吉川 哲史

We present a case of pneumonia with marked pleural effusion caused by Aspergillus infection in a 2-year-old Japanese girl with Down's syndrome. The patient was previously diagnosed with acute myeloid leukemia, developing the pneumonia during induction treatment. Although no pathogens could be isolated from any clinical specimens, 135,000 copies/mL of Aspergillus DNA were detected in the pleural fluid using real time polymerase chain reaction. The copy numbers of DNA decreased rapidly after appropriate antifungal treatment. © 2006 Lippincott Williams &amp Wilkins.

To determine the cell populations in peripheral blood that are infected latently with human herpesvirus 7 (HHV-7), the real-time polymerase chain reaction (PCR) was used to determine the quantities of viral DNA in adherent and non-adherent cells from 71 healthy volunteers. Real-time PCR, which detected the U31 gene of HHV-7, was developed to measure viral load. The majority of non-adherent cells (14/16 87.5%) contained HHV-7 DNA, while most of the adherent cells did not (1/16 6.3%). HHV-7 viral load in non-adherent cells was significantly higher than that in adherent cells (P&lt 0.0001). Then, HHV-7 DNA load was compared between the CD4-positive and -negative cell fractions derived from the non-adherent cells of 26 healthy adults. As in the previous experiment, only 2 (7.7%) of the 26 adherent cell specimens contained small amounts of HHV-7 DNA (27.7 copies/1 × 106 cells and 208.7 copies/1 × 106 cells). In contrast, 88.5% of CD4+ T cell samples (23/26 specimens) were positive for HHV-7 DNA, ranging from 0.4 to 3,542.8 copies/1 × 106 cells. Viral DNA was detected in only 3 (11.5%) of the 26 CD4- T cell specimens, with 8.4, 63.5, and 74.1 copies/1 × 106 cells. HHV-7-positive DNA loads were significantly higher in the CD4+ T cells than those observed in the CD4- T cells (P=0.0005). The relationship between HHV-7 viral loads in non-adherent cells and those in saliva was investigated. Comparison of HHV-7 DNA load between blood CD4+ T cells and saliva revealed that the HHV-7 DNA load in saliva correlated with that present in CD4+ T cells (r=0.415 P=0.0174). © 2005 Wiley-Liss, Inc.

Idiosyncratic generalized skin disorders resembling serious drug hypersensitivities have reportedly occurred after occupational exposure to trichloroethylene. However, factors associated with the disorders remain unknown except for trichloroethylene exposure. This study aimed at clarifying whether infectious diseases contributed to the development of rash or hepatitis in patients with trichloroethylene-related generalized skin disorders. Fifty-nine patients consecutively hospitalized between March 2002 and December 2003 and 59 healthy exposed workers selected on an age-matched basis in the patients' factories were enrolled in the study. Information on possible risk factors for rash and hepatitis was collected with structured checklists. Antibody titers were measured for hepatitis A, B and C viruses, Mycoplasma pneumoniae, herpes simplex viruses 1 and 2, Epstein-Barr virus, cytomegalovirus, human herpesvirus 6, measles and rubella virus. Thirty-six cases (59%) showed exfoliative dermatitis, 17 (28%) erythema multiforme, 4 (7%) Stevens-Johnson syndrome, and 4 (7%) toxic epidermal necrolysis. Before the onset of rash, 16 (27%) cases had received medication prescribed for the preceding fever, a main first symptom of the disorders. Marked increases in anti-human herpesvirus 6 IgG titer (&ge;256), which indicated viral reactivation, were noted in 14 (25%) patients, while no abnormal increase was detected in the controls (p<0.001). Anti-measles IgM titer was positive in 2 (7%) cases but not in the controls (p=0.49). The involvement of other known risk factors of rash or hepatitis was ruled out. These results suggest that part of trichloroethylene-related generalized cutaneous disorders occurring in China and drug-induced hypersensitivity syndrome overlap in terms of human herpesvirus 6 reactivation.

Background. Whether an association exists between infection with β-herpesviruses and connective tissue diseases remains unclear, as are the mechanisms for the regulation of these infections in the salivary glands. Methods. Human herpesvirus (HHV)-7 was isolated and viral DNA was quantified by real-time polymerase chain reaction (PCR) in serially collected saliva samples, to determine whether viral load correlated with infectivity. Then, to examine the role played by β-herpesviruses in connective tissue diseases, cytomegalovirus, HHV-6, and HHV-7 DNA loads were examined by real-time PCR in serially collected saliva samples from 21 patients with connective tissue diseases. Results. Although subjects with frequent HHV-7 shedding were more likely to have a high viral load than were other subjects, high viral loads were detected in saliva samples from a portion of the subjects with low viral shedding rates. No significant difference between the quantity of HHV-7 DNA in saliva samples from which active virus was isolated and that amplified from samples without detectable virus was observed. Patients with adult-onset Still disease consistently had high HHV-7 DNA loads, in contrast to patients with other connective tissue diseases (P = .0003) and healthy adults (P = .0224). The mean HHV-6 (P = .012) and HHV-7 (P &lt .0001) DNA loads in patients with connective tissue diseases were lower than those in healthy adults. Conclusion. These data suggest that a number of host factors in patients with adult-onset Still disease may function to accelerate HHV-7 replication in the salivary glands. © 2005 by the Infectious Diseases Society of America. All rights reserved.

This study compares herpes simplex virus (HSV) type-specific loop-mediated isothermal amplification (LAMP) with virus isolation and real-time PCR. Genital tract specimens were obtained from 25 patients with genital lesions two swab samples were collected from the vulva and cervix of each patient, for a total of 50 specimens. After culturing, 10 of 50 (20%) samples were positive for HSV-1 and 12 of 50 (24%) samples were positive for HSV-2. None of the patients excreted both HSV-1 and HSV-2 virus. An original HSV type-specific LAMP assay (30 min reaction) was compared with virus isolation and HSV type-specific real-time PCR. Viral DNA was detected by LAMP in 9 of 10 HSV-1 isolated samples and 11 of 12 HSV-2 isolated samples. No viral DNA was detected in samples without virus isolation. Thus, if virus isolation was used as the standard method, the LAMP protocol was highly sensitive and specific. In comparing LAMP to real-time PCR, viral DNA was detected by the LAMP method in 9 of 12 HSV-1 DNA positive samples and 11 of 18 HSV-2 DNA positive samples. If real-time PCR was used as the standard method, then, sensitivity of the LAMP method (in particular, for HSV-2) was low. Taking this into consideration, the LAMP reaction was extended to 60 min. This led to an increase in sensitivity, resulting in an additional one and three samplestesting positive for HSV-1 LAMP and HSV-2 LAMP, respectively, compared to the original LAMP protocol. Therefore, the sensitivity of the LAMP method increased to about 80%. © 2005 Wiley-Liss, Inc.

Primers for herpes simplex virus type 1 (HSV 1)-specific loop-mediated isothermal amplification (LAMP) method amplified HSV-1 DNA, while HSV-2-specific primers amplified only HSV-2 DNA no LAMP products were produced by reactions performed with other viral DNAs. The sensitivities of the HSV-1- and HSV-2-specific LAMP methods, determined by agarose gel electrophoresis, reached 500 and 1,000 copies/tube, respectively. The turbidity assay, however, determined the sensitivity of the HSV-1- and HSV-2-specific LAMP methods to be 1,000 and 10,000 copies/tube, respectively. After initial validation studies, 18 swab samples (in sterilized water) collected from patients with either gingivostomatitis or vesicular skin eruptions were examined. HSV-1 LAMP products were detected by agarose gel electrophoresis in the 10 samples that also demonstrated viral DNA detection by real-time PCR. Nine of these 10 samples exhibited HSV-1 LAMP products by turbidity assay. Furthermore, both the agarose gel electrophoresis and the turbidity assay directly detected HSV-1 LAMP products in 9 of the 10 swab samples collected in sterilized water. Next, we examined the reliability of HSV type-specific LAMP for the detection of viral DNA in clinical specimens (culture medium) collected from genital lesions. HSV-2 was isolated from all of the samples and visualized by either agarose gel electrophoresis or turbidity assay.

The aim of this study is to determine whether human herpesvirus 6 (HHV-6) and HHV-7 might play an important role in causing interstitial pneumonia in patients who have not undergone transplantation. HHV-6 and HHV-7 DNAs were quantitated by real-time polymerase chain reaction (PCR) in paraffin embedded lung tissues collected from 24 patients having the disease. Control tissues (without fibrosis) were also collected from 19 of the 24 patients. Statistical analysis was carried out by the Wilcoxon signed rank test or the Mann-Whitney U-test. HHV-6 DNA was detected in 3 (12.5%) of the 24 target tissues and 3 (15.8%) of the 19 control tissues, respectively. In contrast, HHV-7 DNA was detected in 19 (79.2%) of the 24 target tissues and 11 (57.9%) of the 19 control tissues. Neither HHV-6 DNA load (P = 0.6395) nor HHV-7 DNA load (P = 0.5966) in target tissues differed between males and females. Neither HHV-6 DNA load (P = 0.9589) nor HHV-7 DNA load (P = 0.7419) in target tissues differed between cases with and without underlying collagen disease. While HHV-6 DNA load did not differ between the target and control tissues (P &gt 0.9999), the HHV-7 DNA load was significantly higher in the target tissue than in the control tissue (P = 0.0298). This study suggests that HHV-7 may play an important role in causing interstitial pneumonia in patients who are not transplant recipients. © 2005 Wiley-Liss, Inc.

Objectives/Hypothesis: Perilymph and peripheral blood mononuclear cells (PBMCs) from patients with bilateral severe sensorineural hearing loss (SNHL) were evaluated for the presence of DNA from cytomegalovirus (CMV), herpes simplex virus (HSV), and human herpesvirus (HHV)6. Study Design: A prospective clinical study. Methods: The subjects were 14 patients who underwent cochlear implantation and 1 patient who underwent gentamicin injection in the inner ear. We attempted to detect viral DNA from perilymph and PBMCs by real-time polymerase chain reaction (rtPCR). Results: CMV DNA was detected in two perilymph specimens obtained from patients who were diagnosed as congenitally symptomatic CMV infection, although no CMV DNA was detected in PB-MCs. Neither HSV DNA nor HHV6 DNA was detected in any other perilymph specimens. CMV DNA was detected in three PBMC samples, HSV DNA was detected in two samples, and HHV6 DNA was detected in six samples. Conclusion: CMV may persistently infect the inner ear of patients with congenital CMV infection, and rtPCR analysis may prove to be a valuable tool for investigating the etiology of SNHL.

The reliability of varicella-zoster virus (VZV) loop-mediated isothermal amplification (LAMP) was evaluated for rapid diagnosis of viral infection. VZV-specific primers only amplified VZV DNA no LAMP products were observed in reactions performed with other viral DNA templates. The specificity of this method was confirmed by two independent determinations, agarose gel electrophoresis and a turbidity assay. The sensitivity of VZV LAMP, determined by agarose gel electrophoresis, were 500 copies/tube. Detection using the turbidity assay, however, gave a sensitivity of 1,000 copies/tube. After these initial validation studies, reliability of VZV LAMP was evaluated for the detection of viral DNA in clinical specimens. Thirty-two swab samples collected from patients with vesicular skin eruptions were tested for VZV DNA. VZV was confirmed in sample numbers 10-32 by VZV real-time PCR, a previously established technique. VZV LAMP products were detected using turbidity from samples 13 to 32 (sensitivity 87.0%, specificity 100%, positive predictive value 100%, negative predictive value 75%). Although low levels of VZV DNA could be detected in the three samples exhibiting divergent results (samples numbers 10-12), no VZV LAMP product was detected in these samples, indicating a higher detection limit for this assay. Requirement of a DNA extraction step in the VZV LAMP method was examined in next experiment. The turbidity assay detected a VZV LAMP product in all of the 20 positive swab samples (samples numbers 13-32), regardless of DNA extraction. © 2004 Wiley-Liss, Inc.

A case of neonatal human herpesvirus 6 (HHV-6) B infection is presented. Although HHV-6 B was isolated from peripheral blood at the onset of the illness, a significant increase in viral antibody titers was not observed. The patient had a slight fever with generalized maculopapular skin rash and an increased number of atypical lymphocytes, which is quite different from the typical clinical features of exanthem subitum. © 2004 Wiley-Liss, Inc.

We report a case of hemiconvulsion-hemiplegia (HH) syndrome. An 18-month-old female infant had a hemiconvulsion followed by left hemiplegia. Magnetic resonance imaging immediately after the onset of hemiplegia showed high intensity in the right hemisphere in diffusion-weighted images (DWI), while T1- and T2-weighted images were normal. Single photon emission computed tomography showed hypoperfusion of the right hemisphere in the acute phase. Virological analyses proved primary human herpesvirus 7 (HHV-7) infection. DWI are useful for the early evaluation of HH syndrome. Vascular disorders due to HHV-7 infection may have been related to the development of HH syndrome in this patient. © 2004 Elsevier B.V. All rights reserved.

The reliability of loop-mediated isothermal amplification (LAMP), initially developed for the detection of human herpesvirus 7 (HHV-7), was evaluated in this study. Although a LAMP product was detected in HHV-7 DNA, neither HHV-6 nor human cytomegalovirus DNA produced a product. When agarose gel electrophoresis was used for the detection of LAMP products, the sensitivity of a 30-min HHV-7 LAMP reaction reached 250 copies/tube. The use of turbidity for the detection of the LAMP products gave a sensitivity of 500 and 250 copies/tube for 30- and 60-min reactions, respectively. Following these initial validation studies, clinical samples collected from two patients with primary HHV-7 infections were examined by HHV-7 LAMP. By use of agarose gel electrophoresis, HHV-7 LAMP products could be detected in acute-phase plasma samples but no LAMP product was detectable in convalescent-phase plasma samples from either patient. Since a turbidity assay is less sensitive than agarose gel electrophoresis, no HHV-7 LAMP product could be detected in plasma samples after a 30-min LAMP reaction. After a 60-min LAMP reaction, HHV-7 LAMP product could be detected in acute-phase plasma samples.

Background: It is not clear whether quantitative analysis of viral DNA in ocular specimens is correlated with disease activities of acute retinal necrosis (ARN). Objectives: To monitor viral load in ocular specimens collected from patients with ARN by real-time polymerase chain reaction (PCR). Study design: Ocular samples (aqueous humor and vitreous) were serially collected from three patients with ARN. Viral load in those samples was evaluated by real-time PCR. Result and conclusion: In case 1, large amounts of varicella zoster virus (VZV) DNA (4.8×106 to 5.5×106 copies/ml) were detected in aqueous humor during the first 2 weeks after admission. The viral load in vitreous was higher than that in aqueous humor at the time of vitrectomy. As ophthamoscopic findings and visual acuity improved through acyclovir (ACV) treatment, the viral load in aqueous humor decreased dramatically. In case 2, the patient was treated with intravenous ACV at first, but clinical features did not improve. The herpes simplex virus (HSV)-2 viral load in aqueous humor remained stable (2.3×103 to 2.8×103 copies/ml) during the first 3 weeks after admission. The amount of HSV-2 DNA in vitreous was again higher than that in aqueous humor. Although neither clinical features nor viral load had changed by ACV, intra-ocular ganciclovir (GCV) injection improved clinical features, and decreased viral load to undetectable levels. In case 3, the patient developed ARN within 1 month after the onset of varicella and demonstrated only mild clinical symptoms. She was treated with ACV administration alone and recovered quickly. In contrast to case 1, the copy number of VZV DNA at the time of admission was low (9×102 copies/ml), and decreased quickly in response to the treatment. Correlation between viral load in ocular specimens and clinical course of the disease was demonstrated in these patients. © 2003 Elsevier B.V. All rights reserved.

A novel nucleic acid amplification method, termed loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity, efficiency, and rapidity under isothermal conditions, may be a valuable tool for the rapid detection of infectious agents. LAMP was developed for human herpesvirus 6 (HHV-6), and its reliability was evaluated in this study. Although LAMP products were detected in HHV-6 B and HHV-6 A DNA, they were not detected in HHV-7 and human cytomegalovirus DNA. The sensitivity of the original HHV-6 LAMP protocol was 50 copies/tube. In order to increase the method's sensitivity, HHV-6 LAMP was modified by increasing the primer concentration. As a result of the modification, sensitivity increased to 25 copies/tube. After these initial validation studies, 13 patients with fever were tested for HHV-6 by viral isolation, serological analysis, and HHV-6 LAMP. In three of the eight patients with primary HHV-6 infection, HHV-6 DNA was detected in whole blood by the original HHV-6 LAMP protocol in not only the acute phase but also the convalescent phase. HHV-6 DNA was detected by modified HHV-6 LAMP in all eight plasma samples collected in the acute phase however, no HHV-6 DNA was detected in plasma samples collected in the convalescent phase. Although HHV-6 DNA was detected in both the acute and convalescent phases of whole-blood samples in patients with past HHV-6 infection, it was not detected in plasma samples that did not contain latent viral DNA. Thus, detection of HHV-6 DNA in plasma by using this modified HHV-6 LAMP protocol is appropriate for diagnosis of active HHV-6 infection.

Sequential analysis of human herpesvirus 7 (HHV-7) shedding in saliva obtained from healthy adults was performed for 6 months virus was isolated in 92 (34.1%) of the 270 saliva samples obtained. Frequency of HHV-7 shedding in serially obtained saliva samples varied among subjects. Associations between frequency of HHV-7 shedding and age, sex, and virus antibody titer were analyzed, and, although sex was not associated with frequency of shedding, young age (P = .031) and low antibody titer (P = .006) were correlated with frequent viral shedding (5 or 6 times/6 months).

In order to elucidate the pathogenesis of variant B human herpesvirus 6 (HHV-6) infection in skin tissues, an A431 cell line was inoculated with variant B HHV-6. HHV-6 causes abortive infection in the A431 cells, because neither late antigen (OHV-3 antigen) nor progeny virus is produced. Maximum levels of HHV-6 antigen (IEA/ex3 antigen)-positive cells (36.4%) were observed 48 hr after viral infection. Cocultivation of HHV-6-infected cord blood mononuclear cells with A431 cells was necessary for the establishment of a sufficient level of viral infection. Cell-to-cell contact between the infected cord blood mononuclear cells and A431 cells was crucial for increasing infection efficiency. To determine the biological effect of HHV-6 infection, flow cytometric analysis was carried out in HHV-6- and mock-infected A431 cells. Although no alteration was observed in VCAM-1 and ELAM-1 expression, that of HLA-ABC, HLA-DR, and ICAM-1 was upregulated after infection with HHV-6. © 2003 Wiley-Liss, Inc.

Objective: The purpose of the present study was to determine the degree of spread of varicella-zoster virus to the environment (VZV) from varicella patients who received oral acyclovir treatment. Methods: Over a period of 8 months, seven healthy children (two girls and five boys, 23-64 months of age) with varicella who visited Fujita Health University School of Medicine were treated with routine doses of oral acyclovir (ACV) for 5 days, commencing within 24 h after onset of the disease. Swab samples from the throats of patients and their family members as well as from air purifier filters in their houses were collected for 7 days as frequently as possible after starting treatment for the disease. The VZV DNA in the samples was identified by a sensitive polymerase chain reaction amplification assay. Results: The VZV DNA was detected in 33-100% of throat swab samples from varicella patients by day 7 of the disease, in 17-32% of throat swab samples from family members by day 4 and in 29% of filters from air purifiers by day 3. Conclusions: The results suggest a broad spread of VZV, probably by the airborne route, from the patients with varicella even after the administration of oral ACV.

A 9-year-old boy who received allogeneic stem cell transplantation began to vomit from day 10 after transplantation. In addition to vomiting, the patient had a fever (from day 26) and severe headache (from day 34). His cerebrospinal fluid (CSF) (day 41) demonstrated pleocytosis with an absence of leukemic cells. Although the patient's symptoms were resolved with further supportive care, abrupt onset of bilateral decreased vision occurred at day 54. He was diagnosed with bilateral optic neuritis, due to the presence of disc edema and redness. Concomitant with the occurrence of aseptic meningitis, the human herpesvirus 7 (HHV-7) antibody titer increased significantly in this patient. Although neither HHV-6 nor cytomegalovirus (CMV) DNA was detected in CSF collected at day 41, HHV-7 DNA was detected in the sample. Viral DNA was not detected in CSF collected at day 93. © 2003 Wiley-Liss, Inc.

Monitoring of active human herpesvirus 6 (HHV-6) infection is important for distinguishing between reactivation and latency of the virus. The reverse transcription polymerase chain reaction (RTPCR) may be a useful tool in order to distinguish active and latent HHV-6 infection. An RT-PCR assay detecting 4 different HHV-6 gene transcripts was established. Samples of peripheral blood mononuclear cells (PBMCs) were collected from patients with exanthem subitum and used to evaluate the reliability of the assay. After confirming the reliability of the assay, RT-PCR was used to determine whether HHV-6 reactivation occurs in children with hypercytokinemia. Three gene transcripts (U31, U39, and U94) were detected in 90-100% of the PBMC samples collected from febrile period of exanthem subitum patients, from which HHV-6 was isolated. The two gene transcripts encoding the late proteins U31 and U39, however, were not detected in samples collected during the convalescent period that contained no infectious virus. The putative latency associated gene transcript, U94, was detected in 2 (10%) of the 20 convalescent samples, and another immediate early gene transcript, U90, was also detected in 3 (15%) of the 20 convalescent samples. The frequency of HHV-6 reactivation in patients with hypercytokinemia, suggesting monocyte/macrophage activation, was studied. Only 9 of 17 patients diagnosed with Kawasaki disease and 1 patient diagnosed with juvenile rheumatoid arthritis were positive for HHV-6 DNA in their PBMCs samples. Neither the U31 gene nor the U94 gene transcript was detected in any of the 10 samples. An RT-PCR assay screening for both immediate early and late genes may be useful for monitoring active HHV-6 infection. No HHV-6 reactivation was found in patients with hypercytokinemia using the RT-PCR assay. © 2003 Wiley-Liss, Inc.

Although cytomegalovirus (CMV) has been detected in the inner ear fluid of patients who succumbed to the complications of symptomatic congenital CMV infection, it has not been detected in the inner ear fluid of living patients. In this study, real-time polymerase chain reaction (PCR) was used to measure CMV DNA in clinical samples (including perilymph) collected from five patients with deafness. In case 1, diagnosed as a symptomatic congenital CMV infection, 3 copies/μl of CMV DNA were detected in perilymph, although no viral DNA was detected in peripheral blood mononuclear cells (PBMCs) or urine samples. In case 4, a suspected asymptomatic congenital CMV infection, 36 copies/μg of CMV DNA were detected in PBMCs, but neither perilymph nor urine contained viral DNA. Likewise, in case 5, a case of deafness of unknown origin, 48 copies/μg of CMV DNA were detected in the PBMCs, but none in the perilymph or urine. CMV DNA was not detected in the samples obtained from the remaining two cases with deafness of unknown etiology. To our knowledge, this is the first report to detect CMV DNA in an inner ear sample obtained from a living human subject. © 2003 Wiley-Liss, Inc.

Human herpesvirus 6 (HHV-6) infection was studied in 82 bone marrow transplant (BMT) recipients (72 allogeneic, 10 autologous). All recipients and 30 donors were seropositive for HHV-6 antibody at the time of bone marrow transplantation. Thirty-one recipients (37.8%) had HHV-6 viremia 2-4 weeks after transplantation. The incidence of HHV-6 viremia was significantly higher among allogeneic BMT recipients than in autologous BMT recipients (P = .011). Therefore, the following analyses of allogeneic BMT recipients were carried out (n = 72). Geometric mean antibody titers (log10) were significantly higher in recipients without viremia than in those with viremia (1.84 ± 0.39 vs. 1.61 ± 0.42 P = .022). Logistic regression analysis demonstrated that leukemia or lymphoma is an independent risk factor (P = .031) for HHV-6 viremia. Rash occurring within 1 month after transplantation was observed in 17 (54.8%) of 31 recipients with HHV-6 viremia but in only 8 (19.5%) of 41 recipients without HHV-6 viremia (P = .001). © Oxford University Press 2002.

We previously reported the establishment of an HEp2 cell line which expresses the US3 protein kinase (PK) of herpes simplex virus type 2 (HSV-2) upon induction with IPTG. Here we report that expression, phosphorylation and ubiquitination of cytokeratin 17 (CK17) are enhanced in US3-expressing HEp2 cells. In vitro kinase and co-immunoprecipitation assays provided evidence that US3 PK directly phosphorylates CK17. Expression of US3 PK caused a significant decrease in filamentous staining of CK17, suggesting that phosphorylation of CK17 by US3 PK causes a disruption of intermediate filaments. Our observations suggest a role for US3 in the regulation of CKs and intermediate filaments in cells. Moreover, we found that infection of a keratinocyte-derived cell line, A431, with a US3-deficient virus, results in cytopathic effects that are morphologically distinct from those induced by wild-type and revertant viruses, suggesting that US3 PK may be important for interaction between HSV-2 and peripheral epithelial cells.

Fifty-three (96%) of 55 patients with coronary artery stenosis were positive for serum anti-HHV-6 IgG, and 50 (91%) of these patients had anti-HHV-7 IgG. The number of cases sero-positive for HHV-6 and -7 in the 54 age matched control volunteers was 52 (96%) and 53 (98%), respectively. No statistical difference in the seroprevalence of the viruses existed between the patients and the control group. The mean geometric titer (log10) of anti-HHV-6 IgG in both the patients and controls were the same (1.4) (P=0.845), whereas anti-HHV-7 titers were 1.4 and 1.5, respectively (P=0.161). Ten (18%) of the 55 patients had anti-HHV-6 IgM eight (15%) of the 54 control volunteers were also positive (P=0.636). Three (5%) of the 55 patients had anti-HHV-7 IgM, whereas 3 (6%) of the 54 control volunteers had detectable serum antibody (P=0.691). Forty-seven of the 55 patients were examined by follow-up angiographic evaluation to clarify the association between viral infection and restenosis following balloon angioplasty. Fifteen of these patients developed restenosis, as determined by angiography. The mean geometric titer (log10) of anti-HHV-6 IgG were 1.3 and 1.4 in patients with restenosis and those without restenosis, respectively (P=0.724). The mean geometric titer (log10) of anti-HHV-7 IgG in patients with restenosis was not significantly higher (1.5) than in patients without restenosis (1.3) (P=0.099). Three (20%) of the 15 patients affected by restenosis had anti-HHV-6 IgM five (16%) of the 32 control patients also had the antibody (P=0.965). One (7%) of the 15 patients with restenosis and 2 (6%) of the 32 patients without restenosis had anti-HHV-7 IgM (P= 0.558). The present study demonstrates that HHV-6 and -7 reactivation is not associated with the establishment of coronary artery stenosis and restenosis following balloon angioplasty. © 2002 Wiley-Liss, Inc.

To elucidate the roles of human herpesvirus (HHV)-6 and -7 in pregnant women, peripheral blood samples and genital tract secretions were collected serially from pregnant women, and both serological testing and polymerase chain reaction (PCR) were carried out to detect viral DNA in the secretions. HHV-6 or HHV-7 Immunoglobulin(Ig)M antibodies were not detected in 432 plasma samples collected from pregnant women and cord blood, but IgG antibodies against both viruses were detected in all plasma samples. Significant increases in HHV-6 and HHV-7 IgG antibodies were observed in two (1.6%) and three (2.4%) pregnant women respectively of a total of 123 cases. HHV-6 DNA was detected in the genital tract in three (3.7%) of 82 pregnant women at the first trimester, and in 10 (12.2%) of the same women in the third trimester. The detection rate in the third trimester was significantly higher than that in the first trimester (P=0.043). Although HHV-7 DNA was detected in the genital tract of two (2.7%) and seven (9.6%) pregnant women of a total of 73 during the first and third trimesters respectively, there was no statistical difference in the detection rate of the viral DNA between the trimesters. Because a significant increase in HHV-6 IgG antibodies was detected in only two pregnant women, it was not possible to carry out statistical analysis to determine the relationship between HHV-6 infection and associated clinical features. Although there was a significant increase in HHV-7 antibody titers in three pregnant women, a positive correlation between the virus infection and the clinical features was not demonstrated. There was no statistical association between virus shedding in the genital tract and the clinical features examined in this study. © 2002 Wiley-Liss, Inc.

In order to study the pathogenesis of HHV-6 infection in central nervous system disorders, U251 cell line was infected with freshly isolated variant B HHV-6. Although IEA/ex 3 antigen (immediate early protein) was detected in infected cell nuclei, neither the presence of OHV-3 antigen (late antigen) nor production of infectious virus was demonstrated. These results indicate that abortive infection was established in the cells. After viral infection, the viral genome copy in the infected cells gradually decreased and became stable after 6 cell passages. At that point, HHV-6 gene expression was restricted to only 2 immediate early genes (U90 and U94). However, 12-O-tetra-decanoyl (TPA) treatment induced transcription of other genes (U31 and U39) by the 10th cell passage, indicating HHV-6 reactivation. Moreover, production of two proinflamatory cytokines (IL-6 and IL-1β) was up-regulated by the presence of the HHV-6 genome and TPA-induced activation of the viral transcripts. © 2002 Wiley-Liss, Inc.

A female infant developed Guillain-Barré syndrome 20 days after having exanthem subitum confirmed serologically as human herpesvirus 6 infection. DNA of human herpesvirus 6 was detected in peripheral blood mononuclear cells collected on admission.

Patients with zoster are considered to be less contagious than varicella patients because their infection is localised. It is not known, however, when and for how long a spread of varicella-zoster virus (VZV) from a zoster patient begins and continues and the extent of virus spread from the patient. The polymerase chain reaction (PCR) was used to detect VZV DNA in samples obtained from the hands and throat of a patient with zoster and from her room environments including surfaces of the back of a chair, the door handle, the table and the air conditioner filter. VZV DNA was detected on the surfaces of the back of the seat and the table and in peripheral blood mononuclear cells (PBMCs) and serum on Day 4 of the illness. VZV DNAemia persisted for 4 days until Day 7 of the illness. It was also detected in samples collected from throat and the air conditioner filter on Days 6 and 7 of the illness respectively. All of the surfaces, that were examined in her home environment, were contaminated with VZV DNA by Day 7 of the illness. The present study showed rapid and wide spread of VZV DNA in the environment even from a patient with zoster. (C) 2001 Wiley-Liss, Inc.

Placental transfer of maternal human herpesvirus (HHV) 6 and HHV 7 antibodies to infants was examined simultaneously in 69 paired plasma samples by an indirect immunofluorescence assay. All the mothers had antibodies to both viruses. The mean HHV 6 and HHV 7 antibody titers of infants were significantly higher than those of the mothers. The mean ratio of cord blood antibody titer to the maternal titer for both viruses was 1.89, suggesting active transport by placenta.

We detected primary human herpesvirus 6 (HHV-6) infection in 5 infants who received living related liver transplantation from their HHV-6 seropositive mothers. Primary HHV-6 infection was confirmed by demonstrating the seroconversion of HHV-6 antibodies with an immunofluorescence assay, by the isolation of the virus, or both. Seroconversion of HHV-6 immunoglobulin G antibody was demonstrated in all 5 recipients. HHV-6 was isolated from 3 of the 5 recipients between 2 and 3 weeks after transplantation. Moreover, the virus genome was detected in plasma by polymerase chain reaction in 4 of the 5 recipients during the same period. Although the 5 recipients had pyrexia at the time of primary HHV-6 infection, none of the recipients had a skin rash after defervescence. Clinical symptoms disappeared without specific antiviral treatment in all but 1 of the recipients.

Cord blood transplantation, which has lower risk of graft-versus-host disease than bone marrow transplantation, might have higher risk of infections. A system to quantify four herpesviruses, CMV, human herpesvirus 6 (HHV6), EBV, varicella-zoster virus using the real-time PCR assay was established and applied for prospective viral load monitoring in three recipients undergoing cord blood transplantation. CMV and HHV6 were detected in peripheral blood from all three recipients, while EBV was detected in two. Varicella-zoster virus was not detected at all. At the peak of HHV6 or CMV, each patient showed virus-related symptoms. During the pre-transplant period, CMV DNA was detected in two recipients who later developed CMV-related diseases. These observations indicate that our system is not only useful for managing herpesviruses infections in transplant recipients, but also a powerful method for clarifying the relationships between the viral load and clinical symptoms.

The sequences of ~34 kb from the 3' end of the varicella-zoster virus (VZV) Oka vaccine strain and the previously sequenced Dumas strain were compared. Sequence differences were noted in the coding sequences of several VZV open reading frames (ORFs), including ORFs 48, 51, 52, 55, 56, 58, 59, 60, 62, 64, and 68. Tests based on differences in the ORF62 gene and in the ORF64 poly-A region successfully distinguished the Oka vaccine strain from its wild-type parent and from other Japanese and US clinical isolates. These changes remained stable after passage of the virus in humans.

Objective - To clarify clinical characteristics of children with febrile convulsions during primary human herpesvirus 6 (HHV-6) infection. Subjects and methods - The clinical characteristics of first febrile convulsion were compared between those with and without primary HHV-6 infection in 105 children. HHV-6 infection was verified by culture or acute/convalescent anti- HHV-6 antibody titres. Results - Primary infection with HHV-6 was seen in 21 of 105 patients with febrile convulsions (3 upper respiratory infection, 1 lower respiratory infection, and 17 exanthem subitum). 13 of 23 patients &lt 1 year, 19 of 79 patients with first febrile convulsion, and 2 of 15 with second convulsion were infected with HHV-6. The median age of patients with first febrile convulsion and HHV-6 was significantly lower than those without infection. The frequency of clustering seizures, long lasting seizures, partial seizures, and postictal paralysis was significantly higher among those with primary HHV-6 infection than among those without. The frequency of atypical seizures in 19 patients with first febrile convulsion associated with primary infection was significantly higher than in 60 patients without primary infection. The frequency in infants younger than 1 year of age was also significantly higher than that in 10 age matched infants without primary infection. Conclusions - These findings suggest that primary infection with HHV-6 is frequently associated with febrile convulsions in infants and young children and that it often results in the development of a more severe form of convulsions, such as partial seizures, prolonged seizures, and repeated seizures, and might be a risk factor for subsequent development of epilepsy.

Aim - To investigate whether human herpesvirus 6 (HHV-6) can cause latent infection of liver tissue. Methods - Peripheral blood and liver tissue were coRRected from 25 living related liver transplant recipients at the time of transplantation. An avidin-biotin complex peroxidase method was used to identify HHV-6 antigen in the liver tissue. A nested polymerase chain reaction (PCR) was used to detect HHV-6 DNA in the liver tissue and mononuclear cells. Variant of HHV-6 was determined by the presence of the Hind III site in a second PCR product. Results - Immunohistochemical analysis for HHV-6 antigen was negative in all the liver specimens. HHV-6 DNA was not detected in liver tissue. Virus DNA was detected in peripheral blood mononuclear cells in nine of 25 recipients. All nine HHV-6 identified in the mononuclear cells were variant B. Conclusions - HHV-6 variant B latently infects mononuclear cells but not liver tissue.

Objective. To elucidate persistence of human herpesvirus-6 (HHV-6) in the blood and excretion of the virus into several body fluids of patients with exanthem subitum (ES), and to examine serologic and virologic findings of the parents caring for the patients in the family setting. Materials and Methods. During a 15-month period, 20 infants from 20 families (11 boys and 9 girls mean age, 7.7 months range, 4-11 months) with primary HHV-6 infection and a typical clinical course of ES, and 15 parents from the 20 families (2 males and 13 females mean age, 28.2 years range, 21-34 years) were enrolled in the study and examined clinically and virologically. Primary infection with HHV-6 was confirmed by isolation of the virus from peripheral blood mononuclear cells (MNCs), and seroconversion or a significant increase in the antibody titers to HHV-6 by a neutralization test. Viral persistence or excretion was examined by amplifying the viral deoxyribonucleic acid (DNA) in serially collected peripheral blood MNCs, plasma, saliva, stool, and urine samples with a nested polymerase chain reaction method. Data on saliva from the parents were compared with those of 21 age-matched controls. Results. Twenty infants with virologically confirmed ES had HHV-6 DNA in MNCs persistently during and after the disease but in plasma only in the first 5 days of ES. The viral DNA was also detected persistently or intermittently in saliva and stool during and after the disease but rarely in urine. On the other hand, the 15 parents examined of the 20 infants had no HHV-6 viremia nor viral DNA in peripheral blood MNCs and plasma except 1, but half of them excreted viral DNA in saliva during and after ES. The frequency of excretion of viral DNA into saliva was not significantly different from that of 21 control parents. Only 1 of the 15 showed a fourfold increase in antibody titers to HHV-6 after possible exposure from their children. Conclusions. After systemic replication of HHV-6 in the blood of patients with ES during the first 5 days of the disease, the virus is excreted into saliva and stool persistently or intermittently but rarely into urine. The presence of HHV-6 DNA in plasma suggested active infection with the virus. Excretion of the virus into the saliva of infants with ES and their parents suggests the source and transmission route of infection with HHV-6.