Harutaka Katano

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.

Thymus-derived leukemia-lymphoma in mice transgenic for the Tax gene of human T-lymphotropic virus type I

Adult T-cell leukemia-lymphoma (ATLL) is a group of T-cell malignancies caused by infection with human T-lymphotropic virus type I (HTLV-I). Although the pathogenesis of ATLL remains incompletely understood, the viral regulatory protein Tax is centrally involved in cellular transformation. Here we describe the generation of HTLV-I Tax transgenic mice using the Lck proximal promoter to restrict transgene expression to developing thymocytes. After prolonged latency periods, transgenic mice developed diffuse large-cell lymphomas and leukemia with clinical, pathological and immunological features characteristic of acute ATLL. Transgenic mice were functionally immunocompromised and they developed opportunistic infections. Fulminant disease also developed rapidly in SCID mice after engraftment of lymphomatous cells from transgenic mice. Flow cytometry showed that the cells were CD4− and CD8−, but CD44+, CD25+ and cytoplasmic CD3+. This phenotype is indicative of a thymus-derived pre–T-cell phenotype, and disease development was associated with the constitutive activation of NF-κB. Our model accurately reproduces human disease and will provide a tool for analysis of the molecular events in transformation and for the development of new therapeutics.

Identification of Antigenic Proteins Encoded by Human Herpesvirus 8 and Seroprevalence in the General Population and among Patients with and without Kaposi's Sarcoma

ABSTRACT To establish a sensitive and specific antibody assay, potent antigenic proteins encoded by human herpesvirus 8 (HHV8) were studied. Fifteen recombinant HHV8-encoded proteins were produced as glutathioneS-transferase fusion proteins. The sera from AIDS-associated Kaposis sarcoma (KS) patients reacted with four proteins encoded by open reading frames (ORFs) K8.1, 59, 65, and 73 in a Western blot assay. An enzyme-linked immunosorbent assay (ELISA) using these four proteins as antigens (mixed-antigen ELISA) revealed that all 26 sera derived from KS patients (24 with and 2 without human immunodeficiency virus infection) became positive for anti-HHV8 antibodies. The presence of HHV8 was demonstrated in 14 (1.4%) of 1,004 sera from the Japanese general population and 10 (1.9%) of 527 sera from patients without HHV8-associated diseases. The presence of immunoglobulin G (IgG) and IgM antibodies against HHV8 examined further by the mixed-antigen ELISA and Western blotting revealed IgG antibody in all ELISA-positive sera, while IgM antibody against ORF K8.1 was absent. These data suggest that the ORF 73 and 65 proteins are potent antigens for a sensitive serological assay.

Detection of Merkel cell polyomavirus in Merkel cell carcinoma and Kaposi's sarcoma.

Merkel cell carcinoma is a rare malignancy that sometimes occurs in the skin of elderly people. Recently, a new human polyomavirus, Merkel cell polyomavirus (MCPyV) was identified in Merkel cell carcinoma. In the present study, MCPyV‐DNA was detected in 6 of 11 (55%) cases of Merkel cell carcinoma by nested PCR and real‐time PCR. Histologically, MCPyV‐positive cases showed round and vesicular nuclei with a fine granular chromatin and small nucleoli, whereas MCPyV‐negative cases showed polygonal nuclei with diffusely distributed chromatin. Real‐time PCR analysis to detect the MCPyV gene revealed that viral copy numbers ranged 0.04–0.43 per cell in cases of Merkel cell carcinoma. MCPyV was also detected in 3 of 49 (6.1%) cases of Kaposis sarcoma (KS), but not in 192 DNA samples of other diseases including 142 autopsy samples from 20 immunodeficient patients. The MCPyV copy number in KS was lower than that in Merkel cell carcinoma. PCR successfully amplified a full‐length MCPyV genome from a case of KS. Sequence analysis revealed that the MCPyV isolated from KS had 98% homology to the previously reported MCPyV genomes. These data suggest that the prevalence of MCPyV is low in Japan, and is at least partly associated with the pathogenesis of Merkel cell carcinoma. J. Med. Virol. 81:1951–1958, 2009.

Characterization of Quasispecies of Pandemic 2009 Influenza A Virus (A/H1N1/2009) by De Novo Sequencing Using a Next-Generation DNA Sequencer

Pandemic 2009 influenza A virus (A/H1N1/2009) has emerged globally. In this study, we performed a comprehensive detection of potential pathogens by de novo sequencing using a next-generation DNA sequencer on total RNAs extracted from an autopsy lung of a patient who died of viral pneumonia with A/H1N1/2009. Among a total of 9.4×106 40-mer short reads, more than 98% appeared to be human, while 0.85% were identified as A/H1N1/2009 (A/Nagano/RC1-L/2009(H1N1)). Suspected bacterial reads such as Streptococcus pneumoniae and other oral bacteria flora were very low at 0.005%, and a significant bacterial infection was not histologically observed. De novo assembly and read mapping analysis of A/Nagano/RC1-L/2009(H1N1) showed more than ×200 coverage on average, and revealed nucleotide heterogeneity on hemagglutinin as quasispecies, specifically at two amino acids (Gly172Glu and Gly239Asn of HA) located on the Sa and Ca2 antigenic sites, respectively. Gly239 and Asn239 on antigenic site Ca2 appeared to be minor amino acids compared with the highly distributed Asp239 in H1N1 HAs. This study demonstrated that de novo sequencing can comprehensively detect pathogens, and such in-depth investigation facilitates the identification of influenza A viral heterogeneity. To better characterize the A/H1N1/2009 virus, unbiased comprehensive techniques will be indispensable for the primary investigations of emerging infectious diseases.

Establishing and characterizing a CD30-positive cell line harboring HHV-8 from a primary effusion lymphoma.

Primary effusion lymphoma (PEL, or body‐cavity‐based lymphoma [BCBL]) is a new subtype of non‐Hodgkins lymphoma in which tumor cells locate in the body cavity exclusively. PEL/BCBL is widely accepted as one of the neoplastic complications of AIDS, associated mostly with human herpesvirus 8 (HHV‐8/Kaposis sarcoma‐associated herpesvirus [KSHV]) and Epstein‐Barr virus (EBV). We established and characterized a PEL cell line named TY‐1 from a 47‐year‐old patient with AIDS. TY‐1 exhibits indeterminate immunophenotype, expressing CD45 and CD30 cell surface antigens but not expressing B‐ or T‐cell markers. Cytogenetic analysis revealed the representative karyotype of 50,XYq‐,+7,+8,+11,+15. Southern blot analysis demonstrated HHV‐8 and EBV genomes in the original tumor cells obtained from the pericardial effusion, while HHV‐8 but not EBV was detected in TY‐1 using PCR or Southern blot analysis. Tetradecanylphorbol acetate treatment induced some TY‐1 cells to proceed to the reproductive phase. This cell line may be an useful tool for research on PEL and HHV‐8. J. Med. Virol. 58:394–401, 1999.

HHV-8 infection status of AIDS-unrelated and AIDS-associated multicentric Castleman's disease.

Multicentric Castlemans disease (MCD) is a clinicopathologically defined entity characterized by systemic lymphadenopathy with unique pathomorphology such as angiosclerosis, blood vessel proliferation in and around follicles, and plasmacytosis. While its pathogenesis has remained unclarified for many years, identification of the human herpesvirus 8 (HHV‐8) in at least some MCD cases has opened new perspectives in this field. Because previous reports have described many inconsistencies regarding HHV‐8 positivity in MCD, we intended to clarify this issue by the introduction of more convincing methodologies. For this investigation, we introduced two antibodies produced in our laboratories that recognize a latent gene product ORF73 and a lytic gene product ORF59, together with two well‐recognized methods, in situ hybridization for the detection of lytic phase transcript T1.1/nut‐1, and genomic polymerase chain reaction (PCR). Eighty‐two cases of MCD were collected from Japan (n= 75) and France (n= 7). In three cases, the patients were suffering from acquired immunodeficiency syndrome (AIDS). Immunohistochemistry and in situ hybridization showed identical results: only three out of 82 cases were positively stained, and all the positive cases were found to be the patients with AIDS. Genomic PCR was done in 43 cases, and only one case produced positive results: the only AIDS case among the 43 cases studied by genomic PCR. Histopathologically, the HHV‐8‐positive cases showed the highest intensity of angiosclerosis and germinal center / perifollicular vascular proliferation, while plasmacytosis was not severe in the HHV‐8‐positive cases. Some of the HHV‐8‐negative MCD cases displayed similar histopathology, but at a far less intense level, except for the plasmacytosis. These results suggest that: (i) all three of the HHV‐8‐positive MCD patients in the present group are the patients with AIDS; and (ii) HHV‐8‐positive MCD patients develop typical but marked angiosclerosis and vascular proliferation that might be differentiated from HHV‐8‐negative MCD patients, who showed far less intense changes.

Human Herpesvirus 8–Associated Solid Lymphomas that Occur in AIDS Patients Take Anaplastic Large Cell Morphology

Human herpesvirus type 8 (HHV-8; Kaposis sarcoma–associated herpesvirus) is a recently isolated human herpesvirus frequently identified in Kaposis sarcoma, primary effusion lymphoma, and multicentric Castlemans disease. Here we report three cases of HHV-8–bearing solid lymphomas that occurred in AIDS patients (Cases 1–3). All three patients were homosexual men presenting extranodal masses in the lungs (Case 1) or skin (Cases 2 and 3), together with the presence of Kaposis sarcoma (Case 1), primary effusion lymphoma (Case 2), or multicentric Castlemans disease (Case 3). These solid lymphomas exhibited anaplastic large cell morphology and expressed CD30, corresponding to the recent diagnostic criteria of anaplastic large cell lymphoma (ALCL). The chromosomal translocation t(2;5)-associated chimeric protein p80NPM/ALK was not observed in any of these cases. HHV-8 was detected in all of these cases by polymerase chain reaction, immunohistochemistry of HHV-8–encoded ORF73 protein, and in situ hybridization of T1.1. Epstein-Barr virus was detected only in Cases 2 and 3 by in situ hybridization. It is interesting that inoculation of a cell line obtained from a primary effusion lymphoma cell in Case 2 to severe combined immunodeficiency mice produced HHV-8–positive and Epstein-Barr virus–negative tumors in inoculated sites. These tumor cells exhibited phenotypes of ALCL that were identical to the subcutaneous tumor cells of this particular patient. These findings clearly show that HHV-8 can associate with solid lymphomas and that it can take anaplastic large cell morphology. Those lymphomas should be distinguished from the classical ALCL as were defined by the revised European-American classification of lymphoid neoplasms even though morphology and a part of immunophenotype mimic that of classical ALCL.

Expression of p53 and human herpesvirus-8 (HHV-8)–encoded latency-associated nuclear antigen with inhibition of apoptosis in HHV-8–associated malignancies

Kaposi sarcoma (KS) and primary effusion lymphoma (PEL) cells express human herpesvirus‐8 (HHV‐8)–encoded latency‐associated nuclear antigen (LANA) (open reading frame [ORF] 73 protein), suggesting that LANA plays an important role in the pathogenesis of HHV‐8–associated malignancies. Recently, the binding of LANA to p53 was demonstrated in vitro. In the current study, the authors investigated the association between p53 and LANA expression with apoptosis in HHV‐8–associated malignancies in vivo.

Classic type of Kaposi's sarcoma and human herpesvirus 8 infection in Xinjiang, China

We report 17 cases of the classic type of Kaposis sarcoma in Xinjiang, which is located in the north‐western area of China surrounded by Mongolia in the east, Russia in the north and Kazakhstan in the west. Fifteen of the patients were of the Uygur people. All patients were male and did not have acquired immunodeficiency syndrome. Most of the lesions were found in the lower and/or upper extremities, with 16 patients showing multiple lesions. Immunohistochemical examination of the lesions revealed that human herpesvirus 8 (HHV‐8)‐encoded latency‐associated nuclear antigen was expressed in the nuclei of spindle‐shaped tumor cells. HHV‐8 DNA was detected by polymerase chain reaction in all seven cases examined. Phylogenetic tree analysis revealed that DNA sequences of the HHV‐8‐encoded K1 gene in the seven Kaposis sarcoma cases were classified as subtype C that was common in the Mediterranean, the Middle East and East Asian countries. In addition, using immunofluorescence we investigated the seroprevalence of HHV‐8 in 73 Uygur patients with diseases other than Kaposis sarcoma. Surprisingly, the serological study revealed that 34 of the patients (46.6%) were positive for antibodies against HHV‐8, suggesting that HHV‐8 infection is widespread in Xinjiang area. The occurrence of the classic type of Kaposis sarcoma with a high seropositivity rate implies that Xinjiang is the most endemic area for HHV‐8 infection in the world known to date. Considering that Xinjiang is located at the middle point of the Silk Road that used to extend from Rome to China, these data imply that the virus may have been in circulation in this area due to the migration of the people via the Silk Road.