Hitomi Fukumoto

The First Identification and Retrospective Study of Severe Fever With Thrombocytopenia Syndrome in Japan

Abstract Background. Severe fever with thrombocytopenia syndrome (SFTS) is caused by SFTS virus (SFTSV), a novel bunyavirus reported to be endemic in central and northeastern China. This article describes the first identified patient with SFTS and a retrospective study on SFTS in Japan. Methods. Virologic and pathologic examinations were performed on the patients samples. Laboratory diagnosis of SFTS was made by isolation/genome amplification and/or the detection of anti-SFTSV immunoglobulin G antibody in sera. Physicians were alerted to the initial diagnosis and asked whether they had previously treated patients with symptoms similar to those of SFTS. Results. A female patient who died in 2012 received a diagnosis of SFTS. Ten additional patients with SFTS were then retrospectively identified. All patients were aged ≥50 years and lived in western Japan. Six cases were fatal. The ratio of males to females was 8:3. SFTSV was isolated from 8 patients. Phylogenetic analyses indicated that all of the Japanese SFTSV isolates formed a genotype independent to those from China. Most patients showed symptoms due to hemorrhage, possibly because of disseminated intravascular coagulation and/or hemophagocytosis. Conclusions. SFTS has been endemic to Japan, and SFTSV has been circulating naturally within the country.

Mobile and accurate detection system for infection by the 2009 pandemic influenza A (H1N1) virus with a pocket-warmer reverse-transcriptase loop-mediated isothermal amplification

The 2009 pandemic H1N1 influenza A virus spread quickly worldwide in 2009. Since most of the fatal cases were reported in developing countries, rapid and accurate diagnosis methods that are usable in poorly equipped laboratories are necessary. In this study, a mobile detection system for the 2009 H1N1 influenza A virus was developed using a reverse‐transcriptase loop‐mediated isothermal amplification (RT‐LAMP) kit with a disposable pocket‐warmer as a heating device (designated as pwRT‐LAMP). The pwRT‐LAMP can detect as few as 100 copies of the virus—which is nearly as sensitive as real‐time reverse‐transcription polymerase chain reaction (RT‐PCR)—and does not cross‐react with RNA of seasonal influenza viruses. To evaluate the usefulness of the pwRT‐LAMP system, nasal swab samples were collected from 56 patients with flu‐like symptoms and were tested. Real‐time RT‐PCR confirmed that the 2009 H1N1 influenza A virus was present in 27 of the 56 samples. Of these 27 positive samples, QuickVue Influenza A + B immunochromatography detected the virus in only 11 samples (11/27; 40.7%), whereas the pwRT‐LAMP system detected the virus in 26 of the 56 samples (26/27 of the positive samples; 96.3%). These findings indicate that the mobile pwRT‐LAMP system is an accurate diagnostic system for the 2009 H1N1 influenza A virus, and has great potential utility in diagnosing future influenza pandemics. J. Med. Virol. 83:568–573, 2011.

Pathology of Kaposi’s Sarcoma-Associated Herpesvirus Infection

Kaposi’s sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is a human herpesvirus, classified as a gamma-herpesvirus. KSHV is detected in Kaposi’s sarcoma (KS), primary effusion lymphoma (PEL), and some cases of multicentric Castleman’s disease (MCD). Similar to other herpes viruses, there are two phases of infection, latent and lytic. In KSHV-associated malignancies such as KS and PEL, KSHV latently infects almost all tumor cells. Quantitative PCR analysis revealed that each tumor cell contains one copy of KSHV in KS lesions. The oncogenesis by KSHV has remained unclear. Latency-associated nuclear antigen (LANA)-1 plays an important role in the pathogenesis of KSHV-associated malignancies through inhibition of apoptosis and maintenance of latency. Because all KSHV-infected cells express LANA-1, LANA-1 immunohistochemistry is a useful tool for diagnosis of KSHV infection. KSHV encodes some homologs of cellular proteins including cell-cycle regulators, cytokines, and chemokines, such as cyclin D, G-protein-coupled protein, interleukin-6, and macrophage inflammatory protein-1 and -2. These viral proteins mimic or disrupt host cytokine signals, resulting in microenvironments amenable to tumor growth. Lytic infection is frequently seen in MCD tissues, suggesting a different pathogenesis from KS and lymphoma.

Frequent detection of Merkel cell polyomavirus DNA in sera of HIV-1-positive patients

BackgroundMerkel cell polyomavirus (MCPyV), human polyomavirus-6 (HPyV6), and human polyomavirus-7 (HPyV7) were identified as viruses shed from the skin. Serological analysis revealed that these viruses are common among the general population. However, there is little information about the presence of MCPyV, HPyV6, and HPyV7 in the sera and tissues of immunocompromised individuals. The aims of this study are to know if immune status affects the presence of MCPyV, HPyV6, and HPyV7 in the serum, and to reveal the presence of these viruses in diseased tissues of unknown etiology.MethodsSera from HIV-1-positive and -negative patients were examined by real-time PCR and nested PCR detecting MCPyV, HPyV6 and HPyV7. In addition, diseased tissue samples of unknown etiology were examined.ResultsNine out of 23 serum samples (39.1%) from HIV-1-positive patients who had not received anti-retroviral therapy were positive for MCPyV, which is significantly higher than HIV-1-negative patients (6/110, 5.5%, P < 0.01, Chi-square test). MCPyV DNA was detected in tissue samples of Merkel cell carcinoma (22/30 [73%]), encephalitis (4/19 [21%]), pneumonia (3/17 [18%]), and myocarditis (8/14 [57%]). With the exception of Merkel cell carcinoma samples, MCPyV-positive tissues showed low copy numbers of MCPyV DNA by real-time PCR and no expression of the MCPyV large T antigen by immunohistochemistry. HPyV6 and HPyV7 were rarely detected in serum and tissue samples.ConclusionsThese results suggest that MCPyV viremia is associated with host immunity, and that circulation of HPyV6 and HPyV7 in the serum is rare.

Evidence for human herpesvirus-6B infection of regulatory T-cells in acute systemic lymphadenitis in an immunocompetent adult with the drug reaction with eosinophilia and systemic symptoms syndrome: A case report

We describe a fatal case of drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome with human herpesvirus-6B (HHV-6B)-associated lymphadenitis and virus-associated hemophagocytic syndrome triggered by an over-the-counter medication to treat respiratory and influenza-like symptoms. Histologically, the structure of the lymph node was disrupted with infiltration of large lymphocytes carrying intranuclear acidophilic inclusion bodies. Immunohistochemistry and real-time PCR analysis revealed that these large lymphocytes were positive for HHV-6B. Numerous HHV-6 particles were detected in the inclusion body of the lymphocytes by electron microscopy. Interestingly, immunohistochemistry revealed that HHV-6B-infected cells in the lymph node were CD3(+), CD4(+), CD25(+), and FoxP3(+) T cells, indicating a phenotypic resemblance to regulatory T-cells. This case provides direct evidence of HHV-6 infection in CD25(+)/FoxP3(+) T cells in a case of acute lymphadenitis of DRESS syndrome, suggesting a significant role of HHV-6 infection of regulatory T-cells in the pathogenesis of DRESS syndrome.

Seroprevalence of Kaposi's sarcoma-associated herpesvirus among men who have sex with men in Japan.

Kaposis sarcoma‐associated herpesvirus (KSHV), the etiologic agent of Kaposis sarcoma, causes malignancies frequently in patients with acquired immunodeficiency syndrome. In the United States and Europe, KSHV infection is common among men who have sex with men. However, the seroprevalence of KSHV among men who have sex with men in Japan is unknown. In the present study, the seroprevalence of KSHV was investigated among 230 men who have sex with men and 400 age‐ and area of residence‐matched men (controls) using a mixed‐antigen (KSHV‐encoded K8.1, open reading frame 59, 65, and 73 proteins) enzyme‐linked immunosorbent assay and an immunofluorescence assay. Among the Japanese men who have sex with men, serological assays revealed that 27 (11.7%) were seropositive for KSHV; 20 (5%) of the men in the control group were also KSHV seropositive. The seroprevalence of KSHV among men who have sex with men was significantly higher than in the control group (odds ratio = 2.52, 95% confidence intervals = 1.38–4.62, P = 0.0019, Chi‐square test). Infection with the human immunodeficiency virus, Treponema pallidum, or hepatitis B and C virus did not correlate with KSHV infection. Furthermore, the association of KSHV seropositivity with specific sexual activities was not statistically significant. In conclusion, a higher KSHV seroprevalence was found among Japanese men who have sex with men than among the controls, suggesting that the circulation of KSHV infection is more efficient among men who have sex with men in Japan than among men who do not engage in such sexual activities. J. Med. Virol. 85: 1046–1052, 2013.

Seroprevalence of trichodysplasia spinulosa-associated polyomavirus in Japan.

BACKGROUND Trichodysplasia spinulosa-associated polyomavirus (TSV) was identified in, and shown to be the probable cause of, trichodysplasia spinulosa, a rare skin disease. To date, serological analyses have revealed that TSV infection is common among adults in the general population of Europe and Australia. However, there have been no reports of TSV in Asia. OBJECTIVE To study the prevalence of TSV infection in Japan. STUDY DESIGN TSV-VP1 expressed in a recombinant baculovirus expression system in an insect cell line, Tn5, self-assembled into virus-like particles. Overall, 1000 serum samples were examined by enzyme-linked immunosorbent assays using virus-like particles of TSV as antigen. Participants ranged in age from 0 to 94 years. RESULTS Overall, 629 of 1000 serum samples (62.9%) were positive for anti-TSV antibodies. The seropositive rate increased with age and the seroprevalence of TSV significantly increased from 17.1% (25/146) in children aged from 0 to 4 years to 78.7% (472/600) in adults aged over 20 years (odds ratio = 0.056, 95% confidence interval = 0.035-0.900, P = 0.000, Chi-squared test). TSV seropositivity was not different between sera obtained in 1980 and 2012, and was not associated with sex. Competitive assay demonstrated that TSV antibodies did not cross-react with BK virus or Merkel cell polyomavirus. CONCLUSIONS These results provide evidence that TSV circulates widely in the Japanese population, with primary exposure occurring mainly at early childhood, similar to that previously reported in other countries.

Next-generation sequencing of miRNAs in clinical samples of Epstein-Barr virus-associated B-cell lymphomas

Epstein–Barr virus (EBV) encodes 49 microRNAs (miRNAs) in the BART and BHRF1 regions of its genome. Although expression profiles of EBV‐encoded miRNAs have been reported for EBV‐positive cell lines and nasopharyngeal carcinoma, to date there is little information about total miRNA expression, including cellular and viral miRNAs, in the primary tumors of EBV‐associated B‐lymphoproliferative disorders. In this study, next‐generation sequencing and quantitative real‐time reverse transcription‐PCR were used to determine the expression profiles of miRNAs in EBV‐infected cell lines and EBV‐associated B‐cell lymphomas, including AIDS‐related diffuse large B‐cell lymphoma (DLBCL), pyothorax‐associated lymphoma, methotrexate‐associated lymphoproliferative disorder, EBV‐positive DLBCL of the elderly, and Hodgkin lymphoma. Next‐generation sequencing revealed that EBV‐encoded miRNAs accounted for up to 34% of total annotated miRNAs in these cases. Expression of three miR‐BHRF1s was significantly higher in AIDS‐related DLBCL and pyothorax‐associated lymphoma compared with methotrexate‐associated lymphoproliferative disorder and EBV‐positive DLBCL of the elderly, suggesting the association of miR‐BHRF1s expression with latency III EBV infection. Heat map/clustering analysis of expression of all miRNAs, including cellular and EBV miRNAs, by next‐generation sequencing demonstrated that each EBV tumor, except methotrexate‐associated lymphoproliferative disorder, formed an isolated cluster. Principal component analysis based on the EBV‐encoded miRNA expression showed that each EBV tumor formed a distinguished cluster, but AIDS‐related DLBCL and pyothorax‐associated lymphoma formed larger clusters than other tumors. These data suggest that expression of miRNAs, including EBV‐encoded miRNAs, is associated with the tumor type and status of virus infection in these tumors.

Metachronous Merkel cell carcinoma on both cheeks.

Merkel cell carcinoma (MCC), an aggressive skin cancer with neuroendocrine features, has been found to be associated with a new type of human polyomavirus called Merkel cell polyomavirus (MCV). Patients diagnosed with MCC have a significantly increased risk of a second primary cancer. We report here the first case of two primary MCCs arising on the face at different times, associated with MCV infection. The tumour on the patients right cheek was surgically removed, followed by chemoradiation. After a 10-year tumour-free period, a new tumour developed on the patients left cheek. Histological and immunohistochemical findings were consistent with MCC. The tumours had high MCV copy numbers and expressed large T antigen, which may play a major role in MCV-mediated carcinogenesis. This case highlights the close links between MCC and MCV.

Deep-Sequence Identification and Role in Virus Replication of a JC Virus Quasispecies in Patients with Progressive Multifocal Leukoencephalopathy.

ABSTRACT JC virus (JCV) is a DNA virus causing progressive multifocal leukoencephalopathy (PML) in immunodeficient patients. In the present study, 22 genetic quasispecies with more than 1.5% variant frequency were detected in JCV genomes from six clinical samples of PML by next-generation sequencing. A mutation from A to C at nucleotide (nt) 3495 in JCV Mad1 resulting in a V-to-G amino acid substitution at amino acid (aa) position 392 of the large T antigen (TAg) was identified in all six cases of PML at 3% to 19% variant frequencies. Transfection of JCV Mad1 DNA possessing the V392G substitution in TAg into IMR-32 and human embryonic kidney 293 (HEK293) cells resulted in dramatically decreased production of JCV-encoded proteins. The virus DNA copy number was also reduced in supernatants of the mutant virus-transfected cells. Transfection of the IMR-32 and HEK293 cells with a virus genome containing a revertant mutation recovered viral production and protein expression. Cotransfection with equal amounts of wild-type genome and mutated JCV genome did not reduce the expression of viral proteins or viral replication, suggesting that the mutation did not have any dominant-negative function. Finally, immunohistochemistry demonstrated that TAg was expressed in all six pathological samples in which the quasispecies were detected. In conclusion, the V392G amino acid substitution in TAg identified frequently in PML lesions has a function in suppressing JCV replication, but the frequency of the mutation was restricted and its role in PML lesions was limited. IMPORTANCE DNA viruses generally have lower mutation frequency than RNA viruses, and the detection of quasispecies in JCV has rarely been reported. In the present study, a next-generation sequencer identified a JCV quasispecies with an amino acid substitution in the T antigen in patients with PML. In vitro studies showed that the mutation strongly repressed the expression of JC viral proteins and reduced the viral replication. However, because the frequency of the mutation was low in each case, the total expression of virus proteins was sustained in vivo. Thus, JC virus replicates in PML lesions in the presence of a mutant virus which is able to repress virus replication.