Viral complications Inverse relationship between human herpesvirus-6 and -7 detection after allogeneic and autologous stem cell transplantation

Transcription

1 (21) 27, Nature Publishing Group All rights reserved /1 $15. Viral complications Inverse relationship between human herpesvirus-6 and -7 detection after allogeneic and autologous stem cell transplantation H Miyoshi 1, K Tanaka-Taya 1, J Hara 1, H Fujisaki 1, Y Matsuda 1, H Ohta 1, Y Osugi 1, S Okada 1 and K Yamanishi 2 Departments of 1 Developmental Medicine D-5 and 2 Microbiology C-1, Osaka University, Graduate School of Medicine, Suita, Japan Summary: Human herpesvirus-6 (HHV-6) and -7 were analyzed in 25 and 18 patients with allogeneic (allo) and autologous (auto) stem cell transplantation (SCT), respectively, by weekly examination of viral DNA in peripheral mononuclear cells using semiquantitative PCR and serologic tests up to 12 weeks after SCT. HHV-6 DNA was detected in 29.6% and 27.9% of samples after allo- and auto-sct, respectively. The proportions of HHV-6- DNA-positive samples increased in week 3 and 4 after allo-sct, and in week 1 to 3 after auto-sct. The frequency of HHV-7 DNA detection, however, was higher after auto-sct (24.7%) than allo-sct (12.8%) (P.1). Antibody titer elevation was accompanied by HHV-6 infection in eight of 1 patients. That for HHV- 7 was also frequently observed, but not associated with infection in the majority of patients. Five of six patients with 1 2 copies of HHV-6 DNA (/1 5 cells) on two consecutive occasions were allo-sct recipients and three showed clinical episodes. Conversely, three of five patients with continuous reactivation of HHV-7 were auto-sct recipients. Thus, the frequencies of HHV-6 and -7 DNA detection showed an inverse relationship comparing allo- and auto-sct, suggesting a different mechanism may regulate HHV-6 and -7 reactivation. (21) 27, Keywords: HHV-6; HHV-7; stem cell transplantation; PCR; serology Human herpesvirus-6 (HHV-6) and human herpesvirus-7 (HHV-7) belong to -herpesvirus subfamily. HHV-6 was first isolated from patients with immunodeficiency 1 and HHV-7 was isolated from a healthy adult. 2 It is now believed that HHV-6 and HHV-7 are causative agents of exanthem subitum and febrile illnesses in children. 3,4 HHV- 6 infects persistently or latently after the primary infection in salivary glands, lymph nodes, T cells, monocytes/ Correspondence: Dr J Hara, Department of Developmental Medicine D- 5, Osaka University, Graduate School of Medical Science, 2 2 Yamadaoka, Suita, Osaka, , Japan Received 4 April 2; accepted 13 February 21 macrophages, the liver and kidney. 5,6 HHV-6 infection has some association with chronic fatigue syndrome, 7 multiple sclerosis, 8,9 hepatitis, 1 meningoencephalitis, thrombocytopenia, and hemophagocytic syndrome. 5,7 HHV- 7 also infects in infancy or early childhood and infects persistently throughout life. Although HHV-7 was shown to be CD4 + T cell tropic, 2 the cellular localization and its clinical importance have yet to be fully clarified. It is known that HHV-6 is reactivated or reinfective after stem cell transplantation (SCT) and often causes clinically significant diseases including interstitial pneumonitis, delay of engraftment, thrombotic microangiopathy and skin rash. 5,12,16 26 Thus, it appears that HHV-6 is frequently reactivated under immunosuppressive conditions following SCT and plays significant pathologic roles. On the other hand, the behavior of HHV-7 after SCT and its pathogenic roles are still obscure. 23 In this study, we analyzed the behavior of HHV-6 and HHV-7 after allogeneic (allo) and autologous (auto) SCT by examining viral DNA using semiquantitative PCR and serologic tests. Patients and methods Patients Twenty-five and 18 patients with allo- and auto-sct, respectively, were analyzed. Stem cell source was bone marrow in all patients with allo-sct and nine with auto- SCT. The remaining nine patients received autologous peripheral blood stem cell transplantation. Patient characteristics are shown in Table 1. A combination of cyclosporine and methotrexate or methotrexate alone was administered for prophylaxis against graft-versus-host disease (GVHD). Gammaglobulin 2 mg/kg intravenously was administered weekly until discharge. Oral acyclovir (5 mg/m 2 in two divided doses) was administered until discharge for prophylaxis against herpes simplex and herpes zoster viruses. When cytomegalovirus (CMV) infection was suspected based on clinical symptoms, measurement of antibody titer, detection of CMV DNA by polymerase chain reaction (PCR) and isolation of virus were attempted. Informed consent was obtained from patients and/or their parents.

2 166 Table 1 Characteristics of the patients Allogeneic SCT 25 Age (mean) (years) 9.6 (range 1 19) Sex (M:F) 13/12 Diagnosis Leukemia 21 Aplastic anemia 2 Metabolic disease 14 Donors Matched related 14 Matched unrelated 8 Mismatched (unmodified) 3 Conditioning regimen ATG 8 TBI 18 Non-TBI 7 Cyclosporine and methotrexate 21 Methotrexate 4 Acute GVHD None 9 Grade I and II 11 Grade III and IV 4 Not determined 1 Chronic GVHD No 11 Yes 1 Not determined 5 Autologous SCT 18 Age (mean) (years) 5.7 (range 1 16) Sex (M:F) 11/7 Diagnosis Leukemia 1 Solid tumor 17 Samples EDTA blood samples were collected weekly from 2 weeks before and up to 12 weeks after SCT. Altogether, 257 and 19 samples from patients with allo- or auto-sct, respectively, were collected. Mononuclear cells (MNCs) in these samples were assayed for HHV-6 and -7 DNA. Plasma samples collected up to 8 weeks after SCT from 16 patients were assayed for specific antibodies. Serologic studies Specific antibodies to HHV-6 and -7 were determined in 16 patients by an immunofluorescence antibody (IFA) test as described previously. 4,27,28 Serial two-fold dilutions of sera were put on spotted slides on which MNCs infected with HHV-6 (HST strain) or HHV-7 (7-KHR strain) were fixed with cold acetone for 2 min, and goat antibody to human IgG labeled with fluorescein-isothiocyanate (DAKO, Copenhagen, Denmark) was used as a second antibody. Monoclonal antibodies specific to HHV-6 (OHV-1 and -3) 29 and HHV-7 (KR4; unpublished data) were used to identify the virus. 3,4,29 An IFA test confirmed that these antibodies did not react with antigens of other human herpesviruses. DNA analysis PCR for HHV-6 and -7 has been described previously 27,28 and was used with some modifications as follows. The sequences of the outer primers of HHV-6 were: 5 - TTCTCCAGATGTGCCAGGGAAATCC-3 5 -CATCA TTGTTATCGCTTTCACTCTC-3 ; and the inner primers were: 5 -AGTGACAGATCTGGGCGGGCCCTAATAAC TT-3 5 -AGGTGCTGAGTGATCAGTTTCATAACCA AA-3. Those of the outer primers of HHV-7 were: 5 - AGTTCCAGCACTGCAATCG-3 5 -CCAAAAGCGTC GCTATCAA-3 ; and the inner primers were 5 -CGCATA- CACCAACCCTACTG-3 5 -GACTCATTATGGGGAT CGAC-3. The DNA thermal cycler conditions of HHV-6 consisted of a 1-min denaturation step at 94 C, a 2-min annealing step at 62 C, and elongation steps of 3, 4 and 5 min at 72 C for 1 cycles. Conditions for HHV-7 consisted of a 1-min denaturation step at 94 C, a 2-min annealing step at 55 C and a 1-min elongation step at 72 C for 3 cycles. Amplified samples were separated by electrophoresis on 2% agarose gel and were visualized with ethidium bromide staining. To confirm the results of PCR amplification, Southern hybridization with the alkaline-phosphate-conjugated oligonucleotide probes for HHV-6, variant-a and variant-b, and HHV-7 was performed. The sequences were as follows: HHV-6 variant-a, 5 -TAAATCCATTACTGGCCTTGAA- 3 ; HHV-6 variant-b, 5 -AACTCCATCAGCGGCCTCC AG-3 ; HHV-7, 5 -CATTACTCCAGTGACTTCCGATAT- TAATTT-3. The number of copies of viral genome per 1 5 MNCs was estimated based on the semiquantitative PCR as previously described 27 and expressed as follows: (1+), 1 1 copies; (2+), copies; (3+), copies; and (4+), 1 3 copies. Statistics Differences in frequencies of variables were compared with the 2 -test and Fisher s test. Differences were considered as significant for P.5. Results Viral DNA detection During the study period after SCT, HHV-6 DNA was detected in 66/223 samples (29.6%) after allo-sct and 43/154 samples (27.9%) after auto-sct. The strain detected was variant-b; no variant-a was found in any samples. HHV-6 DNA at a high level ( 1 2 copies per 1 5 cells (3+ or 4+)) was detected in 2/223 samples (9.%) after allo-sct and 12/154 samples (7.8%) after auto-sct. HHV-6 DNA was detected in 68% and in 61% analyzed patients after allo- and auto-sct, respectively. After allo- SCT, the proportions of HHV-6-DNA-positive samples increased to around % in week 3 and 4 after SCT and increased again in week 8 11 (Figure 1). After auto-sct, the frequency of HHV-6 DNA detection increased in weeks 1 to 3, earlier than after allo-sct, and decreased thereafter. The detection of more than 1 2 copies per 1 5 cells was sporadic after week 8 (Figure 1). HHV-7 DNA was detected in 27/211 samples (12.8%)

3 Allo Allo 167 % % 2 Week after SCT Sample number Auto % 2 Week after SCT Sample number Auto 2 % 2 Week after SCT Sample number Figure 1 Frequency of HHV-6 DNA detection and the copy number of viral genome in mononuclear cells from allo- and auto-sct recipients., 1 1 copies (1+);, copies (2+);, copies (3+); and +, 1 3 copies (4+) of HHV-6 genome, respectively. Week after SCT Sample number Figure 2 Frequency of HHV-7 DNA detection and the copy number of viral genome in mononuclear cells from allo- and auto-sct recipients., 1 1 copies (1+);, copies (2+);, copies (3+); and +, 1 3 copies (4+) of HHV-7 genome, respectively. after allo-sct and 36/146 samples (24.7%) after auto-sct (P.1). The samples containing more than 1 2 copies per 1 5 cells were 13/211 (6.2%) and 21/146 (13.4%) after allo- and auto-sct, respectively (P.5). However, the proportion of patients with HHV-7 DNA detection was not different after allo- and auto-sct (42% vs 67%, NS). Thus, the detection of HHV-7 DNA was significantly more frequent after auto-sct than after allo-sct, and a high copy number of HHV-7 DNA was rarely observed after allo- SCT (Figure 2). Although the frequencies of HHV-6 and HHV-7 DNA detection were not different after auto-sct, the frequency of HHV-7 DNA detection was significantly lower than that of HHV-6 after allo-sct (P.1). In addition, HHV-7 DNA was detected earlier following auto- SCT than after allo-sct (Figure 2). Association between HHV-6 and HHV-7 antibody titers and viral reactivation These data are shown in Table 2. All 12 patients analyzed before SCT were seropositive for HHV-6 and -7. Among 16 patients analyzed, 1 patients showed a significant increase of HHV-6 antibody titers and the timing of the titer increase was close to that of virus reactivation in eight of these patients. The remaining six patients showed no increase of either HHV-6 antibody titer or infection throughout the study period. HHV-7 antibody titers increased in 13 patients after transplantation, but no apparent correlation with detection of HHV-7 was observed in the most of them. No HHV-7 infection was observed in six of the 13 patients with an increase of antibody titer and in two of the three without an increase in antibody titer. Administration of intravenous gammaglobulin did not appear to influence infection. Clinical symptoms associated with HHV-6 and -7 reactivation There appeared to be no significant relationships between the reactivation of HHV-6 and -7, and the use of antithymocyte globulin and total body irradiation or the degree of GVHD. Six and five patients showed reactivation of HHV- 6 or -7 at a high level (3+ or 4+) on two consecutive occasions, respectively (Table 3). Five of the six patients with reactivation of HHV-6 and two of the five with HHV- 7 reactivation were recipients of allo-sct. In three of six patients with HHV-6 reactivation, in addition to fever, persistent thrombocytopenia, generalized skin rash similar to that observed with exanthem subitum and interstitial pneumonitis were observed coincident with the increase of HHV-6 copy numbers. In all patients, no other pathogens including HHV-7 and CMV were detected. In patient UPN 115, HHV-6 DNA was detected at a high copy number from BM cells, as well as PBMCs. In patient UPN 118, HHV-6 DNA was detected from bronchoalveolar lavage specimens. In patient UPN 133, symptoms developed before engraftment and HHV-6 was isolated from PBMCs on day 12. Patients UPN 118 and 133 received administration of ganciclovir and both showed an improvement of symptoms. However, patient UPN 118 died of multi-organ failure after resolution of pneumonitis. In the remaining three patients and other patients with reactivation of HHV-6 at a lower level, no specific symptoms developed. Persistent thrombocytopenia accompanied with fever and interstitial pneumonitis were observed in other patients. However, no

4 168 Table 2 Patients with significant serologic HHV-6 and/or HHV-7 increase UPN Age Donor HLA HHV-6 a HHV-7 a (years)/sex Time of Time of DNA Time of Time of DNA serologic detection serologic detection increase increase 125 8/M Sibling Matched 5 5 (4+) 4 4 (2+) /M Sibling Matched 2 2 (2+) /M Sibling Matched 4 4 (2+) 4 8 (2+) 133 8/M Sibling Matched 3 2 (3+) /M Unrelated Matched 5 4 (2+) 7 5 (2+) /F Unrelated Mismatched (1-locus) /M Mother Mismatched (2-loci) /F Mother Mismatched (2-loci) /F Auto 2 3 (2+) 2 4 (2+) 2 1/M Auto 2 1 (2+) 2 4 (2+) 262 7/M Auto 2 2 (2+) 2 1 (2+) 263 5/M Auto /M Auto 4 7 (2+) 4 8 (2+) a Numbers indicate the weeks from transplantation. Numbers in parentheses indicate the number of copies of viral DNA. Table 3 Patients with viral DNA at a high copy number UPN Age Donor HLA Acute Weeks after SCT a Associated symptom and diseases (years)/sex GVHD HHV-6 DNA /F Sibling Matched II thrombocytopenia, fever (week 6 1) 125 8/F Sibling Matched I /M Sibling Matched eruption, fever (week 2 3) 9 12/F Father Matched II /F Father Mismatched IV 4 4 interstitial pneumonitis (week 3 4) (1-locus) 2 1/M Auto HHV-7 DNA 125 8/F Sibling Matched I /M Sibling Matched I /M Auto /F Auto /F Auto a Numbers indicate the numbers of copies of viral DNA on a semiquantitative scale from to 4. difference of the frequency of fever and skin rash was observed between patients with and without HHV-6 reactivation, since these symptoms are frequently observed after SCT and caused by GVHD, bacterial and fungal infections as well as HHV-6 infection. In five patients with HHV-7 reactivation, no symptoms associated with the increase of HHV-7 copy number were documented. Thus, in contrast to HHV-7, continuous reactivation of HHV-6 was more often found in allo-sct and frequently was associated with clinical symptoms. Discussion We studied HHV-6 and -7 infection after SCT using semiquantitative PCR and serologic tests and examined the relationship to clinical symptoms. HHV-6 DNA was detected in 68% and 61% of patients in allo- and auto-sct, respectively. These frequencies are similar to those reported by others. 5,21 23 Although the proportions of HHV-6-DNApositive samples were similar between in allo- and auto- SCT, the high frequency of the detection of HHV-6 DNA persisted during the study period in allo-sct in contrast to auto-sct where the increase of the frequency was sporadic. In addition, five of six patients with continuous reactivation of HHV-6 at a high level were after allo-sct and clinical symptoms seemingly attributed to HHV-6 infection were observed in three of these patients. This difference in alloand auto-sct seems to be attributable to the intense immune suppression caused by the more intense pre-conditioning regimen, GVHD and immune suppressive agents in allo-sct allowing reactivation of HHV-6. Although stem cell source was peripheral blood in half of patients with auto-sct, no difference was observed in the fre-

5 quency of viral reactivation between patients with bone marrow and peripheral blood. On the other hand, the frequency and time of the detection of HHV-7 DNA were quite different from those of HHV-6. After allo-sct, the initiation of the HHV-7 DNA detection was later than that of HHV-6 and the frequency was lower. HHV-7 DNA was detected in 42% of patients similar to a previous report. 23 Based on the semiquantitative analysis, patients with high numbers of viral copies (3+ or 4+) were rare. Conversely, after auto-sct, the frequency of the HHV-7 DNA detection was higher, ie HHV-7 DNA was detected in 67% of patients (36/146 samples), and HHV-7 DNA was detected at earlier times than after allo- SCT. In addition, three of five patients with continuous reactivation of HHV-7 at a high level were after auto-sct and no clinical symptoms associated with HHV-7 infection were observed. Since HHV-7 DNA was detected in almost all healthy individuals, 27 it appears that replication of HHV- 7 in MNCs was suppressed, especially in allo-sct. Thus, the presence of HHV-7 was not associated with intense immunosuppression and more intense immune suppression seemed to eliminate HHV-7, suggesting that mechanisms regulating reactivation and/or maintenance of HHV-7 may be different from HHV-6. Indeed, HHV-7 is isolated from CD4 + T cells after phytohemagglutinin stimulation, 2 indicating that T cell activation is required for reactivation of HHV-7. Elucidation of the mechanisms explaining the converse relationship between HHV-6 and -7 reactivation awaits further study. Antibody titers for HHV-6 increased in 1 of 16 patients analyzed and these serologic increases were associated with HHV-6 infection in eight patients, indicating that antibody response to HHV-6 was maintained in a majority of SCT recipients and serologic tests are useful for the diagnosis HHV-6 infection. Although gammaglobulin was administered weekly at a dosage of 2 mg/kg intravenously until discharge, antibody titers to HHV-6 increase only twofold after administration of 2 g/kg of gammaglobulin for Kawasaki disease (unpublished observation). Antibody titers for HHV-7 increased in 13 patients without an apparent association with HHV-7 infection. Although this discrepancy can not be easily explained, one possible explanation is that HHV-7 might reactivate mainly in cells other than blood cells and cellular tropism might be different from that of HHV-6 after SCT. Although HHV-7 was shown to be present in CD4 + T cells, 3 cellular tropism needs to be fully clarified for a better understanding of HHV-7 behavior. In this study, we recognized three patients who had some HHV-6-associated clinical findings after allo-sct. Interstitial pneumonitis developed in two patients. In one of them, the onset, accompanied by skin rash similar to that observed at exanthem subitum, was very early after transplantation and before engraftment. Thus, when early onset of skin rash with a elevation of body temperature is observed, HHV-6 infection should be considered. In patient UPN 115, since persistent low-grade fever and thrombocytopenia were associated with HHV-6 infection and these symptoms were relieved with the disappearance of HHV-6 DNA, it is suggested that this clinical episode was caused by HHV-6 infection. In summary, HHV-6 and -7 DNA was frequently detected in allo- and auto-sct recipients. In some allo-sct recipients who showed HHV-6 reactivation at a high copy number, clinical symptoms associated with HHV-6 were documented. Thus, diseases associated with HHV-6 infection were more frequent than had been thought and semiquantitative PCR analysis, together with serologic tests, has a clinical value for the diagnosis of HHV-6 infection. However, the relationship between HHV-7 reactivation and clinical episodes was not clear. A more detailed study will help our further understanding of pathogenic roles of HHV-7, not only in immunocompromised hosts, but also in the healthy population. Acknowledgements This study was supported in part by a Grant-in Aid from the Ministry of Education, Science and Culture of Japan. We thank T Aono of Toyobo Chemical Co. for the synthesis of DNA primers and probes. References 1 Salahuddin SZ, Ablashi DV, Markham PD et al. Isolation of a new virus, HBLV in patients with lymphoproliferative disorders. Science 1986; 234: Frenkel N, Schirmer EC, Wyatt LS et al. Isolation of a new herpesvirus from CD4 + T cells. Proc Natl Acad Sci USA 199; 87: Yamanishi K, Okuno T, Shiraki K et al. Identification of human herpesvirus-6 as a causal agent for exanthem subitum. Lancet 1988; 1: Tanaka K, Kondo T, Torigoe S et al. Human herpesvirus-7: another causal agent for roseola (exanthem subitum). J Pediatr 1994; 125: Wilborn F, Brinkmann V, Schmidt CA et al. Herpesvirus type 6 in patients undergoing bone marrow transplantation: serologic features and detection by polymerase chain reaction. Blood 1994; 83: Kondo K, Kondo T, Okuno T et al. Latent human herpesvirus 6 infection of human monocytes/macrophages. J Gen Virol 1991; 72: Di Luca D, Dolcetti R, Mirandola P et al. Human herpesvirus 6: a survey of presence and variant distribution in normal peripheral lymphocytes and lymphoproliferative disorders. J Infect Dis 1994; 17: Challoner PB, Smith KT, Parker JD et al. Plaque-associated expression of human herpesvirus 6 in multiple sclerosis. Proc Natl Acad Sci USA 1995; 92: Soldan SS, Berti R, Salem N et al. Association of human herpes virus 6 (HHV-6) with multiple sclerosis: increased IgM response to HHV-6 early antigen and detection of serum HHV-6 DNA. Nature Med 1997; 3: Tajiri H, Nose O, Baba K, Okada S. Human herpesvirus-6 infection with liver injury in neonatal hepatitis. Lancet 199; 335: Hall CB, Long CE, Schnabel KC et al. Human herpesvirus-6 infection in children. A prospective study of complications and reactivation. New Engl J Med 1994; 331: Drobyski WR, Knox KK, Majewski D, Carrigan DR. Brief report: fetal encephalitis due to variant herpesvirus-6 infection in a bone marrow transplant recipient. New Engl J Med 1994; 33:

6 17 13 Ishiguro N, Yamada S, Takahashi T et al. Meningo-encephalitis associated with HHV-6 related exanthem subitum. Acta Paediatr Scand 199; 79: Yanagihara K, Tanaka-Taya K, Itagaki K et al. Human herpesvirus 6 meningoencephalitis with sequelae. Pediatr Infect Dis J 1995; 14: Singh N, Carrigan DR, Gayowski T et al. Variant B human herpesvirus-6-associated febrile dermatosis with thrombocytopenia and encephalopathy in a liver transplantation recipient. Transplantation 1995; : Drobyski WR, Dunne WM, Burd EM et al. Human herpesvirus-6 (HHV-6) infection in allogenic bone marrow transplant recipients: evidence of a marrow suppressive role for HHV-6 in vivo. J Infect Dis 1993; 167: Knox KK, Carrigan DR. Chronic myelosuppression associated with persistent bone marrow infection due to human herpesvirus 6 in bone marrow transplant recipient. Clin Infect Dis 1996; 22: Carrigan DR, Drobyski WR, Russler SK et al. Interstitial pneumonitis associated with herpesvirus-6 infection after marrow transplantation. Lancet 1991; 338: Asano Y, Yoshikawa T, Suga S et al. Reactivation of herpesvirus type 6 in children receiving bone marrow transplantations for leukemia. New Engl J Med 1991; 324: Drobyski WR, Eberle M, Majewski D, Baxter-Lowe LA. Prevalence of human herpesvirus 6 variant A and B infections in bone marrow transplant recipients as determined by polymerase chain reaction and sequence-specific oligonucleotide probe hybridization. J Clin Microbiol 1993; 31: Yoshikawa T, Suga S, Asano Y et al. Human herpesvirus- 6 infection in bone marrow transplantation. Blood 1991; 78: Kadakia MP, Rybka WB, Stewart JA et al. Human herpesvirus 6: infection and disease following autologous and allogeneic bone marrow transplantation. Blood 1996; 87: Wang F-Z, Dahl H, Linde A et al. Lymphotropic herpesviruses in allogeneic bone marrow transplantation. Blood 1996; 88: Cone RW, Hackman RC, Huang M-LW et al. Human herpesvirus 6 in lung tissue from patients with pneumonitis after bone marrow transplantation. New Engl J Med 1993; 329: Singh N, Carrigan DR. Human herpesvirus-6 in transplantation: an emerging pathogen. Ann Intern Med 1996; 124: Matsuda Y, Hara J, Miyoshi H et al. Thrombotic microangiopathy associated with reactivation of human herpesvirus-6 following high-dose chemotherapy with autologous bone marrow transplantation in young children. Bone Marrow Transplant 1999; 24: Tanaka-Taya K, Kondo T, Mukai T et al. Seroepidemiological study of human herpesvirus-6 and -7 in children of different ages and detection of these two viruses in throat swabs by polymerase chain reaction. JMedVirol1996; 48: Yalcin S, Karpuzoglu T, Suleymanlar G et al. Human herpesvirus 6 and human herpesvirus 7 infections in renal transplant recipients and healthy adults in Turkey. Arch Virol 1994; 136: Okuno T, Sao H, Asada H et al. Analysis of a glycoprotein of human herpesvirus 6 (HHV-6) using monoclonal antibodies. Virology 199; 176: Lusso P, Secchiero P, Crowley RW et al. CD4 is a critical component of the receptor for human herpesvirus 7: interference with human immunodeficiency virus. Proc Natl Acad Sci USA 1994; 94: