody results by complement fixation test when tested with a battery of agents (viruses, Mycoplasma pneumoniae, and chlamydia) selected for testing on the basis of the symptoms of the patient. Seventeen serum pairs showed a fourfold or greater rise in titer of antibody to two agents in the battery, and one showed only a twofold rise in titer of antibody to the identified causative agent but an eightfold rise in titer of antibody to a heterologous agent. The 18 serum pairs were tested for IgM antibody to the two involved agents to determine whether IgM antibody tests would better distinguish the probable cause of the current infection. The serum pairs were separated into three groups based on their IgM responses. Group I consisted of six serum pairs with IgM antibody to both agents, four pairs of which showed a fourfold or greater rise in titer of IgM antibody to both agents, and two of which showed a rise in titer of IgM antibody to only one of the two agents. Group II consisted of 10 serum pairs with IgM antibody to one of the two agents, 7 pairs of which showed a fourfold or greater rise in titer of IgM antibody to the agent. Group III consisted of two serum pairs with no IgM antibody to either agent. Results show that determination of presence or absence of IgM antibody per se or demonstration of a fourfold or greater rise in specific IgM antibody titer does not always help in distinguishing the causative agent in current infections. In doubling dilution tests, a fourfold or greater increase in antibody titer between acute-phase (A) and convalescentphase (C) serum samples is considered to be a significant change in antibody concentration between the two samples and evidence for current infection with that agent. In our laboratory, serum samples received for serodiagnosis of viral infection are tested against a battery of antigens which may include, in addition to viral antigens, Mycoplasma pneumoniae and chlamydial antigens. The composition of the battery is chosen on the basis of the symptoms described by the physician on the specimen submittal form of the patient. Occasionally, a fourfold or greater rise in titer of antibody to more than one agent occurs. Such multiple rises could occur because of mixed infections, reactivation of a latent infection, cross-reactivity between antigens, or nonspecific polyclonal activation of memory cells from a previous infection with an unrelated agent. Certain cross-reactions between viruses are known, such as those between mumps virus and the parainfluenza group of viruses (7) and between herpes simplex (HSV) and varicella-zoster (VZV) viruses (26, 30). In other cases, no known cross-reaction between the agents in question is recognized. In addition to a fourfold or greater increase in antibody titer between paired serum samples, presence of IgM antibody to the agent under study is also considered evidence for a current infection. In the present study, paired serum samples showing a rise in titer, by complement fixation (CF), of antibody to two of the agents in the battery were selected for determination of IgM antibody to those agents to see whether the presence or absence of IgM antibody could better identify the causative agent of the current infection. MATERIALS AND METHODS Serum samples. Eighteen serum pairs were studied, 17 showing fourfold or greater rises in CF titers of antibody to two agents in the tested battery, and one showing a twofold * Corresponding author. 468 rise in CF titer of antibody to the causative agent and an eightfold rise in CF titer of antibody to a second agent in the battery. The 18 serum pairs were studied for IgM antibody to those agents by the indirect fluorescent-antibody technique (IFA-M) (11) with fluorescein-labeled sheep anti-human muchain-specific antibody (Wellcome Research Laboratories, Beckenham, England) and by enzyme immunoassay (Rubazyme-M; Abbott Laboratories, North Chicago, Ill.) in those cases in which one of the involved agents was rubella virus (RV). The serum pairs were also tested by indirect fluorescent-antibody technique with fluorescein-labeled rabbit antihuman gamma globulin (IFA-G) (not chain specific, Beckman Instruments, Fullerton, Calif.). The anticomplement immunofluorescence test (ACIF) as described (24), instead of the IFA-G test, was done to test for antibody to viruses of the herpes group to avoid the problem of Fc receptors. Fluorescein-labeled goat anti-guinea pig complement (Cappel Laboratories, Cochranville, Pa.) was used. CF and IFA- G tests were done by standard methods (13, 25). With all the different fluorescent-antibody techniques (IFA-M, IFA-G, and ACIF), cells infected with the agent under study were used for the test antigen in antibody assays, and noninfected cells were used for control antigen. The tests were read by epifluorescence microscopy with Zeiss equipment. All serum samples were absorbed with IgG insolubilized by crosslinking with glutaraldehyde for removal of rheumatoid factor (9) before IgM tests. None of the sera was positive for antinuclear antibody, as determined by lack of fluorescence of nuclei of noninfected cells in the IFA-G and IFA-M tests. In selected cases, a hemagglutination inhibition test (22), an enzyme immunoassay test (Rubazyme; Abbott Laboratories) for antibody to RV, and an enzyme immunoassay test for antibody to HSV, which was previously described (8), modified for use with an enzyme immunoassay PR50 automatic analyzer (Gilford Instrument Laboratories, Inc., Oberlin, Ohio), and performed with alkaline phophatase-conjugated goat F(ab')2 fragment of anti-human gamma-chainspecific antibody (Sigma Chemical Co., St. Louis, Mo.), were done.

2 VOL. 20, 1984 ANOMALOUS ANTIBODY RESPONSES 469 TABLE 1. Time of serum sample collection and age of patients Paitient no. Collection day of sample" after onset of symptoms Age of patient (yr) A ?b Adult 15? "Acute-phase (A) and convalescent-phase (C) samples. "Date of onset of symptoms not given. Day of C sample is days after. collection of A sample. The mean collection day of the A samples from date of onset of symptoms was 5.8 ± 4.1 days, and the mean collection day of the C samples from date of onset of symptoms was days (Table 1). The serum samples were collected aseptically and stored in sterile containers at 4 C. The ages of the donors of the serum pairs ranged from 2 to 74 years (Table 1). Viral isolation. Specimens for virus isolation (two cases) or identification (skin lesion slides, two cases) were submitted along with the corresponding serum samples. Human fetal diploid lung or primary monkey kidney cultures were used for isolation. Identification of the isolates or viral antigen in C the skin lesions was made by the direct fluorescent antibody technique, using fluorescein-labeled anti-hsv antibody or fluorescein-labeled antibody to cytomegalovirus (CMV) prepared in hamsters or mice in our laboratory. Controls included noninfected and infected human fetal diploid lung cells. RESULTS Virus isolation. CMV was isolated from the urine of patient 1, and HSV was isolated from the throat of patient 9 (Table 2). Slides of skin lesions were positive for HSV in patient 8 and negative in patient 12. Tissues for virus isolation or identification from the other patients were not submitted. Serum antibody studies. Tables 2 and 3 show the two agents to which a dual rise (fourfold or greater) in CF antibody titer occurred. CF titers of antibody to other tested agents in each case were either <8 or standing titers. The dual rises (fourfold or greater) in CF antibody titers were observed with IFA-G or ACIF in sera of 13 of the 17 patients; in one case (patient 8), viral antigen was identified in the lesion of the patient, but a rise in CF titer of antibody occurred only to a heterologous virus (Table 3). In four cases, a fourfold-or-greater rise was seen in IFA-G titer of antibody to one agent of the combination, whereas the titer of antibody to the other agent was high and standing (Table 3, patients 4, 9, 15, and 16). The fact that four of the serum pairs showed a high standing antibody titer by IFA-G rather than a rise in antibody titer is not surprising, since antibody detected by CF tends to appear more slowly after antigenic stimulation than does antibody detected by other methods. The 18 serum pairs were separated into three groups based on their IgM response to the two agents involved in the corresponding antibody titer rises (Table 2). Group I consisted of 6 serum pairs with IgM antibody to both agents, four pairs of which showed a fourfold-or-greater rise in titer of IgM antibody to both agents and two of which showed a rise in titer of IgM antibody to only one of the two agents, group II consisted of 10 serum pairs with IgM antibody to one of the two agents, 7 of which showed fourfold-or-greater rise in TABLE 2. IgM antibody in paired serum samples showing fourfold-or-greater rise in CF titer of antibody to two agents Group no." Patient no. Combination of agentsh Clinical description from patient submittal form 1 (HSV-CMV) Fever, sore throat, CMV? 2 (HSV-Flu A) Herpes pharyngitis, fever, lymphadenopathy 3 (Flu A-RSV) Pneumonia, fever, cough 4 (MP)-Flu B Pneumonia, fever, cough 5 MP-(chlamydia) Flu-like symptoms 6 (RSV-parainfluenza virus type 3) Fever, upper respiratory distress II 7 HSV-(CMV) Herpetic lesion, abnormal liver function 8 HSV-(CMV) Genital lesion, lymphadenopathy 9 HSV-mumps Encephalitis, rash 10 HSV-(VZV) Rash, vesicles, ulcer on tongue 11 HSV-VZV Diffuse vesicular eruption, history of VZV as child, VZV? 12 (HSV)-VZV Vaginal and rectal blisters 13 RV-measles Rash, lymphadenopathy 14 (RV)-measles Rash, lymphadenopathy, cough 15 (VZV)-measles Rash, sore throat, chicken pox 16 (MP)-RSV Fever, chills lll 17 HSV-RV Herpes? 18 HSV-VZV Eruption of vesicles, fever, sore throat "Patients were grouped according to their IgM antibody response as follows: group I, IgM response to both agents: group It, IgM response to one agent: and group III, IgM response to neither agent. " Items in parentheses are those to which a fourfold-or-greater rise in IgM antibody titer occurred. IgM antibody was present to the boldfaced agents in one or both serum samples, but a fourfold increase in IgM antibody titer did not occur.

3 470 CREMER ET AL. J. CLIN. MICROBIOL. TABLE 3. Antibody titers on paired serum samples" showing a rise in antibody titer to two agents First agent tested with: Titer of antibody to": Second agent tested with: patient no. Combination of agents CF IFA-G or ACIF IFA-M CF IFA-G or ACIF IFA-M A C A C A C A C A C A C Group I 1 HSV-CMV < , HSV-Flu A <8 128 <8 512 < ,048 < Flu A-RSV < ,096 < < ,096 < MP-Flu B < ,048 <10.40 < MP-chlamydia < <10 10 < RSV-parainfluenza virus type 3 <8 512 <8 512 < Group II 7 HSV-CMV <10 <10 < < HSV-CMV 8 16b < <8 < , HSV-mumps <16 64 <8 < ,096 4, HSV-VZV ,024 <8 <8 <8 128 <8 256 < HSV-VZV <8 8 < <8 <8 12 HSV-VZV <8 128 <8 512 < <10 <10 13 RV-measles <8 16 < ,024.8,192 <8 <8 14 RV-measles <8 16 < 16 1, ,024 8,192 <8 <8 15 VZV-measles <8 128 <8 128 < <8 16 1,024 2,048 <10 <10 16 MP-RSV , < ,048 <10 <10 Group III 17 HSV-RV 8 64 <8 128 <10 < <8 512 <10 <10 18 HSV-VZV <10 <10 < <10 <10 "Acute-phase sample (A) and convalescent-phase sample (C). HSV antigen present in lesion. titer of IgM antibody to the agent. Group III consisted of two serum pairs with no IgM antibody to either agent. The virus most associated with dual antibody titer rises was HSV, which was involved in 10 of the 18 cases, 3 of which, however, were in combination with VZV, with which it shares a common antigen (26, 30). Measles virus, RV, respiratory syncytial virus (RSV), and M. pneumoniae (MP) appeared on the list of dual CF antibody titer rises three times each, influenza A virus (Flu A) appeared two times, and mumps virus, influenza B (Flu B) and parainfluenza virus type 3 appeared one time each. Four paired serum samples not included in the 18 paired samples had rises of fourfold or greater in CF titers of antibody to parainfluenza virus types 1 and 3 and also had IgM antibody to both viruses. This could be expected because of known heterotypic responses between these viruses (7). DISCUSSION Some viruses are known to be nonspecific modulators of the immune response (21, 29), causing enhancement or depression of antibody formation to unrelated antigens. In cases in which clinical symptoms are not characteristic for the suspected viral agent, serological tests, particularly in the absence of viral isolation, assume a significant role in diagnosis. Serodiagnosis becomes complicated, however, when rise in titer of antibody to more than one agent occurs. The incidence of dual antibody titer rises in our laboratory over the past 2 years has been 4.6% (26 cases with dual titer rises of 555 total cases with titer rises). More rarely encountered are multiple (triple or more antibody titer rises). In a recent example, the paired serum samples of a 30-year-old male patient with high fever and rash showed fourfold-orgreater rises in CF titers of antibody to five agents (MP, RSV, CMV, HSV, and measles), a low titer in the C sample (titer 10) of IgM to MP only, and a rise in IFA-G titer of antibody to RSV. The other IFA-G titers were high and standing. The pattern of IFA results for antibody to Epstein- Barr viral antigens indicated a past infection with no evidence of reactivation (14). In this example, the multiple CF antibody titer rises appear to be nonspecific polyclonal activation of antibody formation due to causes unknown. The present study illustrates the difficulties encountered with 18 paired serum samples in the interpretation of results. In the six cases in which IgM antibody to both agents was present (Table 2, group I) the presence of 1gM antibody per se was not helpful in distinguishing the causative agent, nor was the fourfold-or-greater rise in IgM antibody titer in four cases, since it occurred with IgM antibody to both agents. In the two cases (patients 4 and 5) in which there was a rise in titer of IgM antibody to only one of the agents of the pair, the clinical symptoms were compatible with those of an infection with either agent, but IgM serological results (fourfoldor-greater rise in IgM antibody titer) favored MP and chlamydia infections, respectively. Clinical findings in patients 4 and 5, as well as in patients 3 and 6, were also compatible with a mixed infection with the corresponding two agents. In patient 1 (HSV-CMV combination), CMV was isolated from the urine. This then appears to be a CMV infection with either reactivation of HSV infection or polyclonal stimulation of memory cells for IgM and IgG antibody synthesis from a previous HSV infection. The latter may be more likely, for although IgM antibody is reported present in severe secondary HSV infection (15), it is only rarely present in usual recurrent or reactivated HSV infection (10, 27). Examination of the sera for IgM antibody did not help in the diagnosis of the two patients in group III, since IgM antibody to both agents was absent. Patient 17 may have had a recurrent HSV infection with polyclonal activation of IgG antibody to RV. In that particular case a significant change

4 VOL. 20, 1984 in antibody concentration indicative of current infection with RV was found by four tests, hemagglutination inhibition, EIA, IFA-G, and CF; and a change in antibody concentration indicative of current infection with HSV was found by 3 tests, EIA, ACIF, and CF. In 6 of the 10 cases in which IgM antibody to only one agent was present, its presence and clinical findings were compatible with a current infection with that agent (patients 10 and 12 to 16). In the sera of patients 7 and 8 (HSV-CMV combinations) the fourfold or greater increase in titers of IgM antibody to CMV would indicate current CMV infection; however, clinical findings and presence of HSV viral antigen (patient 8) also indicate HSV infection. Patient 7 could have a recurrent HSV infection with a concomitant CMV infection, since abnormal liver function, compatible with CMV infection, was present. IgM antibody responses are reported in cases of primary, recurrent, and reactivated CMV infections (20, 23, 28). In patient 8, HSV infection was diagnosed by fluorescent-antibody staining of HSV antigen in tissue from a skin lesion. Since IgM antibody to HSV was absent, it was probably a recurrent HSV infection with possible concurrent CMV infection or possibly nonspecific polyclonal activation of the CMV antibody response by HSV. Patient 9 (HSV-mumps combination) apparently had a reactivated HSV infection with polyclonal stimulation by HSV of memory cells committed to production of antimumps viral antibody. There was a high standing titer of IgM antibody to mumps virus (A, 128; C, 128), no IgM antibody to HSV was isolated from the throat of this patient, but HSV was. However, it is possible that neither agent was the causative agent of the encephalitis. Latent HSV infection is often reactivated under conditions of stress, which might occur in a sick child, and although HSV can be isolated from the throat, it was not necessarily the causative agent of the encephalitis. HSV is reported to be a mitogen, as demonstrated by [3H]thymidine incorporation by mouse B lymphocytes after reaction with HSV in vitro (16). It is noteworthy that HSV appeared in 6 cases (Table 2) in combination with seemingly unrelated agents. In patient 11, although a low level of IgM antibody to HSV (titer of 8) was present in the C sample, the clinical findings were more compatible with recurrent VZV (zoster) infection. IgM antibody is usually present in cases of infection with herpes zoster virus (3, 5, 27); however, it appears to arise more slowly than in cases of infection with varicella virus (31). In a report by Sundqvist (31), none of the zoster virus-infected patients had IgM antibody earlier than 10 days after onset of symptoms. In that study, 21 of 25 zoster patients became positive for IgM antibody to VZV. In patient 11 (Table 1), the A sample and the C sample were taken 1 and 10 days, respectively after onset of symptoms, possibly too early for an IgM response. The dual rises in CF titer of antibody to HSV and VZV are assumed due to shared antigens. In addition to HSV, other agents appearing in Table 2 are reported to act as mitogenic agents for mouse B lymphocytes. MP acted as a B-cell mitogen for mouse and guinea pig lymphocytes and induced polyclonal antibody formation in mouse spleen cells (4); isolated hemagglutinin glycoproteins from Flu A subtypes HON1, HlNl, H2N2, and H3N2 and from Flu B were mitogenic for mouse lymphocytes (1, 6), as were the isolated hemagglutinin-neuraminidase and fusion glycoproteins of Sendai virus, a paramyxovirus type 1 virus (17). Representatives of these agents appeared in the serum of patients 2, 3, 4, 5, 6, and 16. Recent studies by other investigators report on stimulation ANOMALOUS ANTIBODY RESPONSES 471 of IgM antibody to RV during infection with Epstein-Barr virus (18), stimulation of IgM antibody to HSV types 1 and 2 and CMV during vaccination of a child with VZV, and stimulation of IgM antibody to CMV in HSV type 1 stomatitis (19). Other reports include studies on the production of IgM antibody to CMV in VZV infections (12), rises in CF titers of antibody to measles virus and RV in cases of ulcerative colitis and chronic active hepatitis (32), increased production, by lymphocytes in vitro of patients in cases of measles and varicella, of antibody to the etiological virus as well as to unrelated viruses (varicella, measles, rubella, and mumps viruses) (2). As more is learned about basic mechanisms in isotype switching and the development, expression, and activation of memory cells, anomalous results such as those described here may be explained. This study illustrates the importance of testing for antibody to a battery of likely agents rather than testing for antibody to only one suspected agent; this study also indicates that no one test or combination of tests can always resolve problems in interpretation of laboratory results. ACKNOWLEDGMENT We thank Dana Gallo for consultation on IgM assays. LITERATURE CITED 1. Armstrong, R. B., G. M. Butchko, S. C. Kiley, M. A. Phelan, and F. A. Ennis Mitogenic;ty of influenza hemagglutinin glycoproteins and influenza viruses bearing H2-hemagglutinin. Infect. Immun. 34: Arneborn, P., G. Biberfeld, M. Forsgren, and L. V. Von Stedingk Specific and non-specific B cell activation in measles and varicella. Clin. Exp. Immunol. 51: Arvin, A. M., and C. M. Koropchak Immunoglobulins M and G to varicella-zoster virus measured by solid-phase radioimmunoassay: antibody responses to varicella and herpes zoster infections. J. Clin. Microbiol. 12: Biberfeld, G., and E. Gronowicz Mycoplasma pneumoniae is a polyclonal B-cell activator. Nature (London) 261: Brunell, P. A., A. A. Gershon, S. A. Uduman, and S. Steinberg Varicella-zoster immunoglobulins during varicella, latency and zoster. J. Infect. Dis. 132: Butchko, G. M., R. B. Armstrong, W. J. Martin, and F. A. Ennis Influenza A viruses of the H2N2 subtype are lymphocyte mitogens. Nature (London) 271: Chanock, R. M Parainfluenza viruses, p In E. H. Lennette and N. J. Schmidt (ed.), Viral, rickettsial and chlamydial infections, 5th ed. American Public Health Association, Inc., Washington, 8. Cremer, N. E., C. K. Cossen, C. V. Hanson, and G. R. Shell Evaluation and reporting of enzyme immunoassay determinations of antibody to herpes simplex virus in sera and cerebrospinal fluid. J. Clin. Microbiol. 15: Cremer, N. E., and R. L. Riggs Immunoglobulin classes and viral diagnosis, p In E. H. Lennette and N. J. Schmidt (ed.), Viral, rickettsial, and chlamydial infections, 5th ed. American Public Health Association, Inc., Washington, 10. Doerr, H. W., G. Gross, and H. Schmitz Neutralizing serum IgM antibodies in infections with Herpes simplex Virus Hominis. Med. Microbiol. Immunol. 162: Gallo, D., J. L. Riggs, J. Schachter, and R. W. Emmons Multiple-antigen slide test for detection of immunoglobulin M antibodies in newborn and infant sera by immunofluorescence. J. Clin. Microbiol. 13: Hanshaw, J. B., J. C. Niederman, and L. N. Chessin Cytomegalovirus macroglobulin in cell-associated herpesvirus infections. J. Infect. Dis. 125: Hawkes, R. A General principles underlying laboratory diagnosis of viral infections. p , In E. H. Lennette and N. J. Schmidt, (ed.), Viral, rickettsial, and chlamydial infec-

5 472 CREMER ET AL. tions, 5th ed. American Public Health Association, Inc., Washington, 14. Henle, W., G. Henle, and C. A. Horwitz Infectious mononucleosis and Epstein-Barr virus-associated malignancies, p In E. H. Lennette and N. J. Schmidt (ed.), Diagnostic procedures for viral, rickettsial and chlamydial infections, 5th ed. American Public Health Association, Inc., Washington, 15. Kalimo, K. 0. K., R. J. Marttila, K. Granfors, and M. K. Viljanen Solid-phase radioimmunoassay of human immunoglobulin M and immunoglobulin G antibodies against herpes simplex virus type 1 capsid, envelope, and excreted antigens. Infect. Immun. 15: Kirchner, H., G. Darai, H. M. Hirt, K. Keyssner, and K. Munk In vitro mitogenic stimulation of murine spleen cells by herpes simplex virus. J. Immunol. 120: Kizaka, S., G. Goodman-Snitkoff, and J. J. McSharry Sendai virus glycoproteins are T cell-dependent B cell mitogens. Infect. Immun. 40: Morgan-Capner, P., R. S. Tedder, and J. E. Mace Reactivity for rubella-specific IgM in sera from patients with infectious mononucleosis. Lancet i: Morinet, F., A. Devillechabrolle, B. Fortier, and J. M. Huraux Study of serological cross-reactions in human herpesvirus infections by an enzyme-linked immunosorbent assay (ELISA). Ann. Virol. 133: Nagington, J Cytomegalovirus antibody production in renal transplant patients. J. Hyg. 69: Notkins, A. L., S. E. Mergenhagen, and R. J. Howard Effect of virus infections on function of the immune system. Annu. Rev. Microbiol. 22: Palmer, D. F., J. J. Cavallaro, and K. L. Hermann A procedural guide to the performance of rubella hemagglutination-inhibition tests, p U.S. Department of Health, J. CLIN. MICROBIOL. Education, and Welfare, Public Health Service, Centers for Disease Control, Atlanta, Ga. 23. Pass, R. F., P. D. Griffiths, and A. M. August Antibody response to cytomegalovirus after renal transplantation: comparison of patients with primary and recurrent infections. J. Infect. Dis. 147: Reedman, B. M., and G. Klein Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines. Int. J. Cancer 11: Riggs, J. L Immunofluorescent staining, p In E. H. Lennette and N. J. Schmidt (ed.), Viral, rickettsial, and chlamydial infections, 5th ed. American Public Health Association, Inc., Washington, 26. Schmidt, N. J Further evidence for common antigens in herpes simplex and varicella-zoster viruses. J. Med. Virol. 9: Schmidt, N. J., and D. Gallo Class-specific antibody responses to early and late antigens of varicella and herpes simplex viruses. J. Med. Virol. 13: Schmitz, H., D. Kampa, H. W. Doerr, T. Luthardt, H. G. Hillemanns, and A. Wurtele IgM antibodies to cytomegalovirus during pregnancy. Arch. Virol. 53: Semenov, B. F Viruses as nonspecific modulators of immunological reactivity. Acta Virol. 25: Shiraki, K., T. Okuno, K. Yamanishi, and M. Takahashi Polypeptides of varicella-zoster (VZV) and immunological relationship of VZV and herpes simplex virus (HSV). J. Gen. Virol. 61: Sundqvist, V. A Frequency and specificity of varicella zoster virus IgM response. J. Virol. Methods 5: Triger, D. R., J. B. Kurtz, F. 0. MacCallum, and R. Wright Raised antibody titres to measles and rubella viruses in chronic active hepatitis. Lancet i: Downloaded from on November 20, 2018 by guest