Prolonged viremia and immune response to human herpesvirus 7 in an infant with liver dysfunction

Prolonged viremia and immune response to human herpesvirus 7 in an infant with liver dysfunction

Sadayoshi Torigoe

Human herpesvirus 7 (HHV-7) is the second newest virus in the human herpesvirus group. HHV-7 is closely related to human herpesvirus 6 (HHV-6) in both virologic and clinical aspects. Both viruses are now known to cause so-called exanthem subitum with or without complications.[1,2] The reported complications associated with HHV-6 infection are hepatitis,[3] febrile convulsion,[4] encephalitis,[5] acute hemiplegia,[6] invagination,[7] hemophagocytic syndrome,[8] and idiopathic thrombocytopenic purpura.[9] There are two cases of acute hemiplegia as a complication of exanthem subitum caused by HHV-7.[10] In only one case report was HHV-7 infection the probable cause of hepatitis.[11]

This report describes an infant who had exanthem subitum complicated by liver dysfunction. Because a prolonged viremic phase was observed, the cellular immunities to HHV-6 and HHV-7 were examined.

CASE REPORT

A previously healthy 11-month-old girl visited the outpatient clinic at Shingu Municipal Hospital. Although her general condition was good, she had experienced fever of up to 39[degrees]C for 5 days. Acetaminophen was given to her up to twice a day while she was febrile. When she visited the clinic, her temperature was 38.2[degrees]C. She had mild diarrhea and otitis media. A salmon-pink colored macular rash was scattered mainly on the lower part of the trunk. There was no hepatosplenomegaly. Her throat was inflamed, and several lymph nodes 5 mm in diameter were palpable on the neck and inguinal area. The next day, her fever subsided and she looked fine. The macular skin rash had increased on the lower part of the trunk but disappeared in 3 days. The clinical diagnosis was exanthem subitum complicated by liver dysfunction. The liver dysfunction normalized within 1 month after the onset of the disease. She had been healthy since she became afebrile.

Laboratory findings on day 5 of the illness revealed a white blood cell count of 5800/[mu]L, with 20% neutrophils, 76% lymphocytes, 2% monocytes, and 2% atypical lymphocytes; a platelet count 219 x 10[sup.3]/[mu]L; and liver dysfunction (aspartate aminotransferase, 600 IU/L [normal, 11 to 43 IU/L] and alanine aminotransferase, 618 IU/L [normal, 5 to 33 IU/L]). Hyperbilirubinemia was not observed. The data on day 6 of the illness showed a white blood cell count of 6300/[mu]L, with 16% neutrophils, 74% lymphocytes, 3% monocytes, 1% eosinophils, and 6% atypical lymphocytes; a platelet count 215 x 10[sup.3]/[mu]L; aspartate aminotransferase 251 IU/L; and alanine aminotransferase 395 IU/L. Gammaglobulin levels were normal (immunoglobulin G, 825 mg/dL; immunoglobulin A, 30 mg/dL; immunoglobulin M, 258 mg/dl).

The HHV-7 antibody titers, determined by immunofluorescence assay,[2] rose from [is less than]1:10 on day 5 of the illness to 1:160 on day 15. The HHV-6 antibody titers, however, remained negative. There was no serologic evidence of recent infection with Epstein-Barr virus; cytomegalovirus; or hepatitis A, B, or C. She was HIV-negative.

Isolation of HHV-7

Serial virus isolations were attempted from peripheral blood mononuclear cells (PBMCs) and saliva. PBMCs were separated as described previously.(2) The saliva sample was filtrated with Millex 13 mm HV (0.45 [mu]m filter) (Millipore Corp, Bedford, MA). The samples were cocultured with stimulated umbilical cord blood mononuclear cells (CBMCs) in RPMI 1640 medium with 10% heat-inactivated fetal bovine serum supplement with 2 U/mL interleukin-2 (Life Technologies, Gaithersburg, MD) at 37[degrees]C. The ballooning of the culturing cells was observed 3 weeks after the culture was inoculated. HHV-7 was identified by means of immunofluorescence assay with monoclonal antibodies against HHV-6 and HHV-7.[2] HHV-7 was isolated from PBMCs obtained on days 6 and 35 of the illness and from saliva obtained on days 35, 57, and 118 of the illness. However, HHV-7 was not isolated from PBMCs obtained on day 68 or from saliva obtained on day 16.

T-cell Immune Response

The immunologic response was evaluated by lymphocyte proliferation and the expression of interleukin-2 receptor (IL-2R) in response to HHV-6, HHV-7, and control antigen.[12-16] PBMCs were separated as described previously[2] and suspended in RPMI 1640 medium with 20% autologous plasma at a concentration of 1 x 10[sup.6]/mL. PBMCs were cultured with viral antigens for 6 days, then the uptake of 5-bromo-2′-deoxyuridine (BrdU) and the expression of IL-2R on T-cells were measured. HHV-6, HHV-7, and control antigens were prepared as described by Yakushijin et al.[12] The virus strains used were HHV-7 isolated from the patient and HST strain of HHV-6. Incorporation of BrdU was measured with the Cell Proliferation ELISA, BrdU kit (catalog no. 1 647 229, Boehringer Mannheim, Mannheim, Germany) as described previously.[16] T-cell immune response was examined on day 68 of the illness. The uptakes of BrdU with HHV-7, HHV-6, and control antigens were 0.714, 0.510, and 0.302, respectively, by enzyme-linked immunosorbent assay. The ratios of the uptakes with HHV-7 antigen to control and HHV-6 antigen to control were 2.36 and 1.69, respectively. The ratio of the uptake with HHV-6 antigen to control, measuring four CBMCs, was 1.25 [+ or -] 0.17 (mean [+ or -] SD). When two children who were seropositive and seronegative, respectively, to HHV-7 were evaluated, the ratios of the uptakes with HHV-7 antigen to control were 1.55 and 0.90, respectively. The results indicated that lymphocytes of the patient proliferated to both HHV-7 and HHV-6 antigens. However, the lymphocyte proliferation response was more obvious with HHV-7 antigen than with HHV-6 antigen.

The expression of IL-2R on lymphocytes cultured with viral antigens was examined as described previously[14,15] (Table). The percentages of IL-2R[sup.+] cells in CD4[sup.+] and CD8[sup.+] cells were increased in response to HHV-7 antigen, but there was less change in response to HHV-6 antigen. The increase of the percentages of IL-2R[sup.+] cells in CD45RO[sup.+] cells in response to HHV-6 and HHV-7 antigens suggested that T-cells were activated by both antigens. The ratios of the percentage with HHV-6 or HHV-7 antigen to control were from 1.05 to 3.11. On the other hand, the mean ratios of the percentage with HHV-6 antigen to control calculated from four CBMCs were from 0.83 to 1.4 (data not shown).

DISCUSSION

The clinical features of HHV-7 infection are still not well understood. The previous reports indicate that HHV-7 clearly caused exanthem subitum.[2,17] The infection could be complicated by central nervous system manifestations and hepatitis; however, only two cases of central nervous system manifestations[10] and a case of hepatitis[11] have been reported. This case provided much information on clinical features of HHV-7 infection. The patient had 5 days of fever, a longer period than usual[11] Thus, the skin rash appeared 1 day before the fever subsided. The liver dysfunction was dissolved rapidly, although the maximum value of alanine aminotransferase was 618 IU/L on day 5 of the illness. During the course of the illness, she had mild leukocytopenia and thrombocytopenia, and atypical lymphocytes were observed.

HHV-7 was isolated from PBMC and saliva samples. Notably, HHV-7 was isolated from PBMCs not only on day 6 but also on day 35 of the illness. In HHV-6 infection, it is difficult to isolate a virus from PBMCs after the skin rash has disappeared. It is not clear whether HHV-7 was persistently or intermittently present in PBMCs of this patient. However, we believe that the viremic phase continued for [is greater than] 35 days and had been eliminated by 68 days after the onset of the disease. Although the serum antibody against HHV-7 was already produced on day 15 of the illness, the serum antibody was not sufficient to eliminate the viremia. It is necessary to determine whether the prolonged viremia is a rare or common phenomenon in HHV-7 infection. On the other hand, HHV-7 was not isolated from saliva on day 16 of the illness, but was isolated from saliva on days 35, 57, and 118. The virus is usually not isolated from saliva at an acute phase of HHV-7 infection. Thus, the saliva gland is thought to be a site of reactivation of HHV-7. The result of virus isolation from saliva samples means that the reactivation of HHV-7 into saliva had not started on day 16 of the disease, but the reactivation had started on day 35 of the disease.

Because the viremic phase continued longer than usually thought, the immunologic status of the patient was examined. The antibody response to HHV-7 and immunoglobulin levels was normal. T-cell response to the isolated HHV-7 was also normal at 2 months after the onset of the disease. These results did not show any immunodeficiency in the patient. Interestingly, a weak T-cell response to HHV-6 was observed in the patient, although she had not yet had HHV-6 infection. This may mean that there is a cross-T-cell immune response between HHV-7 and HHV-6 in vivo. The cut-off ratio for HHV-6 lymphocyte proliferation response was 1.59 (the mean ratio of CBMCs + 2 SD). The lymphocyte proliferation to HHV-7 was usually weaker than to HHV-6. In a child who had antibodies to both HHV-7 and HHV-6, the ratios of the uptake with HHV-7 antigen to control and of HHV-6 antigen to control were 1.55 and 3.14, respectively. These ratios of the patient with HHV-7 and HHV-6 antigens were 2.36 and 1.69, respectively, indicating lymphocyte proliferation to both HHV-7 and HHV-6 antigens. However, the lymphocytes proliferated more with HHV-7 antigen than with HHV-6 antigen. It was difficult to decide whether there was an increase of IL-2R expression after HHV-6 antigen stimulation. Some ratios of percentage IL-2R-positive cells after HHV-6 or HHV-7 antigen stimulation to control were [is less than]1.0 when CBMCs or seronegative PBMCs were used. Therefore, we considered that with all the ratios being [is greater than]1.0, this seemed to indicate that there was a significant increase of IL-2R. According to the results of lymphocyte proliferation and IL-2R expression, we believe that there was a cross-T-cell immune response in this patient. An in vitro study indicated a possibility of a cross-reactivity among HHV-6 and HHV-7 in the T-cell immune response.(18) Yasukawa et al described that the CD4[sup.+] T-cell clones generated from PBMCs and stimulated with HHV-7 proliferated in response to both HHV-6 and HHV-7 antigen stimulation, indicating that there were T-cell epitopes common to HHV-6 and HHV-7. If a cross-T-cell reaction between HHV-6 and HHV-7 occurs in vivo, it might affect the clinical manifestations of HHV-6 and HHV-7 infections. For example, we know that HHV-6 infection tends to occur earlier than HHV-7 infection, but we have little knowledge about the clinical features of HHV-7 infection. One possibility is that HHV-7 infection following HHV-6 infection is usually a subclinical infection. The reason may be that the T-cells produced by the first HHV-6 infection inhibit the overt clinical manifestations of HHV-7 infection because of a cross-T-cell immune response. We have observed that in children who had exanthem subitum with HHV-7 infection, only 33.3% had a history of exanthem subitum. However, in children who did not have exanthem subitum with HHV-7 infection, 90% had a history of exanthem subitum.[17] This data may indicate that there is a cross-immunity that protects the second exanthem subitum. Additional study is necessary to verify this interesting phenomenon.

REFERENCES

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[10.] Torigoe S, Koide W, Yamada M, Miyashiro E, Tanaka-Taya K, Yamanishi K. Human herpesvirus 7 infection associated with central nervous system manifestations. J Pediatr. 1996;129:301-305

[11.] Hashida T, Komura E, Yoshida M, et al. Hepatitis in association with human herpesvirus-7 infection. Pediatrics. 1995,96:783,-785

[12.] Yakushijin Y, Yasukawa M, Kobayashi Y. T-cell immune response to human herpesvirus-6 in healthy adults. Microbiol Immunol. 1991;35: 655-660

[13.] Porstmann T, Ternynck T, Avrameas S. Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response. J Immunol Methods. 1985;82:169-179

[14.] Ito M, Nakano T, Kamiya T, et al. Activation of lymphocytes by varicella-zoster virus (VZV): expression of interleukin-2 receptors on lymphocytes cultured with VZV antigen. J Infect Dis. 1992;165: 158-161

[15.] Nakano T, Ito M, Mizuno T, et al. Increase of interleukin 2 receptor and CD45RO antigen on lymphocytes cultured with human cytomegalovirus. Cell Immunol. 1993;147:73-80

[16.] Ito M, Watanabe M, Kamiya H, Sakurai M. Inhibition of natural killer (NK) cell activity against varicella-zoster virus (VZV)-infected fibroblasts and lymphocyte activation in response to VZV antigen by nitric oxide-releasing agents. Clin Exp Immunol. 1996;106:40-44

[17.] Torigoe S, Kumamoto T, Koide W, Taya K, Yamanishi K. Clinical manifestations associated with human herpesvirus 7 infection. Arch Dis Child. 1995;72:518-519

[18.] Yasukawa M, Yakushijin Y, Furukawa M, Fujita S. Specificity analysis of human CD4′ T-cell clones directed against human herpesvirus 6 (HHV-6), HHV-7, and human cytomegalovirus. J Virol. 1993; 67:6259-6264

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