研究者情報

マスター

アカウント（マスター）

• 氏名

  木村　享史(キムラ　タカシ), キムラ　タカシ

所属（マスター）

• 獣医学研究院　獣医学部門　臨床獣医科学分野

所属（マスター）

• 獣医学研究院　獣医学部門　臨床獣医科学分野

独自項目

syllabus

• 2021, 大学院共通授業科目（一般科目）：自然科学・応用科学, Inter-Graduate School Classes(General Subject):Natural and Applied Sciences, 修士課程, 大学院共通科目, ケミカルハザード、食の安全、毒性学、環境汚染、シェルターメディスン、リスクアセスメント、GIS、生態学、薬理学、病理学、免疫毒性学、分子毒性学、地球規模課題、リスクアナリシス、コンピューターシミュレーション、R、ドッキングシミュレーション、分析化学、環境化学
• 2021, 獣医病理学特論, Advanced Lecture on Veterinary Pathology, 博士後期課程, 獣医学院
• 2021, 臨床疾病学特論, Advanced Lecture on Veterinary Clinical Diseases, 博士後期課程, 獣医学院
• 2021, 一般教育演習(ﾌﾚｯｼｭﾏﾝｾﾐﾅｰ), Freshman Seminar, 学士課程, 全学教育, 動物、ヒト、病気、病因、病態、病理、症状、予防、治療
• 2021, 泌尿器病理学, Pathology of the Urinary system, 学士課程, 獣医学部, 腎病理学、生殖器病理学、内分泌病理学、皮膚病理学
• 2021, 神経・運動器病理学, Pathology of the Neurological and Locomotor system, 学士課程, 獣医学部, 神経病理学、感覚器病理学、骨病理学、骨格筋病理学
• 2021, 病理学総論, General Pathology, 学士課程, 獣医学部, 変性、壊死、アポトーシス、炎症、腫瘍、先天異常
• 2021, 病理学総論実習, Practice in General Pathology, 学士課程, 獣医学部, 動物病理学、病理解剖、病理組織学、細胞傷害、細胞・組織の適応、循環障害、炎症、腫瘍
• 2021, 病理学各論実習, Practice in Particular Pathology, 学士課程, 獣医学部, 疾患、病理学、診断学

researchmap

プロフィール情報

学位

• 医学博士（北海道大学）

プロフィール情報

• 姓

  木村, キムラ

• 名

  享史, タカシ

• ID各種

  200901041582949278

業績リスト

論文

• Characterization of a mammalian orthoreovirus isolated from the large flying fox, Pteropus vampyrus, in Indonesia.

  Kittiya Intaruck, Koshiro Tabata, Yukari Itakura, Nijiho Kawaguchi, Mai Kishimoto, Agus Setiyono, Ekowati Handharyani, Hayato Harima, Takashi Kimura, William W Hall, Yasuko Orba, Hirofumi Sawa, Michihito Sasaki

  The Journal of general virology 105 9 2024年09月 

  Fruit bats serve as an important reservoir for many zoonotic pathogens, including Nipah virus, Hendra virus, Marburg virus and Lyssavirus. To gain a deeper insight into the virological characteristics, pathogenicity and zoonotic potential of bat-borne viruses, recovery of infectious viruses from field samples is important. Here, we report the isolation and characterization of a mammalian orthoreovirus (MRV) from a large flying fox (Pteropus vampyrus) in Indonesia, which is the first detection of MRV in Southeast Asia. MRV was recovered from faecal samples of three different P. vampyrus in Central Java. Nucleotide sequence analysis revealed that the genome of the three MRV isolates shared more than 99% nucleotide sequence identity. We tentatively named one isolated strain as MRV12-52 for further analysis and characterization. Among 10 genome segments, MRV12-52 S1 and S4, which encode the cell-attachment protein and outer capsid protein, had 93.6 and 95.1% nucleotide sequence identities with known MRV strains, respectively. Meanwhile, the remaining genome segments of MRV12-52 were divergent with 72.9-80.7 % nucleotide sequence identities. Based on the nucleotide sequence of the S1 segment, MRV12-52 was grouped into serotype 2, and phylogenetic analysis demonstrated evidence of past reassortment events. In vitro characterization of MRV12-52 showed that the virus efficiently replicated in BHK-21, HEK293T and A549 cells. In addition, experimental infection of laboratory mice with MRV12-52 caused severe pneumonia with 75% mortality. This study highlights the presence of pathogenic MRV in Indonesia, which could serve as a potential animal and public health concern.
• Identification of the Promoter Antisense Transcript Enhancing the Transcription of the Equine Herpesvirus-1 Immediate-Early Gene.

  Mayuko Maeda, Miou Abe, Keisuke Aoshima, Atsushi Kobayashi, Hideto Fukushi, Takashi Kimura

  Viruses 16 8 2024年07月25日 

  Equine herpesvirus-1 (EHV-1) causes respiratory diseases, abortion, and encephalomyelitis in horses. The EHV-1 immediate-early (IE) protein, essential for viral replication, is transactivated by the binding of a multiprotein complex including the open reading frame 12 (ORF12) and some host factors to the IE promoter region. Promoter-associated non-coding RNAs (pancRNAs), which are transcribed from bidirectional promoters, regulate the transcription of neighboring genes in mammals and pathogens. In this study, we identified a novel pancRNA transcribed from across the areas of the 5'-untranslated region and a promoter of EHV-1 IE and named it IE pancRNA. IE pancRNA and mRNA were simultaneously expressed in EHV-1-infected RN33B-A68B2M cells. This pancRNA was also transcribed in RK13 and E. Derm cells, which are highly susceptible to EHV-1 infection. Furthermore, IE pancRNA upregulated IE gene expression in the presence of ORF12, and stable expression of IE pancRNA increased the number of EHV-1-infected RN33B-A68B2M cells. These results suggest that IE pancRNAs facilitate EHV-1 proliferation by promoting IE gene expression.
• A point mutation in GPI-attachment signal peptide accelerates the development of prion disease

  Kobayashi A, Hirata T, Shimazaki T, Munesue Y, Aoshima K, Kimura T, Nio-Kobayashi J, Hasebe R, Takeuchi A, Matsuura Y, Kusumi S, Koga D, Iwasaki Y, Kinoshita T, Mohri S, Kitamoto T

  Acta Neuropathol 145 5 637 - 650 2023年03月 [査読有り][通常論文]
   

  A missense variant from methionine to arginine at codon 232 (M232R) of the prion protein gene accounts for ~ 15% of Japanese patients with genetic prion diseases. However, pathogenic roles of the M232R substitution for the induction of prion disease have remained elusive because family history is usually absent in patients with M232R. In addition, the clinicopathologic phenotypes of patients with M232R are indistinguishable from those of sporadic Creutzfeldt-Jakob disease patients. Furthermore, the M232R substitution is located in the glycosylphosphatidylinositol (GPI)-attachment signal peptide that is cleaved off during the maturation of prion proteins. Therefore, there has been an argument that the M232R substitution might be an uncommon polymorphism rather than a pathogenic mutation. To unveil the role of the M232R substitution in the GPI-attachment signal peptide of prion protein in the pathogenesis of prion disease, here we generated a mouse model expressing human prion proteins with M232R and investigated the susceptibility to prion disease. The M232R substitution accelerates the development of prion disease in a prion strain-dependent manner, without affecting prion strain-specific histopathologic and biochemical features. The M232R substitution did not alter the attachment of GPI nor GPI-attachment site. Instead, the substitution altered endoplasmic reticulum translocation pathway of prion proteins by reducing the hydrophobicity of the GPI-attachment signal peptide, resulting in the reduction of N-linked glycosylation and GPI glycosylation of prion proteins. To the best of our knowledge, this is the first time to show a direct relationship between a point mutation in the GPI-attachment signal peptide and the development of disease.
• バロキサビルマルボキシルのニワトリへの4週間反復経口投与による安全性評価

  三木 万梨子, 尾原 涼, 西村 享平, 岡 良子, 宍戸 貴雄, 福島 民雄, 吉本 淳, 中山 翔太, 木村 享史, 池中 良徳, 日尾野 隆大, 磯田 典和, 迫田 義博

  日本獣医学会学術集会講演要旨集 165回 [DV1A - 08] (公社)日本獣医学会 2022年09月
• キタキツネ及びタヌキからの高病原性鳥インフルエンザウイルスの分離

  日尾野 隆大, 磯田 典和, 小林 篤史, 小林 大樹, 鈴木 玲海, 佐竹 優樹, 松野 啓太, 佐鹿 万里子, 伴 日向子, 小林 茉弥, 高谷 文仁, 冨士田 裕子, 木村 享史, 迫田 義博

  日本獣医学会学術集会講演要旨集 165回 [DV1A - 09] (公社)日本獣医学会 2022年09月
• 高病原性鳥インフルエンザウイルスに感染したキタキツネの病理学的解析とウイルス受容体の検出

  小林 大樹, 小林 篤史, 日尾野 隆大, 原田 里桜, 鈴木 玲海, 松野 啓太, 磯田 典和, 高谷 文仁, 冨士田 裕子, 木村 享史, 迫田 義博

  日本獣医学会学術集会講演要旨集 165回 [DV1A - 10] (公社)日本獣医学会 2022年09月
• 鼻疽の診断抗原の探索

  市川 世識, 飯沼 由希帆, 岡川 朋弘, 前川 直也, 村田 史郎, 今内 覚, 木下 優太, 丹羽 秀和, 青島 圭佑, 小林 篤史, 大橋 和彦, 木村 享史

  日本獣医学会学術集会講演要旨集 165回 [DI1A - 09] (公社)日本獣医学会 2022年09月
• ヒストンアセチル化の改変はイヌ血管肉腫細胞に抗腫瘍効果をもたらす

  鈴木 玲海, 青島 圭佑, 山崎 淳平, 小林 篤史, 木村 享史

  日本獣医学会学術集会講演要旨集 165回 [B2P - 08] (公社)日本獣医学会 2022年09月
• The expression of histone lysine demethylase 2B in canine hemangiosarcoma is associated with disease progression

  Kevin Christian, M. Gulay, Keisuke Aoshima, Atsushi Kobayashi, Takashi Kimura

  Veterinary and Comparative Oncology 20 2 529 - 534 2022年06月 [査読有り][通常論文]
  
• Manipulating Histone Acetylation Leads to Antitumor Effects in Hemangiosarcoma Cells.

  Tamami Suzuki, Keisuke Aoshima, Jumpei Yamazaki, Atsushi Kobayashi, Takashi Kimura

  Veterinary and comparative oncology 2022年05月14日 

  Canine hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. No effective treatment has yet been developed because of the lack of understanding of its pathogenesis. Histone acetylation, an epigenetic modification, is highly associated with cancer pathogenesis. Manipulating histone acetylation by histone deacetylase inhibitors (HDACi) or bromodomain and extraterminal domain inhibitors (BETi) is one approach to treat various cancers. However, the role of histone acetylation in HSA remains unknown. This study aimed to investigate how histone acetylation functions in HSA pathogenesis using two HDACi, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), and one BETi, JQ1, in vitro and in vivo. Histone acetylation levels were high in cell lines and heterogeneous in clinical cases. SAHA and JQ1 induced apoptosis in HSA cell lines. HSA cell lines treated with SAHA and VPA upregulated inflammatory-related genes and attracted macrophage cell line RAW264 cells, which suggests that SAHA and VPA can affect immune responses. JQ1 stimulated autophagy and inhibited the cell cycle in HSA cell lines. Finally, we demonstrated that JQ1 suppressed HSA tumor cell proliferation in vivo although SAHA and VPA did not affect tumor growth. These results suggest that BETi can be alternative drugs for HSA treatment. Although further research is required, our study indicated that dysregulation of histone acetylation is likely to be involved in HSA malignancy. This article is protected by copyright. All rights reserved.
• Malignant oligoastrocytoma in the spinal cord of a cat

  Dai HASEGAWA, Keisuke AOSHIMA, Kazuyoshi SASAOKA, Atsushi KOBAYASHI, Mitsuyoshi TAKIGUCHI, Takashi KIMURA

  Journal of Veterinary Medical Science 84 9 1277 - 1282 2022年
• Manipulating Histone Acetylation Leads to Adverse Effects in Hemangiosarcoma Cells

  Tamami Suzuki, Keisuke Aoshima*, Jumpei Yamazaki, Atsushi Kobayashi, Takashi Kimura, *Corresponding author

  bioRxiv 2021年12月11日 [査読無し]
  
• Potential for transmission of sporadic Creutzfeldt–Jakob disease through peripheral routes

  Atsushi Kobayashi, Yoshiko Munesue, Taishi Shimazaki, Keisuke Aoshima, Takashi Kimura, Shirou Mohri, Tetsuyuki Kitamoto

  Laboratory Investigation 101 10 1327 - 1330 2021年10月 [査読有り]
   

  Five sporadic Creutzfeldt-Jakob disease (CJD) strains have been identified to date, based on differences in clinicopathological features of the patients, the biochemical properties of abnormal prion proteins, and transmission properties. Recent advances in our knowledge about iatrogenic transmission of sporadic CJD have raised the possibility that the infectivity of sporadic CJD strains through peripheral routes is different from that of intracranial infection. To test this possibility, here we assessed systematically the infectivity of sporadic CJD strains through the peripheral route for the first time using a mouse model expressing human prion protein. Although the infectivity of the V2 and M1 sporadic CJD strains is almost the same in intracerebral transmission studies, the V2 strain infected more efficiently than the M1 strain through the peripheral route. The other sporadic CJD strains examined lacked infectivity. Of note, both the V2 and M1 strains showed preference for mice with the valine homozygosity at the PRNP polymorphic codon. These results indicate that the V2 strain is the most infectious sporadic CJD strain for infection through peripheral routes. In addition, these findings raise the possibility that individuals with the valine homozygosity at the PRNP polymorphic codon might have higher risks of infection through peripheral routes compared with the methionine homozygotes. Thus, preventive measures against the transmission of the V2 sporadic CJD strain will be important for the eradication of iatrogenic CJD transmission through peripheral routes.
• Duck Tembusu virus induces stronger cellular responses than Japanese encephalitis virus in primary duck neurons and fibroblasts

  Sittinee Kulprasertsri, Shintaro Kobayashi, Keisuke Aoshima, Atsushi Kobayashi, Takashi Kimura

  Microbiology and Immunology 65 11 481 - 491 2021年09月08日 [査読有り]
   

  Duck Tembusu virus (DTMUV) and Japanese encephalitis virus (JEV) are mosquito-borne flaviviruses. These two viruses infect ducks; however, they show different neurological outcomes. The mechanism of DTMUV- and JEV-induced neuronal death has not been well investigated. In the present study, we examined the differences in the mechanisms involved in virus-induced cell death and innate immune responses between DTMUV KPS54A61 strain and JEV JaGAr-01 strain using primary duck neurons (DN) and duck fibroblasts (CCL-141). DN and CCL-141 were permissive for the infection and replication of these two viruses, which upregulated the expression of innate immunity genes. Both DTMUV and JEV induced cell death via a caspase-3-dependent manner; however, DTMUV triggered more cell death than JEV did in both CCL-141 and DN. These findings suggest that DTMUV infection causes apoptosis in duck neurons and fibroblasts more strongly than JEV. Levels of the mRNA expression of innate immunity-related genes after DTMUV infection were generally higher than levels after JEV infection, suggesting that DTMUV-induced immune response in duck cells may exhibit toxic effect rather than protective effects. This article is protected by copyright. All rights reserved.
• Systemic mucoid degeneration of the arterial tunica intima in a young dog

  Yoshiki Ichikawa, Mizuki Heishima, Kristin Vyhnal, Keisuke Aoshima, Kazuyoshi Sasaoka, Ryohei Kinoshita, Atsushi Kobayashi, Mitsuyoshi Takiguchi, Takashi Kimura

  Journal of Veterinary Diagnostic Investigation 34 1 104063872110425 - 104063872110425 2021年09月04日 [査読有り]
   

  A 27-mo-old, spayed female mixed-breed dog was presented with left forelimb pain, which progressed to full thickness necrosis of the soft tissues of multiple limbs. Clinical imaging and postmortem examination suggested multiple large arterial thromboemboli. Histologic examination of vascular lesions revealed markedly thickened tunica intima with polypoid intraluminal projections, which partially to entirely occluded the arterial lumen. The expanded tunica intima was comprised of intimal accumulation of Alcian blue–positive matrix with scattered spindle-to-satellite cells. These cells were positive for von Willebrand factor and vimentin but negative for α–smooth muscle actin, suggesting endothelial origin. Deposition of the intimal mucoid matrix was observed in the elastic and muscular arteries associated with regional ischemic changes. Mucoid emboli, likely from fragmentation of proliferative intimal tissue, were identified in smaller vessels supplied by affected arteries. Based on these findings, we diagnosed systemic mucoid degeneration of the arterial tunica intima. Such systemic arterial degeneration characterized by deposition of mucoid matrix in the tunica intima has not been reported previously in dogs, to our knowledge, and should be distinguished from thromboembolism and other degenerative vascular diseases.
• Hemangiosarcoma cells induce M2 polarization and PD-L1 expression in macrophages

  Kevin Christian M. Gulay, Keisuke Aoshima, Naoya Maekawa, Satoru Konnai, Atsushi Kobayashi, Takashi Kimura

  2021年08月01日 

  Hemangiosarcoma (HSA) is a malignant tumor derived from endothelial cells. Tumor-associated macrophages are one of the major components of tumor microenvironment and crucial for cancer development. The presence and function of macrophages in HSA have not been studied because there is no syngeneic model for HSA. In this study, we evaluated two mouse HSA cell lines and one immortalized mouse endothelial cells for their usefulness as syngeneic models for canine HSA. Our results show that the ISOS-1 cell line develops tumors with similar morphology to canine HSA. ISOS-1 cells highly express KDM2B and have similar KDM2B target expression patterns with canine HSA. Moreover, we determine that in both ISOS-1 and canine HSA tumors, macrophages are present as a major constituent of the tumor microenvironment. These macrophages are positive for CD204, an M2 macrophage marker, and express PD-L1. ISOS-1-conditioned medium can induce M2 polarization and PD-L1 expression in RAW264.7 mouse macrophage cell line. These results show that ISOS-1 can be used as a syngenic model for canine HSA and suggest that macrophages play an important role in immune evasion in HSA. Using the syngeneic mouse model for canine HSA, we can further study the role of immune cells in the pathology of HSA.
• Peliosis Hepatis with Chylous Ascites in a Dog

  Kevin Christian Montecillo Gulay, Noriyuki Nagata, Keisuke Aoshima, Nozomi Shiohara, Atsushi Kobayashi, Mitsuyoshi Takiguchi, Takashi Kimura

  Journal of Comparative Pathology 187 63 - 67 2021年08月 [査読有り]
   

  A 6-year-old spayed female Toy Poodle dog was referred to the Hokkaido University Veterinary Teaching Hospital for abdominal distension. Abdominocentesis yielded ascitic fluid that had a mildly increased total protein concentration and a 2.7-fold higher triglyceride concentration than plasma, and was interpreted as chylous ascites. The patient had an enlarged liver, which contained multiple, small, nodular masses and cyst-like structures. Microscopically, these lesions were multifocal dilated spaces containing lymphocytes, endothelial cells, fibrin and islands of hepatocytes. Increased α-smooth muscle actin-positive cells were observed in hepatic sinusoids. Based on these findings, we diagnosed peliosis hepatis with chylous ascites, which is likely to have been due to lymphangiectasia and disrupted hepatic sinusoids. Neither Bartonella spp DNA nor mutations in ACVRL1 and MTM1 genes were detected, although there was a 47-fold increase in hepatic ACVRL1 expression compared with age-matched control liver. To the authors' knowledge, this is the first report of chylous ascites resulting from peliosis hepatis in any species.
• Analysis of lead distribution in avian organs by LA-ICP-MS: Study of experimentally lead-exposed ducks and kites

  Ryouta Torimoto, Chihiro Ishii, Hiroshi Sato, Keisuke Saito, Yukiko Watanabe, Kohei Ogasawara, Ayano Kubota, Takehisa Matsukawa, Kazuhito Yokoyama, Atsushi Kobayashi, Takashi Kimura, Shouta M.M. Nakayama, Yoshinori Ikenaka, Mayumi Ishizuka

  Environmental Pollution 283 117086 - 117086 2021年08月 [査読有り]
   

  Lead poisoning of wild birds by ingestion of lead ammunition occurs worldwide. Histopathological changes in organs of lead-intoxicated birds are widely known, and lead concentration of each organ is measurable using mass spectrometry. However, detailed lead localization at the suborgan level has remained elusive in lead-exposed birds. Here we investigated the detailed lead localization in organs of experimentally lead-exposed ducks and kites by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). In both the ducks and kites, lead accumulated diffusely in the liver, renal cortex, and brain. Lead accumulation was restricted to the red pulp in the spleen. With regard to species differences in lead distribution patterns, it is noteworthy that intensive lead accumulation was observed in the arterial walls only in the kites. In addition, the distribution of copper in the brain was altered in the lead-exposed ducks. Thus, the present study shows suborgan lead distribution in lead-exposed birds and its differences between avian species for the first time. These findings will provide fundamental information to understand the cellular processes of lead poisoning and the mechanisms of species differences in susceptibility to lead exposure.
• KDM2B promotes cell viability by enhancing DNA damage response in canine hemangiosarcoma

  Kevin Christian Montecillo Gulay, Keisuke Aoshima, Yuki Shibata, Hironobu Yasui, Qin Yan, Atsushi Kobayashi, Takashi Kimura

  Journal of Genetics and Genomics 48 7 618 - 630 2021年07月 [査読有り]
   

  Epigenetic regulators have been implicated in tumorigenesis of many types of cancer; however, their roles in endothelial cell cancers such as canine hemangiosarcoma (HSA) have not been studied. In this study, we find that lysine-specific demethylase 2b (KDM2B) is highly expressed in HSA cell lines compared with normal canine endothelial cells. Silencing of KDM2B in HSA cells results in increased cell death in vitro compared with the scramble control by inducing apoptosis through the inactivation of the DNA repair pathways and accumulation of DNA damage. Similarly, doxycycline-induced KDM2B silencing in tumor xenografts results in decreased tumor sizes compared with the control. Furthermore, KDM2B is also highly expressed in clinical cases of HSA. We hypothesize that pharmacological KDM2B inhibition can also induce HSA cell death and can be used as an alternative treatment for HSA. We treat HSA cells with GSK-J4, a histone demethylase inhibitor, and find that GSK-J4 treatment also induces apoptosis and cell death. In addition, GSK-J4 treatment decreases tumor size. Therefore, we demonstrate that KDM2B acts as an oncogene in HSA by enhancing the DNA damage response. Moreover, we show that histone demethylase inhibitor GSK-J4 can be used as a therapeutic alternative to doxorubicin for HSA treatment.
• Minocycline prevents primary duck neurons from duck Tembusu virus-induced death

  Sittinee KULPRASERTSRI, Keisuke AOSHIMA, Atsushi KOBAYASHI, Takashi KIMURA

  Journal of Veterinary Medical Science 83 4 734 - 741 2021年 [査読有り]
  
• Long interspersed nucleotide element‐1 hypomethylation in canine malignant mucosal melanoma

  Teita Ishizaki, Jumpei Yamazaki, Shinji Meagawa, Nozomu Yokoyama, Keisuke Aoshima, Mitsuyoshi Takiguchi, Takashi Kimura

  Veterinary and Comparative Oncology 18 4 854 - 860 2020年12月 [査読有り][通常論文]
   

  Canine malignant melanoma is a common cancer with a high mortality rate and is a clinically important disease. DNA methylation has been considered to be a potential tumorigenic mechanism through aberrant DNA methylation at promoter region which represses gene transcription. Global hypomethylation could also facilitate chromosome instability. There are few reports regarding DNA methylation in canine malignant melanoma; therefore, the purpose of this study was to examine DNA methylation status of long interspersed nucleotide element-1 (LINE-1) to be a surrogate marker of genome-wide methylation changes in this disease. We measured levels of DNA methylation of two adjacent cytosine-guanine sites on CpG island (CGI) at the putative promoter of canine LINE-1 sequence by bisulphite-pyrosequencing in 41 canine melanoma patient samples as well as six cell lines compared with normal mucosae. The survival rates were obtained from owners or medical records. We found DNA methylation levels of LINE-1 in normal mucosae were methylated. Interestingly, both melanoma cell lines and clinical melanoma samples showed remarkable hypomethylation. In addition, patients with lower LINE-1 methylation showed worse prognosis than those with higher LINE-1 methylation, though the difference did not reach statistical significance (P = .09). Here, we demonstrate that hypomethylation of LINE-1 is an epigenetically aberrant feature in canine melanoma with possible prognostic value.
• Pathological and Immunohistochemical Analyses of Naturally Occurring Equine Glanders Using an Anti-BpaB Antibody

  Ochbayar Erdemsurakh, Baatarjargal Purevdorj, Khurtsbaatar Ochirbat, Altanchimeg Adilbish, Batbaatar Vanaabaatar, Keisuke Aoshima, Atsushi Kobayashi, Takashi Kimura

  Veterinary Pathology 57 6 807 - 811 2020年11月 [査読有り]
   

  Glanders is caused by the gram-negative bacterium Burkholderia mallei. In this study, we investigated the histopathology and immunohistochemical localization of B. mallei in natural cases of equine glanders. Four horses showing clinical signs of nasal discharge and multiple cutaneous nodules or papulae in the hindlimbs and abdomen were reported in Mongolia. They tested positive for B. mallei infection on complement fixation, Rose Bengal agglutination, and mallein tests. Gross and histological lesions observed in these cases were similar to those previously reported in equine glanders. Immunohistochemistry using a monoclonal antibody to B. mallei BpaB showed localization of the bacterial antigen in the cytoplasm of neutrophils, macrophages, epithelioid cells, and multinucleated giant cells in the pyogranulomas and abscesses in target organs. Some alveolar type II cells and bronchiolar epithelial cells also contained the antigen. These results suggest that the anti-BpaB antibody is useful for identifying B. mallei–infected cell types in naturally infected horses.
• Wobbly hedgehog syndrome with disseminated histiocytic sarcoma and lateral ventricular meningioma in an African pygmy hedgehog

  Lesa A. Thompson, Atsuya Morita, Shoko Murakami, Noboru Sasaki, Miou Murashita, Ryou Yamazaki, Atsushi Kobayashi, Takashi Kimura, Mitsuyoshi Takiguchi

  J Vet Diagn Invest 32 6 953 - 956 2020年11月 [査読有り][通常論文]
   

  An 8-mo-old male African pygmy hedgehog was anorectic and ataxic; physical examination revealed tetraparesis and a gangrenous left hindlimb. Analgesic and supportive care were administered, but the animal died 3 d after presentation. Postmortem examination revealed a histiocytic sarcoma in a mesenteric lymph node with metastasis to several organs, multifocal vacuolation in the cerebral and cerebellar white matter, and a meningioma in the left lateral ventricle. We diagnosed wobbly hedgehog syndrome (WHS) with disseminated histiocytic sarcoma and lateral ventricular meningioma. Ventricular meningioma, a rare neoplasm in veterinary and human patients, has not been reported previously in hedgehogs, to our knowledge. The neurologic signs in our case were probably caused by the WHS-related vacuolar lesions and are consistent with those of reported WHS cases. Duration of illness was shorter than is typical of WHS cases, which might be related to the disseminated histiocytic sarcoma. Clinical relevance of the lateral ventricular meningioma was not evident because the ventricular mass was localized and not invasive.
• Genome-wide DNA methylation analysis identifies promoter hypermethylation in canine malignant melanoma

  T. Ishizaki, J. Yamazaki, J. Jelinek, K. Aoshima, T. Kimura

  Research in Veterinary Science 132 521 - 526 2020年10月 [査読有り]
   

  Canine malignant melanoma is a common cancer with a high mortality rate. Although previous studies have evaluated various aspects of this tumour, the exact mechanism of tumourigenesis remains unknown. Epigenetic mechanisms, such as DNA methylation, have recently gained attention as aetiological factors for neoplasia in humans. This study aimed to analyse genome-wide DNA methylation patterns in canine malignant melanoma based on next-generation sequencing data. A total of 76,213 CpG sites, including 29,482 sites in CpG islands (CGIs), were analysed using next-generation sequencing of methylation-specific signatures, obtained by sequential digestion with enzymes, to compare normal oral mucosal samples from four healthy dogs, four canine melanoma cell lines (3 oral cavity and 1 skin), and five clinical samples of oral canine melanoma. Malignant melanoma showed increased methylation at thousands of normally unmethylated CpG sites in CGIs and decreased methylation at normally methylated CpG sites in non-CGIs. Interestingly, the promoter regions of 81-393 genes were hypermethylated; 23 of these genes were present in all melanoma cell lines and melanoma clinical samples. Among these 23 genes, six genes with "sequence-specific DNA binding" annotation were significantly enriched, including three Homeobox genes-HMX2, TLX2, and HOXA9-that may be involved in the tumourigenesis of canine malignant melanoma. This study revealed widespread alterations in DNA methylation and a large number of hypermethylated genes in canine malignant melanoma.
• Susceptibility of rat immortalized neuronal cell line Rn33B to equine herpesvirus-1 infection is differentiation-dependent

  Minato E, Kobayashi A, Aoshima K, Fukushi H, Kimura T

  Microbiol Immunol 64 2 123 - 132 2020年02月 [査読有り][通常論文]
   

  Equine herpesvirus-1 (EHV-1), which causes encephalomyelitis in horses, shows endotheliotropism in the central nervous system of horses, and generally does not infect neurons. However, little is known about the mechanism underlying the resistance of neuron to EHV-1, due to the lack of convenient cell culture systems. In this study, we examined EHV-1 infection in immortalized Rn33B rat neuronal cells, which differentiate into neurons when cultured under nonpermissive conditions. Because murine cell lines are resistant to EHV-1 infections due to the lack of functional entry receptors for EHV-1, we used an Rn33B-derived cell line that stably expresses the equine MHC class 1 molecule, which acts as EHV-1 entry receptor (Rn33B-A68B2M cells). EHV-1 infected undifferentiated Rn33B-A68B2M cells more efficiently than differentiated cells, resulting in the production of progeny virus in the former but not in the latter. By contrast, both differentiated and undifferentiated cells infected with herpes simplex virus-1 produced infectious viral progeny. While EHV-1 infection induced stronger expression of IFN alpha gene in differentiated cells than in undifferentiated cells, downstream IFN responses, including phosphorylation of STAT1 (signal transducer and activator of transcription 1) and expression of IFN-stimulated genes, were not activated regardless of whether cells were differentiated or not. These results suggest that neuronal differentiation of RN33B-A68B2M cells reduced their susceptibility to EHV-1, which is not due to different IFN responses. This culture system may be useful as an in vitro model for studying neuron-specific resistance to EHV-1, by investigating viral and host factors responsible for the difference in susceptibility between differentiated and undifferentiated cells.
• Seroprevalence of equine glanders in horses in the central and eastern parts of Mongolia

  Ochbayar ERDEMSURAKH, Khurtsbaatar OCHIRBAT, Ulziisaikhan GOMBOSUREN, Batbold TSERENDORJ, Baatarjargal PUREVDORJ, Batbaatar VANAABAATAR, Keisuke AOSHIMA, Atsushi KOBAYASHI, Takashi KIMURA

  Journal of Veterinary Medical Science 82 9 1247 - 1252 2020年 [査読有り][通常論文]
   

  Glanders is a contagious and fatal equine disease caused by the gram-negative bacterium Burkholderia mallei.B. mallei is prevalent among horse populations in Asia, the Middle East, and South America. More than four million horses have been registered in Mongolia in 2020. However, the resent prevalence of glanders has not been well investigated. In this study, we aimed to investigate the seropositivity of B. mallei in horse populations in Mongolia using the complement fixation test (CFT) and Rose Bengal plate agglutination test (RBT). We randomly collected blood samples from horses in central and eastern Mongolia between 2018 and 2019. Of 337 horses, 26 (7.7%) and 28 (8.3%) were seropositive using RBT and CFT, respectively. Interestingly, seropositivity in horses resulting from crossbreeding of Mongolian native horses with thoroughbred horses was higher than that in Mongolian native horses. Our observations suggest that equine glanders are still endemic to Mongolia.
• High drug efflux pump capacity and low DNA damage response induce doxorubicin resistance in canine hemangiosarcoma cell lines.

  Atsuya Morita, Keisuke Aoshima, Kevin Christian Montecillo Gulay, Shinichi Onishi, Yuki Shibata, Hironobu Yasui, Atsushi Kobayashi, Takashi Kimura

  Research in veterinary science 127 1 - 10 2019年12月 [査読有り][通常論文]
   

  * Corresponding author
• Exogenous Expression of Equine MHC Class I Molecules in Mice Increases Susceptibility to Equine Herpesvirus 1 Pulmonary Infection.

  Minato E, Aoshima K, Kobayashi A, Ohnishi N, Sasaki N, Kimura T

  Veterinary pathology 56 5 703 - 710 2019年09月 [査読有り][通常論文]
   

  Equine herpesvirus 1 (EHV-1) uses equine major histocompatibility complex class I (MHC class I) as an entry receptor. Exogenous expression of equine MHC class I genes in murine cell lines confers susceptibility to EHV-1 infection. To examine the in vivo role of equine MHC class I as an entry receptor for EHV-1, we generated transgenic (Tg) mice expressing equine MHC class I under the control of the CAG promoter. Equine MHC class I protein was expressed in the liver, spleen, lung, and brain of Tg mice, which was confirmed by Western blot. However, equine MHC class I antigen was only detected in bronchiolar epithelium and not in other tissues, using the immunofluorescence method employed in this study. Both Tg and wild-type (WT) mice developed pneumonia 3 days after intranasal infection with EHV-1. The bronchiolar epithelial cells of Tg mice showed more severe necrosis, compared with those in WT mice. In addition, the number of virus antigen-positive cells in the lungs was higher in Tg mice than in WT mice. These results suggest that exogenous expression of equine MHC class I renders mice more susceptible to EHV-1 infection.
• A novel combination of prion strain co-occurrence in patients with sporadic Creutzfeldt-Jakob disease.

  Kobayashi A, Iwasaki Y, Takao M, Saito Y, Iwaki T, Qi Z, Torimoto R, Shimazaki T, Munesue Y, Isoda N, Sawa H, Aoshima K, Kimura T, Kondo H, Mohri S, Kitamoto T

  The American journal of pathology 189 6 1276 - 1283 2019年06月 [査読有り][通常論文]
   

  Six subgroups of sporadic Creutzfeldt-Jakob disease have been identified by distinctive clinicopathologic features, genotype at polymorphic codon 129 [methionine (M)/valine (V)] of the PRNP gene, and type of abnormal prion proteins (type 1 or 2). In addition to the pure subgroups, mixed neuropathologic features and the coexistence of two types of abnormal prion proteins in the same patient also have been reported. Here, we found that a portion of the patients previously diagnosed as MM1 had neuropathologic characteristics of the MM2 thalamic form (ie, neuronal loss of the inferior olivary nucleus of the medulla). Furthermore, coexistence of biochemical features of the MM2 thalamic form also was confirmed in the identified cases. In addition, in transmission experiments using prion protein-humanized mice, the brain material from the identified case showed weak infectivity and generated characteristic abnormal prion proteins in the inoculated mice resembling those after inoculation with brain material of MM2 thalamic form. Taken together, these results show that the co-occurrence of MM1 and MM2 thalamic form is a novel entity of sporadic Creutzfeldt-Jakob disease prion strain co-occurrence. The present study raises the possibility that the co-occurrence of the MM2 thalamic form might have been overlooked so far because of the scarcity of abnormal prion protein accumulation and restricted neuropathology.
• Ganglioside synthase knock-out reduces prion disease incubation time in mouse models.

  Kobayashi A, Qi Z, Shimazaki T, Munesue Y, Miyamoto T, Isoda N, Sawa H, Aoshima K, Kimura T, Mohri S, Kitamoto T, Yamashita T, Miyoshi I

  The American journal of pathology 189 3 677 - 686 2019年03月 [査読有り][通常論文]
   

  Localization of the abnormal and normal isoforms of prion proteins to detergent-resistant membrane microdomains, lipid rafts, is important for the conformational conversion. Lipid rafts are enriched in sialic acid-containing glycosphingolipids (namely, gangliosides). Alteration in the ganglioside composition of lipid rafts can affect the localization of lipid raft-associated proteins. To investigate the role of gangliosides in the pathogenesis of prion diseases, we performed intracerebral transmission study of a scrapie prion strain Chandler and a Gerstmann-Sträussler-Scheinker syndrome prion strain Fukuoka-1 using various knockout mouse strains ablated with ganglioside synthase gene (ie, GD2/GM2 synthase, GD3 synthase, or GM3 synthase). After challenge with the Chandler strain, GD2/GM2 synthase knockout mice showed 20% reduction of incubation time, reduced prion protein deposition in the brain with attenuated glial reactions, and reduced localization of prion proteins to lipid rafts. These results raise the possibility that the gangliosides may have an important role in prion disease pathogenesis by affecting the localization of prion proteins to lipid rafts.
• Stat3 is indispensable for damage‐induced crypt regeneration but not for Wnt‐driven intestinal tumorigenesis

  Hiroko Oshima, Sau-Yee Kok, Mizuho Nakayama, Kazuhiro Murakami, Dominic Chih-Cheng Voon, Takashi Kimura, Masanobu Oshima

  The FASEB Journal 33 2 1873 - 1886 2019年02月 [査読有り]
   

  Signal transducer and activator of transcription 3 (Stat3) has been shown to play a role in intestinal regeneration and colitis-associated colon carcinogenesis. However, the role of Stat3 in the Wnt-driven sporadic intestinal tumorigenesis remains poorly understood. We examined the roles of Stat3 in intestinal regeneration and tumorigenesis by organoid culture experiments using Stat3∆IEC mouse-derived intestinal epithelial cells in which Stat3 was disrupted. The regeneration of intestinal mucosa and organoid formation were significantly suppressed by Stat3 disruption, which was compensated by Wnt activation. Furthermore, once organoids were recovered, Stat3 was no longer required for organoid growth. These results indicate that Stat3 and Wnt signaling cooperatively protect epithelial cells at the early phase of intestinal regeneration. In contrast, intestinal tumorigenesis was not suppressed by Stat3 disruption in adenomatous polyposis coli ( Apc) Δ716 and Apc∆716 Tgfbr2∆IEC mice, thus indicating that Stat3 is not required for Wnt activation-driven intestinal tumorigenesis. Mechanistically, Itga5 and Itga6 were down-regulated by Stat3 disruption, and focal adhesion kinase (FAK) activation was also suppressed. Notably, FAK inhibitor suppressed the organoid formation of wild-type epithelial cells. These results indicate that Stat3 is indispensable for the survival of epithelial cells through the activation of integrin signaling and the downstream FAK pathway; however, it is not required for the Wnt signaling-activated normal or tumor epithelial cells.-Oshima, H., Kok, S.-Y., Nakayama, M., Murakami, K., Voon, D. C.-C., Kimura, T., Oshima, M. Stat3 is indispensable for damage-induced crypt regeneration but not for Wnt-driven intestinal tumorigenesis.
• Notch2 signal is required for the maintenance of canine hemangiosarcoma cancer stem cell-like cells

  Keisuke Aoshima, Yuki Fukui, Kevin Christian Montecillo Gulay, Ochbayar Erdemsurakh, Atsuya Morita, Atsushi Kobayashi, Takashi Kimura

  BMC Veterinary Research 14 1 301  Springer Nature America, Inc 2018年12月 [査読有り][通常論文]
   

  * Corresponding author
• Generation and validation of novel anti-bovine CD163 monoclonal antibodies ABM-1A9 and ABM-2D6

  Shimamoto Y, Nio-Kobayashi J, Watarai H, Nagano M, Saito N, Takahashi E, Higuchi H, Kobayashi A, Kimura T, Kitamura H

  Vet Immunol Immunopathol 198 6 - 13 2018年04月 [査読有り][通常論文]
  
• Detection of novel gammaherpesviruses from fruit bats in Indonesia.

  Wada Y, Sasaki M, Setiyono A, Handharyani E, Rahmadani I, Taha S, Adiani S, Latief M, Kholilullah ZA, Subangkit M, Kobayashi S, Nakamura I, Kimura T, Orba Y, Sawa H

  Journal of medical microbiology 67 3 415 - 422 2018年03月01日 [査読有り][通常論文]
   

  Bats are an important natural reservoir of zoonotic viral pathogens. We previously isolated an alphaherpesvirus in fruit bats in Indonesia, and here establish the presence of viruses belonging to other taxa of the family Herpesviridae. We screened the same fruit bat population with pan-herpesvirus PCR and discovered 68 sequences of novel gammaherpesvirus, designated 'megabat gammaherpesvirus' (MgGHV). A phylogenetic analysis of approximately 3.4 kbp of continuous MgGHV sequences encompassing the glycoprotein B gene and DNA polymerase gene revealed that the MgGHV sequences are distinct from those of other reported gammaherpesviruses. Further analysis suggested the existence of co-infections of herpesviruses in Indonesian fruit bats. Our findings extend our understanding of the infectious cycles of herpesviruses in bats in Indonesia and the phylogenetic diversity of the gammaherpesviruses.
• Development of a quick bioassay for the evaluation of transmission properties of acquired prion diseases.

  Munesue Y, Shimazaki T, Qi Z, Isoda N, Sawa H, Aoshima K, Kimura T, Mohri S, Kitamoto T, Kobayashi A

  Neuroscience letters 668 43 - 47 2018年03月 [査読有り][通常論文]
   

  Evaluation of transmission properties is important for the differential diagnosis of a subgroup of acquired Creutzfeldt-Jakob disease (CJD) with methionine homozygosity at polymorphic codon 129 of the PRNP gene, an intermediate type abnormal prion protein (PrP), and kuru plaques, denoted as acquired CJD-MMiK. The present study aimed to develop a quick evaluation system of the transmission properties of acquired CJD-MMiK. In the PrP-humanized mice intraperitoneally inoculated with brain homogenates from an acquired CJD-MMiK patient, accumulation of abnormal PrP was observed in follicular dendritic cells of the spleen at 75 days post-inoculation. The transmission properties of acquired CJD-MMiK were quite different from those of sporadic CJD with the same PRNP codon 129 genotype. Moreover, even at 14 days post-inoculation, the characteristic transmission properties of acquired CJD-MMiK could be detected. These findings suggest that the bioassay using follicular dendritic cells of the spleen, named as a FDC assay, can be an easy, time-saving, and useful method to distinguish acquired CJD-MMiK from sporadic CJD.
• Dembo-PCR technique for the detection of bovine abortion, diarrhea, and respiratory disease complex infectious agents in potential vectors and reservoirs

  Rahpaya SS, Tsuchiaka S, Kishimoto M, Oba M, Katayama Y, Nunomura Y, Kokawa S, Kimura T, Kobayashi A, Kirino Y, Okabayashi T, Nonaka N, Mekata H, Aoki H, Shiokawa M, Umetsu M, Morita T, Hasebe A, Otsu K, Asai T, Yamaguchi T, Makino S, Murata Y, Abi AJ, Omatsu T, Mizutani T

  J Vet Sci 19 3 350 - 350 2018年 [査読有り][通常論文]
   

  Bovine abortion, diarrhea, and respiratory disease complexes, caused by infectious agents, result in high and significant economic losses for the cattle industry. These pathogens are likely transmitted by various vectors and reservoirs including insects, birds, and rodents. However, experimental data supporting this possibility are scarce. We collected 117 samples and screened them for 44 bovine abortive, diarrheal, and respiratory disease complex pathogens by using Dembo polymerase chain reaction (PCR), which is based on TaqMan real-time PCR. Fifty-seven samples were positive for at least one pathogen, including bovine viral diarrhea virus, bovine enterovirus, Salmonella enterica ser. Dublin, Salmonella enterica ser. Typhimurium, and Neospora caninum; some samples were positive for multiple pathogens. Bovine viral diarrhea virus and bovine enterovirus were the most frequently detected pathogens, especially in flies, suggesting an important role of flies in the transmission of these viruses. Additionally, we detected the N. caninum genome from a cockroach sample for the first time. Our data suggest that insects (particularly flies), birds, and rodents are potential vectors and reservoirs of abortion, diarrhea, and respiratory infectious agents, and that they may transmit more than one pathogen at the same time.
• Cellular atypia is negatively correlated with immunohistochemical reactivity of CD31 and vWF expression levels in canine hemangiosarcoma.

  Aprilia MAHARANI, Keisuke AOSHIMA, Shinichi ONISHI, Kevin Christian Montecillo GULAY, Atsushi KOBAYASHI, Takashi KIMURA

  The Journal of veterinary medical science 80 2 213 - 218 2018年 [査読有り][通常論文]
   

  * Corresponding author
• The full genome sequences of 8 equine herpesvirus type 4 isolates from horses in Japan

  Satoko IZUME, Rikio KIRISAWA, Kenji OHYA, Aiko OHNUMA, Takashi KIMURA, Tsutomu OMATSU, Yukie KATAYAMA, Tetsuya MIZUTANI, Hideto FUKUSHI

  Journal of Veterinary Medical Science 79 1 206 - 212 2017年 [査読有り][通常論文]
   

  Equine herpesvirus type 4 (EHV-4) is one of the most important pathogens in horses. To clarify the key genes of the EHV-4 genome that cause abortion in female horses, we determined the whole genome sequences of a laboratory strain and 7 Japanese EHV-4 isolates that were isolated from 2 aborted fetuses and nasal swabs of 5 horses with respiratory disease. The full genome sequences and predicted amino acid sequences of each gene of these isolates were compared with of the reference EHV-4 strain NS80567 and Australian isolates that were reported in 2015. The EHV-4 isolates clustered in 2 groups which did not reflect their pathogenicity. A comparison of the predicted amino acid sequences of the genes did not reveal any genes that were associated with EHV-4-induced abortion.
• Use of Histone K-M Mutants for the Analysis of Transcriptional Regulation in Mouse Zygotes.

  Aoshima K, Kimura T, Okada Y

  Methods in molecular biology (Clifton, N.J.) 1605 259 - 270 2017年 [査読有り][通常論文]
  
• Rapid and broad detection of H5 hemagglutinin by an immunochromatographic kit using novel monoclonal antibody against highly pathogenic avian influenza virus belonging to the genetic clade 2.3.4.4.

  Nguyen LT, Nakaishi K, Motojima K, Ohkawara A, Minato E, Maruyama J, Hiono T, Matsuno K, Okamatsu M, Kimura T, Takada A, Kida H, Sakoda Y

  PloS one 12 8 e0182228  2017年 [査読有り][通常論文]
   

  Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds.
• Divergent bufavirus harboured in megabats represents a new lineage of parvoviruses.

  Sasaki M, Gonzalez G, Wada Y, Setiyono A, Handharyani E, Rahmadani I, Taha S, Adiani S, Latief M, Kholilullah ZA, Subangkit M, Kobayashi S, Nakamura I, Kimura T, Orba Y, Ito K, Sawa H

  Scientific reports 6 1 24257 - 24257 Nature Publishing Group 2016年04月 [査読有り][通常論文]
   

  Bufavirus is a recently recognized member of the genus Protoparvovirus in the subfamily Parvovirinae. It has been reported that human bufavirus was detected predominantly in patients with diarrhoea in several countries. However, little is known about bufavirus or its close relatives in nonhuman mammals. In this study, we performed nested-PCR screening and identified bufavirus from 12 megabats of Pteropus spp. in Indonesia. Furthermore, we determined nearly the full genome sequence of a novel megabat-borne bufavirus, tentatively named megabat bufavirus 1. Phylogenetic analyses showed that megabat bufavirus 1 clustered with known protoparvoviruses, including human bufavirus but represented a distinct lineage of bufavirus. Our analyses also inferred phylogenetic relationships among animal-borne bufaviruses recently reported by other studies. Recombination analyses suggested that the most common recent ancestor of megabat bufavirus 1 might have arisen from multiple genetic recombination events. These results characterized megabat bufavirus 1 as the first protoparvovirus discovered from megabats and indicates the high genetic divergence of bufavirus.
• Detection of novel polyomaviruses in fruit bats in Indonesia.

  Kobayashi S, Sasaki M, Nakao R, Setiyono A, Handharyani E, Orba Y, Rahmadani I, Taha S, Adiani S, Subangkit M, Nakamura I, Kimura T, Sawa H

  Archives of virology 160 4 1075 - 1082 4 2015年04月 [査読有り][通常論文]
  
• Detection of coronavirus genomes in Moluccan naked-backed fruit bats in Indonesia.

  Anindita PD, Sasaki M, Setiyono A, Handharyani E, Orba Y, Kobayashi S, Rahmadani I, Taha S, Adiani S, Subangkit M, Nakamura I, Sawa H, Kimura T

  Archives of virology 160 4 1113 - 1118 4 2015年04月 [査読有り][通常論文]
   

  Bats have been shown to serve as natural reservoirs for numerous emerging viruses including severe acute respiratory syndrome coronavirus (SARS-CoV). In the present study, we report the discovery of bat CoV genes in Indonesian Moluccan naked-backed fruit bats (Dobsonia moluccensis). A partial RNA-dependent RNA polymerase gene sequence was detected in feces and tissues samples from the fruit bats, and the region between the RdRp and helicase genes could also be amplified from fecal samples. Phylogenetic analysis suggested that these bat CoVs are related to members of the genus Betacoronavirus.
• Autophagy inhibits viral genome replication and gene expression stages in West Nile virus infection.

  Kobayashi S, Orba Y, Yamaguchi H, Takahashi K, Sasaki M, Hasebe R, Kimura T, Sawa H

  Virus research 191 83 - 91 Elsevier 2014年10月 [査読有り][通常論文]
   

  Autophagy is a lysosomal degradation pathway that is implicated in many viral infections. However, its role in West Nile virus (WNV) infection remains controversial. In the present study, we examined the relationship between WNV infection and autophagy in infected cells. We demonstrated that LC3-II expression, a molecular marker for autophagosomal membranes, was enhanced in WNV-infected cells 6 h post-infection. LC3-II expression was further enhanced in WNV-inoculated cells when treated with a lysosomal protease inhibitor. Meanwhile, WNV replication in cells lacking Atg5, an essential factor for autophagy, was increased compared with replication in wild-type cells. In addition, WNV replication was inhibited in cells lacking Atg5 when they were transfected with an ATG5 expression plasmid. These results suggest an antiviral role for autophagy in WNV-infected cells. We also examined which viral replication stages were affected by autophagy by using a Tat-beclin 1 peptide to induce autophagy and pseudo-infectious WNV reporter virus particles (WNV-RVPs) that monitor viral genome replication and gene expression stages via GFP expression. We found that autophagy induction in HeLa cells by Tat-beclin 1 peptide 3 h after WNV inoculation inhibited viral replication, and GFP expression was significantly inhibited in wild-type cells when compared with cells lacking Atg5. Taken together, these results suggest that autophagy is induced by WNV infection, and that this induction inhibits WNV replication at the viral genome replication and gene expression stages. (C) 2014 Elsevier B.V. All rights reserved.
• Isolation and characterization of a novel alphaherpesvirus in fruit bats.

  Sasaki Michihito, Setiyono Agus, Handharyani Ekowati, Kobayashi Shintaro, Rahmadani Ibenu, Taha Siswatiana, Adiani Sri, Subangkit Mawar, Nakamura Ichiro, Sawa Hirofumi, Kimura Takashi

  Journal of virology 88 17 9819 - 9829 American Society for Microbiology 2014年09月01日 [査読有り]
   

  Bats are known to harbor emerging RNA viruses. Recent studies have used high-throughput sequencing technology to identify various virus species, including DNA viruses that are harbored by bats; however, little is known about the nature of these potentially novel viruses. Here, we report the characterization of a novel herpesvirus isolated from an Indonesian pteropodid bat. The virus, tentatively named fruit bat alphaherpesvirus 1 (FBAHV1), has a double-stranded DNA genome of 149,459 bp. The phylogenetic analyses suggested that FBAHV1 is phylogenetically grouped with simplexviruses within the subfamily Alphaherpesvirinae. Inoculation of FBAHV1 into laboratory mice caused a lethal infection. Virus infection was observed in lung, liver, and brain tissue. Serological and PCR screening revealed that fruit bats infected with FBAHV1 or its related virus are widely distributed in Indonesia. The identification of FBAHV1 makes a considerable contribution to our understanding of simplexviruses associated with bats.
• Role of the C-terminal region of vervet monkey polyomavirus 1 VP1 in virion formation.

  Yamaguchi H, Kobayashi S, Maruyama J, Sasaki M, Takada A, Kimura T, Sawa H, Orba Y

  The Journal of veterinary medical science / the Japanese Society of Veterinary Science 76 5 637 - 644 5 2014年05月 [査読有り][通常論文]
   

  Recently, we detected novel vervet monkey polyomavirus 1 (VmPyV) in a vervet monkey. Among amino acid sequences of major capsid protein VP1s of other polyomaviruses, VmPyV VP1 is the longest with additional amino acid residues in the C-terminal region. To examine the role of VmPyV VP1 in virion formation, we generated virus-like particles (VLPs) of VmPyV VP1, because VLP is a useful tool for the investigation of the morphological characters of polyomavirus virions. After the full-length VmPyV VP1 was subcloned into a mammalian expression plasmid, the plasmid was transfected into human embryonic kidney 293T (HEK293T) cells. Thereafter, VmPyV VLPs were purified from the cell lysates of the transfected cells via sucrose gradient sedimentation. Electron microscopic analyses revealed that VmPyV VP1 forms VLPs with a diameter of approximately 50 nm that are exclusively localized in cell nuclei. Furthermore, we generated VLPs consisting of the deletion mutant VmPyV VP1 (ΔC VP1) lacking the C-terminal 116 amino acid residues and compared its VLP formation efficiency and morphology to those of VLPs from wild-type VmPyV VP1 (WT VP1). WT and ΔC VP1 VLPs were similar in size, but the number of ΔC VP1 VLPs was much lower than that of WT VP1 VLPs in VP1-expressing HEK293T cells. These results suggest that the length of VP1 is unrelated to virion morphology; however, the C-terminal region of VmPyV VP1 affects the efficiency of its VLP formation.
• Molecular epidemiology of paramyxoviruses in frugivorous Eidolon helvum bats in Zambia.

  Muleya W, Sasaki M, Orba Y, Ishii A, Thomas Y, Nakagawa E, Ogawa H, Hang'ombe B, Namangala B, Mweene A, Takada A, Kimura T, Sawa H

  The Journal of veterinary medical science 76 4 611 - 614 JAPANESE SOCIETY OF VETERINARY SCIENCE 2014年04月 [査読有り][通常論文]
   

  In this study, we describe the detection of novel paramyxoviruses from the Eidolon helvum species of fruit bats. We extracted RNA from 312 spleen samples from bats captured in Zambia over a period of 4 years (2008–2011). Semi-nested RT-PCR detected a total of 25 (8%) positive samples for paramyxoviruses which were then directly sequenced and analyzed using phylogenetic analysis. Among the positive samples, seven novel paramyxoviruses were detected. Five viruses were closely related to the genus Henipavirus, while two viruses were related to the unclassified Bat paramyxoviruses from Ghana and Congo Brazzaville. Our study identified novel Henipavirus-related and unrelated viruses using RT-PCR in fruit bats from Kansaka National Park and indicated the presence of similar Bat paramyxoviruses originating from wide geographic areas, suggesting the ability of bats to harbor and transmit viruses. The presence of these viruses in fruit bats might pose a public health risk.
• Molecular epidemiology of paramyxoviruses in Zambian wild rodents and shrews.

  Sasaki M, Muleya W, Ishii A, Orba Y, Hang'ombe BM, Mweene AS, Moonga L, Thomas Y, Kimura T, Sawa H

  The Journal of general virology 95 2 325 - 330 Pt 2 2014年02月 [査読有り][通常論文]
   

  Rodents and shrews are known to harbour various viruses. Paramyxoviruses have been isolated from Asian and Australian rodents, but little is known about them in African rodents. Recently, previously unknown paramyxovirus sequences were found in South African rodents. To date, there have been no reports related to the presence and prevalence of paramyxoviruses in shrews. We found a high prevalence of paramyxoviruses in wild rodents and shrews from Zambia. Seminested reverse transcription-PCR assays were used to detect paramyxovirus RNA in 21% (96/ 462) of specimens analysed. Phylogenetic analysis revealed that these viruses were novel paramyxoviruses and could be classified as morbillivirus- and henipavirus-related viruses, and previously identified rodent paramyxovirus-related viruses. Our findings suggest the circulation of previously unknown paramyxoviruses in African rodents and shrews, and provide new information regarding the geographical distribution and genetic diversity of paramyxoviruses.
• Full genome sequences of zebra-borne equine herpesvirus type 1 isolated from zebra, Onager and Thomson’s gazelle

  Xiaoqin Guo, Satoko Izume, Ayaka Okada, Kenji Ohya, Kenji Ohya, Takashi Kimura, Hideto Fukushi, Hideto Fukushi

  Journal of Veterinary Medical Science 76 9 1309 - 1312 JAPANESE SOCIETY OF VETERINARY SCIENCE 2014年01月 [査読有り][通常論文]
   

  A strain of equine herpesvirus type 1 (EHV-1) was isolated from zebra. This strain, called “zebra-borne EHV-1”, was also isolated from an onager and a gazelle in zoological gardens in U.S.A. The full genome sequences of the 3 strains were determined. They shared 99% identities with each other, while they shared 98% and 95% identities with the horse derived EHV-1 and equine herpesvirus type 9, respectively. Sequence data indicated that the EHV-1 isolated from a polar bear in Germany is one of the zebra-borne EHV-1 and not a recombinant virus. These results indicated that zebra-borne EHV-1 is a subtype of EHV-1.
• Establishment of tracking system for West Nile virus entry and evidence of microtubule involvement in particle transport.

  Makino Y, Suzuki T, Hasebe R, Kimura T, Maeda A, Takahashi H, Sawa H

  Journal of virological methods 195 250 - 257 2014年01月 [査読有り][通常論文]
  
• Characterization of Japanese encephalitis virus infection in an immortalized mesencephalic cell line, CSM14.1.

  Kimura T, Okumura M, Kim E, Sasaki M, Orba Y, Sawa H

  Microbiology and immunology 57 10 723 - 731 10 2013年10月 [査読有り][通常論文]
  
• ラット中脳由来神経細胞株CSM14.1における日本脳炎ウイルス感染様式

  木村 享史, 奥村 恵, 金 恩美, 佐々木 道仁, 大場 靖子, 澤 洋文

  日本獣医学会学術集会講演要旨集 156回 214 - 214 (公社)日本獣医学会 2013年08月
• オートファジーによるウエストナイルウイルス増殖抑制機構の解明

  小林 進太郎, 大場 靖子, 山口 宏樹, 佐々木 道仁, 長谷部 理絵, 木村 享史, 澤 洋文

  日本獣医学会学術集会講演要旨集 156回 267 - 267 (公社)日本獣医学会 2013年08月
• インドネシア共和国に生息するオオコウモリから分離した新規ヘルペスウイルスの性状解析

  佐々木 道仁, Agus Setiyono, Ekowati Handharyani, 中村 一郎, 澤 洋文, 木村 享史

  日本獣医学会学術集会講演要旨集 156回 283 - 283 (公社)日本獣医学会 2013年08月
• Identification of a novel polyomavirus from vervet monkeys in Zambia

  Hiroki Yamaguchi, Shintaro Kobayashi, Akihiro Ishii, Hirohito Ogawa, Ichiro Nakamura, Ladslav Moonga, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa, Yasuko Orba

  Journal of General Virology 94 6 1357 - 1364 2013年06月01日 [査読有り][通常論文]
   

  To examine polyomavirus (PyV) infection in wildlife, we investigated the presence of PyVs in Zambia with permission from the Zambia Wildlife Authority. We analysed 200 DNA samples from the spleens and kidneys (n5100 each) of yellow baboons and vervet monkeys (VMs) (n550 each). We detected seven PyV genome fragments in 200 DNA samples using a nested broadspectrum PCR method, and identified five full-length viral genomes using an inverse PCR method. Phylogenetic analysis of virally encoded proteins revealed that four PyVs were closely related to either African green monkey PyV or simian agent 12. Only one virus detected from a VM spleen was found to be related, with relatively low nucleotide sequence identity (74 %), to the chimpanzee PyV, which shares 48% nucleotide sequence identity with the human Merkel cell PyV identified from Merkel cell carcinoma. The obtained entire genome of this virus was 5157 bp and had large T- and small t-antigens, and VP1 and VP2 ORFs. This virus was tentatively named vervet monkey PyV 1 (VmPyV1) as a novel PyV. Comparison with other PyVs revealed that VmPyV1, like chimpanzee PyV, had a longer VP1 ORF. To examine whether the VmPyV1 genome could produce viral proteins in cultured cells, the whole genome was transfected into HEK293T cells. We detected VP1 protein expression in the transfected HEK293T cells by immunocytochemical and immunoblot analyses. Thus, we identified a novel PyV genome from VM spleen. © 2013 SGM.
• Downregulation of miRNA-31 induces taxane resistance in ovarian cancer cells through increase of receptor tyrosine kinase MET

  T. Mitamura, H. Watari, L. Wang, H. Kanno, M. K. Hassan, M. Miyazaki, Y. Katoh, T. Kimura, M. Tanino, H. Nishihara, S. Tanaka, N. Sakuragi

  Oncogenesis 2 2013年 [査読有り][通常論文]
   

  Ovarian cancer is one of the most aggressive female reproductive tract tumors. Paclitaxel (PTX) is widely used for the treatment of ovarian cancer. However, ovarian cancers often acquire chemotherapeutic resistance to this agent. We investigated the mechanism of chemoresistance by analysis of microRNAs using the ovarian cancer cell line KFr13 and its PTX-resistant derivative (KFr13Tx). We found that miR-31 was downregulated in KFr13Tx cells, and that re-introduction of miR31 re-sensitized them to PTX both in vitro and in vivo. miR-31 was found to bind to the 3′-UTR of mRNA of MET, and the decrease in MET correlated to higher sensitivity to PTX. Furthermore, co-treatment of KFr13Tx cells with MET inhibitors sensitized the tumor cells to PTX both in vitro and in vivo. In addition, lower levels of miR31 and higher expression of MET in human ovarian cancer specimens were significantly correlated with PTX chemoresistance and poor prognosis. This study demonstrated miR31-dependent regulation of MET for chemoresistance of ovarian cancer, raising the possibility that combination therapy with a MET inhibitor and PTX will increase PTX efficacy. © 2013 Macmillan Publishers Limited All rights reserved.
• Heterosubtypic antiviral activity of hemagglutinin-specific antibodies induced by intranasal immunization with inactivated influenza viruses in mice.

  Muramatsu M, Yoshida R, Miyamoto H, Tomabechi D, Kajihara M, Maruyama J, Kimura T, Manzoor R, Ito K, Takada A

  PloS one 8 8 e71534  Public Library of Science 2013年 [査読有り][通常論文]
   

  Influenza A virus subtypes are classified on the basis of the antigenicity of their envelope glycoproteins, hemagglutinin (HA; H1-H17) and neuraminidase. Since HA-specific neutralizing antibodies are predominantly specific for a single HA subtype, the contribution of antibodies to the heterosubtypic immunity is not fully understood. In this study, mice were immunized intranasally or subcutaneously with viruses having the H1, H3, H5, H7, H9, or H13 HA subtype, and cross-reactivities of induced IgG and IgA antibodies to recombinant HAs of the H1-H16 subtypes were analyzed. We found that both subcutaneous and intranasal immunizations induced antibody responses to multiple HAs of different subtypes, whereas IgA was not detected remarkably in mice immunized subcutaneously. Using serum, nasal wash, and trachea-lung wash samples of H9 virus-immunized mice, neutralizing activities of cross-reactive antibodies were then evaluated by plaque-reduction assays. As expected, no heterosubtypic neutralizing activity was detected by a standard neutralization test in which viruses were mixed with antibodies prior to inoculation into cultured cells. Interestingly, however, a remarkable reduction of plaque formation and extracellular release of the H12 virus, which was bound by the H9-induced cross-reactive antibodies, was observed when infected cells were subsequently cultured with the samples containing HA-specific cross-reactive IgA. This heterosubtypic plaque reduction was interfered when the samples were pretreated with anti-mouse IgA polyclonal serum. These results suggest that the majority of HA-specific cross-reactive IgG and IgA antibodies produced by immunization do not block cellular entry of viruses, but cross-reactive IgA may have the potential to inhibit viral egress from infected cells and thus to play a role in heterosubtypic immunity against influenza A viruses.
• Cysteine residues in the major capsid protein, Vp1, of the JC virus are important for protein stability and oligomer formation.

  Kobayashi S, Suzuki T, Igarashi M, Orba Y, Ohtake N, Nagakawa K, Niikura K, Kimura T, Kasamatsu H, Sawa H

  PloS one 8 10 e76668  10 2013年 [査読有り][通常論文]
   

  The capsid of the human polyomavirus JC virus (JCV) consists of 72 pentameric capsomeres of a major structural protein, Vp1. The cysteine residues of the related Vp1 of SV40 are known to contribute to Vp1 folding, pentamer formation, pentamer-pentamer contacts, and capsid stabilization. In light of the presence of a slight structural difference between JCV Vp1 and SV40 counterpart, the way the former folds could be either different from or similar to the latter. We found a difference: an important contribution of Vp1 cysteines to the formation of infectious virions, unique in JCV and absent in SV40. Having introduced amino acid substitution at each of six cysteines (C42, C80, C97, C200, C247, and C260) in JCV Vp1, we found that, when expressed in HeLa cells, the Vp1 level was decreased in C80A and C247A mutants, and remained normal in the other mutants. Additionally, the C80A and C247A Vp1-expressing cell extracts did not show the hemagglutination activity characteristic of JCV particles. The C80A and C247A mutant Vp1s were found to be less stable than the wild-type Vp1 in HeLa cells. When produced in a reconstituted in vitro protein translation system, these two mutant proteins were stable, suggesting that some cellular factors were responsible for their degradation. As determined by their sucrose gradient sedimentation profiles, in vitro translated C247A Vp1 formed pentamers, but in vitro translated C80A Vp1 was entirely monomeric. When individually incorporated into the JCV genome, the C80A and C247A mutants, but not the other Vp1 cysteine residues mutants, interfered with JCV infectivity. Furthermore, the C80A, but not the C247A, mutation prevented the nuclear localization of Vp1 in JCV genome transfected cells. These findings suggest that C80 of JCV Vp1 is required for Vp1 stability and pentamer formation, and C247 is involved in capsid assembly in the nucleus.
• Human parainfluenza virus type 3 in wild nonhuman primates, Zambia.

  Sasaki M, Ishii A, Orba Y, Thomas Y, Hang'ombe BM, Moonga L, Mweene AS, Ogawa H, Nakamura I, Kimura T, Sawa H

  Emerging infectious diseases 19 9 1500 - 1503 Centers for Disease Control and Prevention 2013年 [査読有り][通常論文]
   

  Human parainfluenza virus type 3 (HPIV3) genome was detected in 4 baboons in Zambia. Antibody for HPIV3 was detected in 13 baboons and 6 vervet monkeys in 2 distinct areas in Zambia. Our findings suggest that wild nonhuman primates are susceptible to HPIV3 infection.
• Population genetic analysis and sub-structuring of Theileria parva in the northern and eastern parts of Zambia

  Walter Muleya, Boniface Namangala, Martin Simuunza, Ryo Nakao, Noboru Inoue, Takashi Kimura, Kimihito Ito, Chihiro Sugimoto, Hirofumi Sawa

  PARASITES & VECTORS 5 255  2012年11月 [査読有り][通常論文]
   

  Background: Theileriosis, caused by Theileria parva, is an economically important disease in Africa. It is a major constraint to the development of the livestock industry in some parts of eastern, central and southern Africa. In Zambia, theileriosis causes losses of up to 10,000 cattle annually. Methods: Cattle blood samples were collected for genetic analysis of Theileria parva from Isoka and Petauke districts in Zambia. Microsatellite analysis was then performed on all Theileria parva positive samples for PCR using a panel of 9 microsatellite markers. Microsatellite data was analyzed using microsatellite toolkit, GenAlEx ver. 6, Fstat ver. 2.9.3.2, and LIAN computer softwares. Results: The combined percentage of positive samples in both districts determined by PCR using the p104 gene primers was 54.9% (95% CI: 46.7 - 63.1%, 78/142), while in each district, it was 44.8% (95% CI: 34.8 - 54.8%) and 76.1% (95% CI = 63.9 - 88.4%) for Isoka and Petauke districts, respectively. We analyzed the population genetic structure of Theileria parva from a total of 61 samples (33 from Isoka and 28 from Petauke) using a panel of 9 microsatellite markers encompassing the 4 chromosomes of Theileria parva. Wright's F index (F-ST = 0.178) showed significant differentiation between the Isoka and Petauke populations. Linkage disequilibrium was observed when populations from both districts were treated as a single population. When analyzed separately, linkage disequilibrium was observed in Kanyelele and Kalembe areas in Isoka district, Isoka district overall and in Petauke district. Petauke district had a higher multiplicity of infection than Isoka district. Conclusion: Population genetic analyses of Theileria parva from Isoka and Petauke districts showed a low level of genotype exchange between the districts, but a high level of genetic diversity within each district population, implying genetic and geographic sub-structuring between the districts. The sub-structuring observed, along with the lack of panmixia in the populations, could have been due to low transmission levels at the time of sampling. However, the Isoka population was less diverse than the Petauke population.
• Molecular detection of a novel paramyxovirus in fruit bats from Indonesia

  Michihito Sasaki, Agus Setiyono, Ekowati Handharyani, Ibenu Rahmadani, Siswatiana Taha, Sri Adiani, Mawar Subangkit, Hirofumi Sawa, Ichiro Nakamura, Takashi Kimura

  VIROLOGY JOURNAL 9 240  2012年10月 [査読有り][通常論文]
   

  Background: Fruit bats are known to harbor zoonotic paramyxoviruses including Nipah, Hendra, and Menangle viruses. The aim of this study was to detect the presence of paramyxovirus RNA in fruit bats from Indonesia. Methods: RNA samples were obtained from the spleens of 110 fruit bats collected from four locations in Indonesia. All samples were screened by semi-nested broad spectrum reverse transcription PCR targeting the paramyxovirus polymerase (L) genes. Results: Semi-nested reverse transcription PCR detected five previously unidentified paramyxoviruses from six fruit bats. Phylogenetic analysis showed that these virus sequences were related to henipavirus or rubulavirus. Conclusions: This study indicates the presence of novel paramyxoviruses among fruit bat populations in Indonesia.
• Role of JC virus agnoprotein in virion formation

  Tadaki Suzuki, Shingo Semba, Yuji Sunden, Yasuko Orba, Shintaro Kobayashi, Kazuo Nagashima, Takashi Kimura, Hideki Hasegawa, Hirofumi Sawa

  MICROBIOLOGY AND IMMUNOLOGY 56 9 639 - 646 2012年09月 [査読有り][通常論文]
   

  JC virus (JCV) belongs to the polyomavirus family of double-stranded DNA viruses and causes progressive multifocal leukoencephalopathy in humans. JCV encodes early proteins (large T antigen, small T antigen, and T' antigen) and four late proteins (agnoprotein, and three viral capsid proteins, VP1, VP2, and VP3). In the current study, a novel function for JCV agnoprotein in the morphogenesis of JC virion particles was identified. It was found that mature virions of agnoprotein-negative JCV are irregularly shaped. Sucrose gradient sedimentation and cesium chloride gradient ultracentrifugation analyses revealed that the particles of virus lacking agnoprotein assemble into irregularly sized virions, and that agnoprotein alters the efficiency of formation of VP1 virus-like particles. An in vitro binding assay and immunocytochemistry revealed that agnoprotein binds to glutathione S-transferase fusion proteins of VP1 and that some fractions of agnoprotein colocalize with VP1 in the nucleus. In addition, gel filtration analysis of formation of VP1-pentamers revealed that agnoprotein enhances formation of these pentamers by interacting with VP1. The present findings suggest that JCV agnoprotein plays a role, similar to that of SV40 agnoprotein, in facilitating virion assembly.
• Bilateral Segmental Aplasia with Unilateral Uterine Horn Torsion in a Pomeranian Bitch

  Kensuke Nakamura, Masahiro Yamasaki, Tomohiro Osaki, Hiroshi Ohta, Noboru Sasaki, Keisuke Aoshima, Takashi Kimura, Mitsuyoshi Takiguchi

  JOURNAL OF THE AMERICAN ANIMAL HOSPITAL ASSOCIATION 48 5 327 - 330 2012年09月 [査読有り][通常論文]
   

  Bilateral segmental aplasia of the uterine horns with unilateral pyometra and uterine horn torsion were diagnosed in a Pomeranian bitch that presented with chronic abdominal distension and an acute onset of anorexia and lethargy. Because radiographic and ultrasonographic findings revealed the presence of markedly enlarged bilateral uterine horns filled with fluid in the caudal abdomen, a tentative diagnosis of either pyometra or hydrometra with uterine horn torsion was made. Exploratory laparotomy showed bilateral, segmentally distended uterine horns with unilateral uterine horn torsion. Ovariohysterectomy was performed, and bilateral segmental aplasia of the uterine horns with the development of unilateral uterine horn torsion was diagnosed histopathologically. To the authors' knowledge, this is the first report of uterine horn torsion in conjunction with segmental aplasia of the uterine horn in a bitch. (J Am Anim Hosp Assoc 2012; 48:327-330. DOI 10.5326/JAAHA-MS-5771)
• Pathological Examination of Lung Tissues in Influenza A Virus-Infected Mice

  Nilton Akio Muto, Yuji Sunden, Tomoe Hattori, Daisuke Fujikura, Yosuke Nakayama, Tadaaki Miyazaki, Mitsuo Maruyama, Takashi Kimura, Hirofumi Sawa

  JAPANESE JOURNAL OF INFECTIOUS DISEASES 65 5 383 - 391 2012年09月 [査読有り][通常論文]
   

  This study examined pathological changes in the lung tissues of young and aged mice infected with influenza virus. Young mice inoculated with influenza virus showed body weight loss at 4 days post-infection (dpi), meanwhile body weight decrease started from 9 dpi in the aged mice. We histopathologically examined the lungs of these mice. Immunohistochemical examination revealed that viral antigen-positive bronchiolar and alveolar epithelial cell numbers at 3 dpi were significantly higher in young mice than in the aged ones. Further, viral antigen-positive cells were observed at 9 dpi in the aged mice, but not in the young ones. Diffuse and severe bronchointerstitial pneumonia characterized by the accumulation of polymorphonuclear leukocytes (PMNs) was observed in young mice at 6 dpi. Histopathological changes in the aged mice were milder than those in the young mice. Moreover, T cell and macrophage accumulation in the lungs was significantly higher in the young mice than in the aged mice at 9 dpi. These results suggest that there may be a correlation between the relatively low level of infiltration of PMNs, macrophages, and T lymphocytes and the delayed body weight loss and longer lasting infections observed in the lungs of the aged mice. These findings provide detailed insights into the age-specific course of infection in young and aged populations with associated differences in lung pathology.
• Accumulation of ubiquitinated proteins is related to West Nile virus-induced neuronal apoptosis

  Shintaro Kobayashi, Yasuko Orba, Hiroki Yamaguchi, Takashi Kimura, Hirofumi Sawa

  NEUROPATHOLOGY 32 4 398 - 405 2012年08月 [査読有り][通常論文]
   

  West Nile virus (WNV) belongs to the Flaviviridae family of viruses and has emerged as a significant cause of viral encephalitis in humans, animals and birds. It has been reported that WNV replication directly induces neuronal injury, followed by neuronal cell death proven as apoptosis. Therefore, it is important to understand the mechanism of neuronal apoptosis caused by this virus to develop strategies to control its pathogenicity. Accumulation of ubiquitinated abnormal proteins has been reported to be associated with neuronal apoptosis in some pathological conditions. A lot of cellular stresses prevent cellular protein quality control mechanisms, resulting in the accumulation of ubiquitinated abnormal proteins. To obtain a better understanding of the mechanisms of WNV-induced neuronal apoptosis, we evaluated the accumulation of ubiquitinated proteins in the WNV-infected neuronal cells. We have observed that WNV infection caused massive neuronal injury in the brains of mice. Viral antigen was detected in the neuronal cytoplasm of the cells exhibiting neuronal apoptosis. Notably, ubiquitinated proteins were detected in WNV-infected neuronal cells. In addition, accumulation of ubiquitinated proteins was markedly enhanced in mouse neuroblastoma, Neuro-2a cells after WNV infection. Our histopathological and in vitro studies suggest that accumulation of ubiquitinated proteins in neuronal cells might be associated with neuronal apoptosis caused by WNV infection.
• Inhibitory effects of an M2-specific monoclonal antibody on different strains of influenza A virus

  Nilton Akio Muto, Reiko Yoshida, Tadaki Suzuki, Shintaro Kobayashi, Hiroichi Ozaki, Daisuke Fujikura, Rashid Manzoor, Mieko Muramatsu, Ayato Takada, Takashi Kimura, Hirofumi Sawa

  JAPANESE JOURNAL OF VETERINARY RESEARCH 60 2-3 71 - 83 2012年08月 [査読有り][通常論文]
   

  New approaches to the treatment of influenza have been designed based on the highly conserved antigenicity of the M2 envelope protein among influenza A virus strains. The present study examined the anti-viral activities of an anti-M2 ectodomain (M2e) monoclonal antibody (clone rM2ss23), which binds to the M2 proteins of the influenza A virus strains A/Aichi/2/68 (H3N2) (Aichi) and A/PR/8/34 (H1N1) (PR8). The results showed that rM2ss23 bound to both Aichi and PR8 M2 proteins expressed on the cell surface. While the antibody did not prevent virus entry into cells, it significantly inhibited plaque formation by the Aichi strain in a dose-dependent manner when infected cells were cultured in the presence of the antibody. By contrast, the growth of PR8 (H1N1) was not affected by the antibody. A reverse genetics approach revealed that the inhibitory effect of rM2ss23 on the Aichi virus was abolished by replacing the genes encoding the HA and/or M proteins with those of the PR8 strain. These results suggest that rM2ss23 prevents virus release from infected cells and further suggest that the mechanisms underlying the virus budding mediates by HA and M2 proteins might differ between the Aichi and PR8 strains.
• Molecular epidemiology and a loop-mediated isothermal amplification method for diagnosis of infection with rabies virus in Zambia

  Walter Muleya, Boniface Namangala, Aaron Mweene, Luke Zulu, Paul Fandamu, Douglas Banda, Takashi Kimura, Hirofumi Sawa, Akihiro Ishii

  VIRUS RESEARCH 163 1 160 - 168 2012年01月 [査読有り][通常論文]
   

  The National Livestock Epidemiology and Information Center (NALEIC) in Zambia reported over 132 cases of canine rabies diagnosed by the direct fluorescent antibody test (DFAT) from 2004 to 2009. In this study, the lineage of rabies virus (RABV) in Zambia was determined by phylogenetic analysis of the nucleoprotein (N) and glycoprotein (G) gene sequences. Total RNA was extracted from 87-DFAT brain specimens out of which only 35 (40%) were positive on nested reverse transcription polymerase chain reaction (RT-PCR) for each gene, and 26 being positive for both genes. Positive specimens for the N (n =33) and G (n = 35) genes were used for phylogenetic analysis. Phylogenetic analysis of the N gene showed two phylogenetic clusters in Zambia belonging to the Africa 1b lineage present in eastern and southern Africa. While one cluster exclusively comprised Zambian strains, the other was more heterogeneous regarding the RABV origins and included strains from Tanzania, Mozambique and Zambia. Phylogenetic analysis of the G gene revealed similar RABV strains in different hosts and regions of Zambia. We designed primers for reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay from the consensus sequence of the N gene in an attempt to improve the molecular diagnosis of RABV in Zambia. The specificity and reproducibility of the RI-LAMP assay was confirmed with actual clinical specimens. Therefore, the RI-LAMP assay presented in this study may prove to be useful for routine diagnosis of rabies in Zambia. (C) 2011 Elsevier B.V. All rights reserved.
• Single Amino Acid Residue in the A2 Domain of Major Histocompatibility Complex Class I Is Involved in the Efficiency of Equine Herpesvirus-1 Entry

  Michihito Sasaki, Eunmi Kim, Manabu Igarashi, Kimihito Ito, Rie Hasebe, Hideto Fukushi, Hirofumi Sawa, Takashi Kimura

  JOURNAL OF BIOLOGICAL CHEMISTRY 286 45 39370 - 39378 2011年11月 [査読有り][通常論文]
   

  Equine herpesvirus-1 (EHV-1), an alpha-herpesvirus of the family Herpesviridae, causes respiratory disease, abortion, and encephalomyelitis in horses. EHV-1 utilizes equine MHC class I molecules as entry receptors. However, hamster MHC class I molecules on EHV-1-susceptible CHO-K1 cells play no role in EHV-1 entry. To identify the MHC class I molecule region that is responsible for EHV-1 entry, domain exchange and site-directed mutagenesis experiments were performed, in which parts of the extracellular region of hamster MHC class I (clone C5) were replaced with corresponding sequences from equine MHC class I (clone A68). Substitution of alanine for glutamine at position 173 (Q173A) within the alpha 2 domain of the MHC class I molecule enabled hamster MHC class I C5 to mediate EHV-1 entry into cells. Conversely, substitution of glutamine for alanine at position 173 (A173Q) in equine MHC class I A68 resulted in loss of EHV-1 receptor function. Equine MHC class I clone 3.4, which possesses threonine at position 173, was unable to act as an EHV-1 receptor. Substitution of alanine for threonine at position 173 (T173A) enabled MHC class I 3.4 to mediate EHV-1 entry into cells. These results suggest that the amino acid residue at position 173 of the MHC class I molecule is involved in the efficiency of EHV-1 entry.
• ウマMHCクラスI遺伝子導入マウスのウマヘルペスウイルス1型に対する感受性

  木村 享史, 佐々木 道仁, 金 亨振, 福士 秀人, 澤 洋文

  日本獣医学会学術集会講演要旨集 152回 184 - 184 (公社)日本獣医学会 2011年08月
• Unique heparan sulfate from shrimp heads exhibits a strong inhibitory effect on infections by dengue virus and Japanese encephalitis virus

  Jiancheng Chen, Shuhei Yamada, Yoshiki Hama, Ajaya Kumar Shetty, Takanari Kobayashi, Hiroshi Oda, Kosuke Seiki, Eunmi Kim, Takashi Kimura, Naonori Takahashi, Kazuya I. P. J. Hidari, Takashi Suzuki, Yasuo Suzuki, Kazuyuki Sugahara

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 412 1 136 - 142 2011年08月 [査読有り][通常論文]
   

  The structure and biological activities of a highly sulfated heparan sulfate (HS) extracted from shrimp (Penaeus brasiliensis) heads were characterized. Structurally the shrimp HS was more heterogenous than heparin, although it is still highly sulfated. The molecular mass of the shrimp HS preparation was determined to be 32.3 kDa by gel filtration HPLC. Analysis by surface plasmon resonance demonstrated that various growth/differentiation factors specifically bound to the shrimp HS with comparable affinity. Notably, the shrimp HS had a greater inhibitory effect against infections by dengue virus type 2 as well as Japanese encephalitis virus than heparin. Experiments on anticoagulant activity indicated that the shrimp HS exhibited significant anti-thrombin activity, but less than the commercial heparin. Hence, the HS preparation from shrimp heads, an industrial waste, is a prospective agent for a variety of clinical applications. (C) 2011 Elsevier Inc. All rights reserved.
• Paradoxical effects of chondroitin sulfate-E on Japanese encephalitis viral infection

  Eunmi Kim, Megumi Okumura, Hirofumi Sawa, Tadaaki Miyazaki, Daisuke Fujikura, Shuhei Yamada, Kazuyuki Sugahara, Michihito Sasaki, Takashi Kimura

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 409 4 717 - 722 2011年06月 [査読有り][通常論文]
   

  Glycosaminoglycans (GAGs) have diverse functions in the body and are involved in viral infection. The purpose of this study was to evaluate the possible roles of the E-disaccharide units GlcA beta 1-3Gal-NAc(4,6-O-disulfate) of chondroitin sulfate (CS), a GAG involved in neuritogenesis and neuronal migration, in Japanese encephalitis virus (JEV) infection. Soluble CS-E (sCS-E) derived from squid cartilage inhibited JEV infection in African green monkey kidney-derived Vero cells and baby hamster kidney-derived BHK cells by interfering with viral attachment. In contrast, sCS-E enhanced viral infection in the mouse neuroblastoma cell line Neuro-2a, despite the fact that viral attachment to Neuro-2a cells was inhibited by sCS-E. This enhancement effect in Neuro-2a cells seemed to be related to increased viral RNA replication and was also observed in a rat infection model in which intracerebral coadministration of sCS-E with JEV in 17-day-old rats resulted in higher brain viral loads than in rats infected without sCS-E administration. These results show the paradoxical effects of sCS-E on JEV infection in different cell types and indicate that potential use of sCS-E as an antiviral agent against JEV infection should be approached with caution considering its effects in the neuron, the major target of JEV. (C) 2011 Elsevier Inc. All rights reserved.
• Detection and characterization of a novel polyomavirus in wild rodents

  Yasuko Orba, Shintaro Kobayashi, Ichiro Nakamura, Akihiro Ishii, Bernard M. Hang'ombe, Aaron S. Mweene, Yuka Thomas, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF GENERAL VIROLOGY 92 4 789 - 795 2011年04月 [査読有り][通常論文]
   

  To investigate polyomavirus infection in wild rodents, we analysed DNA samples from the spleens of 100 wild rodents from Zambia using a broad-spectrum PCR-based assay. A previously unknown polyomavirus genome was identified in a sample from a multimammate mouse (Mastomys species) and the entire viral genome of 4899 bp was :subsequently sequenced. This viral genome contained potential ORFs for the capsid proteins, VP1, VP2 and VP3, and early proteins, small t antigen and large T antigen. Phylogenetic analysis showed that it was a novel member of the family Polyomaviridae, and thus the virus was tentatively named mastomys polyomavirus. After transfection of the viral genome into several mammalian cell lines, transient expression of the VP1 and large T antigen proteins was confirmed by immunoblotting and immunocytochemical analyses. Comparison of large T antigen function in mastomys polyomavirus with that in rhesus monkey polyomavirus SV40 and human polyomavirus JC virus revealed that the large T antigen from mastomys polyomavirus interacted with the tumour suppressor protein pRb, but not with p53.
• Equine major histocompatibility complex class I molecules act as entry receptors that bind to equine herpesvirus-1 glycoprotein D

  Michihito Sasaki, Rie Hasebe, Yoshinori Makino, Tadaki Suzuki, Hideto Fukushi, Minoru Okamoto, Kazuya Matsuda, Hiroyuki Taniyama, Hirofumi Sawa, Takashi Kimura

  GENES TO CELLS 16 4 343 - 357 2011年04月 [査読有り][通常論文]
   

  The endotheliotropism of equine herpesvirus-1 (EHV-1) leads to encephalomyelitis secondary to vasculitis and thrombosis in the infected horse central nervous system (CNS). To identify the host factors involved in EHV-1 infection of CNS endothelial cells, we performed functional cloning using an equine brain microvascular endothelial cell cDNA library. Exogenous expression of equine major histocompatibility complex (MHC) class I heavy chain genes conferred susceptibility to EHV-1 infection in mouse NIH3T3 cells, which are not naturally susceptible to EHV-1 infection. Equine MHC class I molecules bound to EHV-1 glycoprotein D (gD), and both anti-gD antibodies and a soluble form of gD blocked viral entry into NIH3T3 cells stably expressing the equine MHC class I heavy chain gene (3T3-A68 cells). Treatment with an anti-equine MHC class I monoclonal antibody blocked EHV-1 entry into 3T3-A68 cells, equine dermis (E. Derm) cells and equine brain microvascular endothelial cells. In addition, inhibition of cell surface expression of MHC class I molecules in E. Derm cells drastically reduced their susceptibility to EHV-1 infection. These results suggest that equine MHC class I is a functional gD receptor that plays a pivotal role in EHV-1 entry into equine cells.
• Chondroitin sulfate-E induced enhancement of Japanese encephalitis virus infection

  Eunmi Kim, Megumi Okumura, Michihito Sasaki, Daisuke Fujikura, Tadaaki Miyazaki, Hirofumi Sawa, Takashi Kimura

  JOURNAL OF NEUROVIROLOGY 16 44 - 44 2010年10月 [査読有り][通常論文]
  
• Flavivirus Encephalitis: Pathological Aspects of Mouse and Other Animal Models

  T. Kimura, M. Sasaki, M. Okumura, E. Kim, H. Sawa

  VETERINARY PATHOLOGY 47 5 806 - 818 2010年09月 [査読有り][通常論文]
   

  Encephalitic flaviviruses are important arthropod-borne pathogens of humans and other animals. In particular, the recent emergence of the West Nile virus (WNV) and Japanese encephalitis virus (JEV) in new geographic areas has caused a considerable public health alert and international concern. Among the experimental in vivo models of WNV and JEV infection, mice and other laboratory rodents are the most thoroughly studied and well-characterized systems, having provided data that are important for understanding the infectious process in humans. Macaca monkeys have also been used as a model for WNV and JEV infection, mainly for the evaluation of vaccine efficacy, although a limited number of published studies have addressed pathomorphology. These animal models demonstrate the development of encephalitis with many similarities to the human disease; however, the histological events that occur during infection, especially in peripheral tissues, have not been fully characterized.
• Functional analysis of an alpha-helical antimicrobial peptide derived from a novel mouse defensin-like gene

  Akira Kawaguchi, Tadaki Suzuki, Takashi Kimura, Naoki Sakai, Tokiyoshi Ayabe, Hirofumi Sawa, Hideki Hasegawa

  BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 398 4 778 - 784 2010年08月 [査読有り][通常論文]
   

  Gene-encoded antimicrobial peptides (AMPS) are an essential component of the innate immune system in many species. Analysis of beta-defensin gene expression in mouse tissue using primers that were specific for conserved sequences located outside of the beta-defensin translated region identified a novel small gene. The novel gene had an open reading frame of 114 bp and encoded a predicted protein of 37 amino acid residues. A search of the genome database revealed that the gene locus and the sequence of exon 1 of this novel gene were similar to subgroup 1 mouse beta-defensins. A small peptide, K17 (FSPQMLQDIIEKKTKIL), derived from the amino acid sequence of this novel gene was synthesized. Circular dichroism (CD) spectroscopic analysis of chemically synthesized peptide demonstrated that the peptide exhibited random coil conformation in aqueous solution, but the peptide adopted helical conformation in the presence of trifluoroethanol or sodium dodecyl sulfate, a membrane-mimicking environment. The peptide exhibited bactericidal activity against Salmonella enterica serovar Typhimurium (Gram negative) and Staphylococcus aureus (Gram positive); it was not cytotoxic in cultures of mammalian cells or hemolytic in cultures of erythrocytes. These results suggested that K17 may be a candidate therapeutic for the treatment of bacterial infection. (C) 2010 Elsevier Inc. All rights reserved.
• Natalizumab Has No Direct Biological Effect on JC Virus Infectivity in Permissive Human Neural Cell Lines

  Tadaki Suzuki, Satoko Yamanouchi, Yuji Sunden, Yasuko Orba, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF MEDICAL VIROLOGY 82 7 1229 - 1235 2010年07月 [査読有り][通常論文]
   

  The human polyomavirus JC virus (JCV) infects 70-80% of humans and establishes latent infection in the kidney. In immunosuppressed patients, JCV reactivates and causes a fatal and progressive neurological disease known as progressive multifocal leukoencephalopathy (PML). Over the past three decades, PML has become an important neurological complication in acquired immunodeficiency syndrome (AIDS) patients. Recently, it was reported that patients treated with therapeutics that target the integrin receptor very late antigen (VLA)-4 are at increased risk of developing PML. However, the relationship between Natalizumab and this unexpected onset of PML has yet to be elucidated. Here, we investigated the effect of Natalizumab on the growth of JCV in the permissive human neural cell line IMR-32 following viral inoculation. Natalizumab had no effect either on the expression levels of viral proteins as determined by immunoblot analysis using specific antibodies or on the hemagglutination activity of cellular lysates from infected cells. These results suggest that there is no direct effect of Natalizumab on JCV infectivity in IMR-32 cells in vitro. J. Med. Virol. 82:1229-1235, 2010. (C) 2010 Wiley-Liss, Inc.
• Transcellular transport of West Nile virus-like particles across human endothelial cells depends on residues 156 and 159 of envelope protein

  Rie Hasebe, Tadaki Suzuki, Yoshinori Makino, Manabu Igarashi, Satoko Yamanouchi, Akihiko Maeda, Motohiro Horiuchi, Hirofumi Sawa, Takashi Kimura

  BMC MICROBIOLOGY 10 165  2010年06月 [査読有り][通常論文]
   

  Background: West Nile virus (WNV) causes viremia after invasion to the hosts by mosquito bite. Endothelial cells could play an important role in WNV spread from the blood stream into the central nervous system and peripheral tissues. Here, we analyzed the capacity of virus-like particles (VLPs) of the highly virulent NY99 6-LP strain (6-LP VLPs) and the low virulence Eg101 strain (Eg VLPs) to cross cultured human endothelial cells. Results: 6-LP VLPs were transported from the apical to basolateral side of endothelial cells, whereas Eg VLPs were hardly transported. The localization of tight junction marker ZO-1 and the integrity of tight junctions were not impaired during the transport of 6-LP VLPs. The transport of 6-LP VLPs was inhibited by treatment with filipin, which prevents the formation of cholesterol-dependent membrane rafts, suggesting the involvement of raft-associated membrane transport. To determine the amino acid residues responsible for the transport of VLPs, we produced mutant VLPs, in which residues of E protein were exchanged between the 6-LP and Eg strains. Double amino acid substitution of the residues 156 and 159 greatly impaired the transport of VLPs. Conclusion: Our results suggest that a transcellular pathway is associated with 6-LP VLPs transport. We also showed that the combination of the residues 156 and 159 plays an important role in the transport of VLPs across endothelial cells.
• The Human Polyoma JC Virus Agnoprotein Acts as a Viroporin

  Tadaki Suzuki, Yasuko Orba, Yuki Okada, Yuji Sunden, Takashi Kimura, Shinya Tanaka, Kazuo Nagashima, William W. Hall, Hirofumi Sawa

  PLOS PATHOGENS 6 3 e1000801  2010年03月 [査読有り][通常論文]
   

  Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca(2+) homeostasis leading to membrane dysfunction and enhancement of virus release.
• Large T Antigen Promotes JC Virus Replication in G(2)-arrested Cells by Inducing ATM- and ATR-mediated G(2) Checkpoint Signaling

  Yasuko Orba, Tadaki Suzuki, Yoshinori Makino, Kanako Kubota, Shinya Tanaka, Takashi Kimura, Hirofumi Sawa

  JOURNAL OF BIOLOGICAL CHEMISTRY 285 2 1544 - 1554 2010年01月 [査読有り][通常論文]
   

  Large T antigen (TAg) of the human polyomavirus JC virus (JCV) possesses DNA binding and helicase activities, which, together with various cellular proteins, are required for replication of the viral genome. We now show that JCV-infected cells expressing TAg accumulate in the G(2) phase of the cell cycle as a result of the activation of ATM- and ATR-mediated G(2) checkpoint pathways. Transient transfection of cells with a TAg expression vector also induced G(2) checkpoint signaling and G(2) arrest. Analysis of TAg mutants with different subnuclear localizations suggested that the association of TAg with cellular DNA contributes to the induction of G(2) arrest. Abrogation of G(2) arrest by inhibition of ATM and ATR, Chk1, and Wee1 suppressed JCV genome replication. In addition, abrogation of the G(2)-M transition by Cdc2 depletion disabled Wee1 depletion induced suppression of JCV genome replication, suggesting that JCV replication is facilitated by G(2) arrest resulting from G(2) checkpoint signaling. Moreover, inhibition of ATM and ATR by caffeine suppressed JCV production. The observation that oligodendrocytes productively infected with JCV in vivo also undergo G(2) arrest suggests that G(2) checkpoint inhibitors such as caffeine are potential therapeutic agents for JCV infection.
• Inhibition of the SDF-1 alpha-CXCR4 axis by the CXCR4 antagonist AMD3100 suppresses the migration of cultured cells from ATL patients and murine lymphoblastoid cells from HTLV-I Tax transgenic mice

  Akira Kawaguchi, Yasuko Orba, Takashi Kimura, Hidekatsu Iha, Masao Ogata, Takahiro Tsuji, Akira Ainai, Tetsutaro Sata, Takashi Okamoto, William W. Hall, Hirofumi Sawa, Hideki Hasegawa

  BLOOD 114 14 2961 - 2968 2009年10月 [査読有り][通常論文]
   

  Adult T-cell leukemia (ATL) is a T-cell malignancy caused by human T lymphotropic virus type I, and presents as an aggressive leukemia with characteristic widespread leukemic cell infiltration into visceral organs and skin. The molecular mechanisms associated with leukemic cell infiltration are poorly understood. We have used mouse models of ATL to investigate the role of chemokines in this process. Transfer of splenic lymphomatous cells from transgenic to SCID mice reproduces a leukemia and lymphoma that is histologically identical to human disease. It could be shown that lymphomatous cells exhibit specific chemotactic activity in response to stromal cell-derived factor-1 alpha (SDF-1 alpha). Lymphomatous cells exhibited surface expression of CXCR4, the specific receptor of SDF-1 alpha. AMD3100, a CXCR4 antagonist, was found to inhibit both SDF-1 alpha-induced migration and phosphorylation of extracellular signal-related kinase 1/2. Investigation of cultured cells from human ATL patients revealed identical findings. Using the SCID mouse model, it could be demonstrated that AMD3100 inhibited infiltration of lymphomatous cells into liver and lung tissues in vivo. These results demonstrate the involvement of the SDF-1 alpha/CXCR4 interaction as one mechanism of leukemic cell migration and this may provide a novel target as part of combination therapy for ATL. (Blood. 2009; 114: 2961-2968)
• Infectious entry of equine herpesvirus-1 into host cells through different endocytic pathways

  Rie Hasebe, Michihito Sasaki, Hirofumi Sawa, Ryuichi Wada, Takashi Umemura, Takashi Kimura

  VIROLOGY 393 2 198 - 209 2009年10月 [査読有り][通常論文]
   

  We investigated the mechanism by which equine herpesvirus-1 (EHV-1) enters primary Cultured equine brain microvascular endothelial cells (EBMECs) and equine dermis (E. Derm) cells. EHV-1 colocalized with caveolin in EBMECs and the infection was greatly reduced by the expression of a dominant negative form of equine caveolin-1 (ecavY14F), suggesting that EHV-1 enters EBMECs via caveolar endocytosis. EHV-1 entry into E. Derm cells was significantly reduced by ATP depletion and treatments with lysosomotropic agents. Enveloped virions were detected from E. Derm cells by infectious virus recovery assay after vital internalization, suggesting that EHV-1 enters E. Derm cells via energy- and pH-dependent endocytosis. These results suggest that EHV-1 utilizes multiple endocytic pathways in different cell types to establish productive infection. (C) 2009 Elsevier Inc. All rights reserved.
• Pharmacological cdk inhibitor R-Roscovitine suppresses JC virus proliferation

  Yasuko Orba, Yuji Sunden, Tadaki Suzuki, Kazuo Nagashima, Takashi Kimura, Shinya Tanaka, Hirofumi Sawa

  VIROLOGY 370 1 173 - 183 2008年01月 [査読有り][通常論文]
   

  The human Polyomavirus JC virus (JCV) utilizes cellular proteins for viral replication and transcription in the host cell nucleus. These cellular proteins represent potential targets for antiviral drugs against the JCV. In this study, we examined the antiviral effects of the pharmacological cyclin-dependent kinase (cdk) inhibitor R-Roscovitine, which has been shown to have antiviral activity against other viruses. We found that Roscovitine significantly inhibited the viral production and cytopathic effects of the JCV in a JCV-infected cell line. Roscovitine attenuated the transcriptional activity of JCV late genes, but not early genes, and also prevented viral replication via inhibiting phosphorylation of the viral early protein, large T antigen. These data suggest that the JCV requires cdks to transcribe late genes and to replicate its own DNA. That Roscovitine exhibited antiviral activity in JCV-infected cells suggests that Roscovitine might have therapeutic utility in the treatment of progressive multifocal leukoencephalopathy (PML). (c) 2007 Elsevier Inc. All rights reserved.
• 犬末梢血単核球におけるCD56mRNA発現とその定量法の確立

  星野 有希, 高木 哲, 木村 享史, 奥村 正裕, 藤永 徹

  日本獣医学会学術集会講演要旨集 143回 217 - 217 (公社)日本獣医学会 2007年03月
• Cerebellar hypoplasia associated with an avian leukosis virus inducing fowl glioma

  T Toyoda, K Ochiai, H Hatai, M Murakami, E Ono, T Kimura, T Umemura

  VETERINARY PATHOLOGY 43 3 294 - 301 2006年05月 [査読有り][通常論文]
   

  Fowl glioma-inducing virus (FGV), which belongs to subgroup A of avian leukosis virus (ALV), shows tumorigenicity and pathogenicity, mainly in the nervous system, and causes astrocytoma and perineurioma. Apart from these neoplasms, cerebellar anomaly was found in chickens infected with FGV in ovo. The study reported here describes the morphologic characteristics of the affected cerebellum. Specific-pathogen-free chickens (C/O) were inoculated with FGV through the yolk sac on the 7th day of incubation. The cerebellar anomaly included diffuse depletion of granular cells of the internal granular layer (IGL), remnants of the external granular layer (EGL), and disorganization of the Purkinje cell layer. These cerebellar changes were observed in all birds except one. In the infected embryos, the EGL was thicker and had an irregular arrangement with a thin molecular layer (ML) and IGL, compared with the control. The granular cells were immunohistochemically positive for ALV common antigen. Immunohistochemical analysis for vimentin revealed disarrangement and decreased number of Bergmann's fibers. Use of the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method and electron microscopy indicated that apoptotic granular cells were frequently observed in the EGL and ML. These results suggested that the cerebellar anomaly was hypoplasia, principally resulting from the apoptosis of granular cells in the EGL and ML caused by FGV infection and that the cell loss induced obstruction of granular cell migration and disarrangement of Bergmann's fibers in the ML.
• Differential susceptibility of equine and mouse brain microvascular endothelial cells to equine herpesvirus 1 infection

  R Hasebe, T Kimura, K Nakamura, K Ochiai, K Okazaki, R Wada, T Umemura

  ARCHIVES OF VIROLOGY 151 4 775 - 786 2006年04月 [査読有り][通常論文]
   

  Equine herpesvirus 1 (EHV-1) shows endotheliotropism in the central nervous system (CNS) of infected horses. However, infection of endothelial cells has not been observed in the CNS of infected mice. To explore the basis for this difference in endotheliotropism, we compared the susceptibility of equine brain microvascular endothelial cells (EBMECs) and mouse brain microvascular endothelial cells (MBMECs) to EHV-1 infection. The kinetics of viral growth in EBMECs was typical of a fully productive infection whereas viral infection in MBMECs seemed to be nonproductive. Immunofluorescence microscopy using anti-EHV-1 polyclonal antibody demonstrated viral antigen in infected EBMECs, but not infected MBMECs. EHV-1 immediate early (IE), early (ICP0), and late (gB, gD and gK) transcripts were expressed in infected EBMECs. However, none of these genes was detected in infected MBMECs by reverse transcription-polymerase chain reaction. Electron microscopic examination at the stage of viral entry showed that viral particles were present within uncoated vesicles in the cytoplasm of EBMECs, but absent from those of MBMECs. These results suggest that viral entry is an important determinant of the susceptibility of EBMECs and MBMECs to EHV-1 infection.
• Efficacy of Intracerebral Immunization against Pseudorabies Virus in Mice

  Shin, J, Sakoda, Y, Kim, J.-H, Tanaka, T, Kida, H, Kimura, T, Ochiai, K, Umemura, T

  Microbiology and Immunology 50 823 - 830 2006年 [査読無し][通常論文]
  
• Efficacy of intracerebral immunization against pseudorabies virus in mice

  Jae-Ho Shin, Yoshihiro Sakoda, Jae Hoon Kim, Tomohisa Tanaka, Hiroshi Kida, Takashi Kimura, Kenji Ochiai, Takashi Umemura

  MICROBIOLOGY AND IMMUNOLOGY 50 10 823 - 830 2006年 [査読有り][通常論文]
   

  To evaluate the efficacy of intracerebral (IQ immunization, mice were immunized with formalin-inactivated pseudorabies virus (PRV) by either subcutaneous (SQ or IC injection, and then 10(6) plaque-forming units of PRV were introduced into the hindleg of the immunized or non-immunized mice by intramuscular injection. The antibody titer in serum was elevated and boosted by additional immunization via both the SC and IC routes, but was higher after IC immunization. Intracerebrally immunized mice were completely protected from mortality and neurological signs, whereas all the non-immunized and 80% of the subcutaneously immunized mice died after developing neurological signs. In mouse models, IC immunization is more effective at inducing a protective immune response against the transneural spread of PRV than SC immunization.
• Nested polymerase chain reaction for detection of the avian leukosis virus causing so-called fowl glioma

  H Hatai, K Ochiai, Y Tomioka, T Toyoda, K Hayashi, M Anada, M Kato, A Toda, K Ohashi, E Ono, T Kimura, T Umemura

  AVIAN PATHOLOGY 34 6 473 - 479 2005年12月 [査読有り][通常論文]
   

  The complete nucleotide sequence of the avian leukosis virus causing so-called fowl glioma has been previously determined. Primers were designed for detection of the fowl glioma-causal virus (FGV) based on the 3' untranslated region of the viral genome. The provirus and viral RNA of FGV were specifically detected in various organs and tissues, including feather pulp, from experimentally infected birds using nested polymerase chain reaction (PCR) and reverse transcription nested PCR. The prevalence of FGV was evaluated in 131 Japanese fowls of a zoological garden in Japan based on the detection of the FGV genome in feather pulp using PCR and the detection of viral antigen in faeces by enzyme-linked immunosorbent assay. FGV proviral DNA was detected in feather pulp of 52 birds (39.7%) by nested PCR. Later, nine dead birds from among the 52 were histologically diagnosed as having fowl glioma and found to have the proviral DNA in the affected brain. These results demonstrated that the PCR-based detection of FGV in feather pulp is useful for epidemiological studies on fowl glioma.
• In vitro demonstration of neural transmission of avian influenza A virus

  K Matsuda, T Shibata, Y Sakoda, H Kida, T Kimura, K Ochiai, T Umemura

  JOURNAL OF GENERAL VIROLOGY 86 4 1131 - 1139 2005年04月 [査読有り][通常論文]
   

  Neural involvement following infections of influenza viruses can be serious. The neural transport of influenza viruses from the periphery to the central nervous system has been indicated by using mouse models. However, no direct evidence for neuronal infection has been obtained in vitro and the mechanisms of neural transmission of influenza viruses have not been reported. In this study, the transneural transmission of a neurotropic influenza A virus was examined using compartmentalized cultures of neurons from mouse dorsal root ganglia, and the results were compared with those obtained using the pseudorabies virus, a virus with well-established neurotransmission. Both viruses reached the cell bodies of the neurons via the axons. This is the first report on axonal transport of influenza A virus in vitro. In addition, the role of the cytoskeleton (microtubules, microfilaments and intermediate filaments) in the neural transmission of influenza virus was investigated by conducting cytoskeletal perturbation experiments. The results indicated that the transport of avian influenza A virus in the neurons was independent of microtubule integrity but was dependent on the integrity of intermediate filaments, whereas pseudorabies virus needed both for neural spread.
• The vagus nerve is one route of transneural invasion for intranasally inoculated influenza A virus in mice

  Matsuda K, Shibata T, Sakoda Y, Kida H, Kimura T, Ochiai K, Umemura T

  Journal of General Virology 86 1131 - 1139 2005年04月 [査読有り][通常論文]
  
• Multiple perineuriomas in chicken (Gallus gallus domesticus)

  T Toyoda, K Ochiai, K Ohashi, Y Tomioka, T Kimura, T Umemura

  VETERINARY PATHOLOGY 42 2 176 - 183 2005年03月 [査読有り][通常論文]
   

  Intraneural perineurioma is an extremely rare condition characterized by perineurial cell proliferation within peripheral nerve (PN) sheaths. In the veterinary field, this entity has been reported only in a dog. We examined multiple enlargements of PNs in 11 chickens (Gallus gallus domesticus) (9 Japanese bantams and 2 specific pathogen-free White Leghorn), which were inoculated with an avian leukosis virus (ALV) causing so-called fowl glioma. All chickens clinically exhibited progressive leg paralysis. Lumbosacral plexus, brachial plexus, and/or spinal ganglion were commonly affected, and these nerves contained a diffuse proliferation of spindle cells arranged concentrically in characteristic onion bulb-like structures surrounded by residual axons and myelin sheaths. The spindle cells were immunohistochemically negative for S-100alpha/beta protein. Electron microscopy revealed that these cells were characterized by short bipolar cytoplasmic processes, occasional cytoplasmic pinocytotic vesicles, and discontinuous basal laminae. These features are consistent with those of intraneural perineurioma. Furthermore, the specific sequence of the ALV was detected in the PN lesions of 8/11 (73%) birds by polymerase chain reaction. These results indicate that the multiple intraneural perineuriomas of chicken may be associated with the ALV-A causing fowl glioma.
• トリの神経膠腫原因ウイルス接種鶏に見られた小脳異形成

  豊田 武士, 落合 謙爾, 畑井 仁, 村上 真理子, 木村 享史, 梅村 孝司

  日本獣医学会学術集会講演要旨集 139回 203 - 203 (公社)日本獣医学会 2005年03月 [査読有り][通常論文]
  
• Different chemokine expression in lethal and non-lethal murine West Nile virus infection

  K Shirato, T Kimura, T Mizutani, H Kariwa, Takashima, I

  JOURNAL OF MEDICAL VIROLOGY 74 3 507 - 513 2004年11月 [査読有り][通常論文]
   

  West Nile (WN) virus is a mosquito-borne flavivirus that can cause lethal encephalitis in humans and horses. The WN virus endemic in New York City (NY) in 1999 caused large-scale mortality of wild birds that was not evident in endemic areas in other parts of the world, and the pathogenesis of the WN virus strain isolated in NY (NY strain) appears to differ from that of previously isolated strains. However, the pathogenesis of NY strain infection remains unclear. This study examined CC (RANTES/CCL5, MIP-1alpha/CCL3, MIP-1beta/CCL4) and CXC (IP-10/CXCL10, B lymphocyte chemoattractant (BLC/CXCL13), and B cell- and monocyte-activating chemokine (BMAC/CXCL14)) chemokine expression during lethal NY strain and non-lethal Eg101 strain infection in mice. We found that the mRNA of the CC chemokines, RANTES, MIP-1alpha, MIP-1beta, and IP-10 was highly up-regulated in the brain of NY strain-infected mice. By contrast, BLC mRNA was not detected in either group of mice, and BMAC mRNA was highly up-regulated in late stage of infection with the non-lethal Eg101 strain relative to levels in NY strain-infected mice. (C) 2004 Wiley-Liss, Inc.
• Odontogenic cysts in three dogs: One odontogenic keratocyst and two dentigerous cysts

  K Watanabe, T Kadosawa, T Ishiguro, S Takagi, K Ochiai, T Kimura, M Okumura, T Fujinaga

  JOURNAL OF VETERINARY MEDICAL SCIENCE 66 9 1167 - 1170 2004年09月 [査読有り][通常論文]
   

  Odontogenic cysts, which showed cystic radiolucency in the jaw bone by radiographic examination and computed tomography, were enucleated by operation in 3 dogs. One dog had a odontogenic keratocyst in the incisive bone of the right maxilla and another 2 cases revealed dentigerous cysts in the mandible. These cyst walls were enucleated or transpired by semiconductor laser. Afterwards, osteogenesis was confirmed at the defective part of jaw bone by extirpation of the cyst in all cases, and no recurrence has been noted in any cases. Odontogenic cyst is a disease which should be treated by surgical extirpation or transpiration.
• Ｈ５強毒インフルエンザウイルスの神経病原性と解熱剤投与の影響に関する実験的研究

  梅村孝司, 松田一哉, 朴天鍋, 寸田祐嗣, 木村享史, 落合謙爾, 喜田宏

  日本病理学会会誌 93 1 175  2004年04月 [査読無し][通常論文]
  
• Genome sequence analysis of the avian retrovirus causing so-called fowl glioma and the promoter activity of the long terminal repeat

  Y Tomioka, K Ochiai, K Ohashi, E Ono, T Toyoda, T Kimura, T Umemura

  JOURNAL OF GENERAL VIROLOGY 85 3 647 - 652 2004年03月 [査読有り][通常論文]
   

  So-called fowl glioma is a retroviral infectious disease caused by avian leukosis virus subgroup A (ALV-A). We determined the complete nucleotide sequence of the virus genome. The full-length sequence was consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The coding sequences were well conserved with those of replication-competent viruses, but the 3' noncoding regions including LTR were most related to those of replication-defective sarcoma viruses. The U3 region of the LTR had a few deletions and several point mutations compared to that of other ALVs. The promoter activities of the LTRs of glioma-inducing ALV and ALV-A standard strain, RAV-1, were equivalent in chick embryo fibroblasts (CEF), while that of glioma-inducing ALV was significantly lower than that of RAV-1 in human astrocytic cells. These subtle differences of the promoter activity of the LTR may be related to the induction of glial neoplasm.
• The vagus nerve is one route of transneural invasion for intranasally inoculated influenza A virus in mice

  K Matsuda, CH Park, Y Sunden, T Kimura, K Ochiai, H Kida, T Umemura

  VETERINARY PATHOLOGY 41 2 101 - 107 2004年03月 [査読有り][通常論文]
   

  Intranasally inoculated neurotropic influenza viruses in mice infect not only the respiratory tract but also the central nervous system (CNS), mainly the brain stem.(26,34) Previous studies suggested that the route of invasion of virus into the CNS was via the peripheral nervous system, especially the vagus nerve. To evaluate the transvagal transmission of the virus, we intranasally inoculated unilaterally vagectomized mice with a virulent influenza virus (strain 24a5b) and examined the distribution of the viral protein and genome by immunohistochemistry and in situ hybridization over time. An asymmetric distribution of viral antigens was observed between vagal (nodose) ganglia: viral antigen was detected in the vagal ganglion of the vagectomized side 2 days later than in the vagal ganglion of the intact side. The virus was apparently transported from the respiratory mucosa to the CNS directly and decussately via the vagus nerve and centrifugally to the vagal ganglion of the vagectomized side. The results of this study, thus, demonstrate that neurotropic influenza virus,:,an, travels to the CNS mainly via the vagus nerve.
• Nutritional secondary hyperparathyroidism and osteodystrophia fibrosa in a Hodgson's hawk-eagle (Spizaetus nipalensis)

  T Toyoda, K Ochiai, M Komatsu, T Kimura, T Umemura

  AVIAN PATHOLOGY 33 1 9 - 12 2004年02月 [査読有り][通常論文]
   

  A Hodgson's hawk- eagle ( Spizaetus nipalensis) reared by a falconer showed severe weakness with multiple fractures of bone. It had a history of being fed an all- meat diet. Serological examination revealed a hypocalcaemia ( 72.0 mug/ ml), and hypophosphataemia ( 29.0 mug/ ml). Gross and microscopic examinations demonstrated severe osteodystrophia fibrosa ( fibrous osteodystrophy) characterized by osteoclastic bone resorption and intertrabecular fibrosis with unmineralized trabecular bone containing a large amount of unmineralized osteoid. There was also hypertrophy and hyperplasia of the parathyroid glands, which is consistent with nutritional secondary hyperparathyroidism.
• Canine ganglioneuroblastoma in the oral mucosa

  K Nakamura, K Ochiai, T Kadosawa, T Kimura, T Umemura

  JOURNAL OF COMPARATIVE PATHOLOGY 130 2-3 205 - 208 2004年02月 [査読有り][通常論文]
   

  A ganglioneuroblastoma of the oral cavlity in a dog was examined histologically and immunohistochemically This rare neoplasm was considered to be derived from ectopic neural crest cells. This is the first report of a canine ectopic ganglioneuroblastoma located in the oral mucosa. (C) 2003 Elsevier Ltd. All rights reserved.
• Decreased expression of equine herpesvirus-1 early and late genes in the placenta of naturally aborted equine fetuses

  T Kimura, R Hasebe, R Mukaiya, K Ochiai, R Wada, T Umemura

  JOURNAL OF COMPARATIVE PATHOLOGY 130 1 41 - 47 2004年01月 [査読有り][通常論文]
   

  Intrauterine infection with equine herpesvirus-1 (EHV-1) has been considered to be the consequence of transplacental transmission of the virus following maternal cell-associated viraemia. In this study the state of EHV-1 gene expression in the placenta of seven naturally aborted equine fetuses was examined. Neither lesions nor viral antigens were detected in the placenta of the fetuses. The amount of infectious virus in the placentas was considerably lower than that in the fetal lungs, which showed pneumonia and typical herpesvirus\ inclusions. Quantitative dot blot hybridization with probes specific for immediate-early (IE), early (ICP0), and late (gD and gK) genes revealed that the placentas expressed the IE gene at a level comparable with that in the lungs; however, expression of the ICP0, gD and gK genes was significantly weaker in the placentas than in the lungs. In-situ hybridization demonstrated that both IE and gK RNAs were distributed mainly in the cytoplasm of trophoblasts. These results suggest that the low level of early and late gene transcription may be related to the limited production of viral progeny and the lack of immunoreactivity for viral antigen in trophoblasts infected with EHV-1. (C) 2003 Elsevier Ltd. All rights reserved.
• Persistence of viral RNA segments in the central nervous system of mice after recovery from acute influenza A virus infection

  CH Park, K Matsuda, Y Sunden, A Ninomiya, A Takada, H Ito, T Kimura, K Ochiai, H Kida, T Umemura

  VETERINARY MICROBIOLOGY 97 3-4 259 - 268 2003年12月 [査読有り][通常論文]
   

  One-hundred thirty-seven BALB/c mice were intranasally inoculated with neurotropic avian influenza A virus (H5N3). Thirty-nine of these mice died within 16 days post-inoculation (PID) and 98 of the mice recovered from the infection. To investigate whether viral antigens and genomes persist in the central nervous system (CNS) of recovered mice, immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR) methods were performed. Histopathologically, mild interstitial pneumonia and non-suppurative encephalomyelitis restricted to the basal part of the frontal lobe of the cerebrum, brain stem and thoracic spinal cord were observed in BALB/c mice until 40 PID. Small amounts of viral antigens were detected in the brain and spinal cord and some viral RNA segments (NA, NP, M, PA, HA, NS, PB1) were intermittently detected in the CNS until 48 PID. Immunosuppression of these mice by dexamethazone (DEX) treatment did not increase the frequency of detection of the lesions, viral antigens or genomes. These findings suggest that viral genomes of neurovirulent influenza virus persist with restricted transcriptive activity in the CNS of the mice even after clinical recovery from the infection. (C) 2003 Elsevier B.V. All rights reserved.
• In ovo infection with an avian leukosis virus causing fowl glioma: viral distribution and pathogenesis

  Y Tomioka, K Ochiai, K Ohashi, T Kimura, T Umemura

  AVIAN PATHOLOGY 32 6 617 - 624 2003年12月 [査読有り][通常論文]
   

  We have previously isolated an avian leukosis virus (ALV) from a chicken affected with so-called fowl glioma. A resistance-inducing factor test indicated that the isolate was classified into a subgroup A. The distribution and pathogenicity were investigated in C/O specific pathogen free chickens infected in ovo with this virus. Histologically, 11 of 12 (92%) infected birds had non-suppurative encephalitis and three birds (25%) showed the characteristic nodules of fowl glioma at 50 or 100 days of age. Non-suppurative myocarditis with matrix inclusions and atypical myocytes were also noted in nine (75%) of the birds and the ALV antigens were immunohistochemically detected in various general organs as well as the central nervous system and heart. The semi-quantitative determination of the proviral DNA and viral RNA supported the immunohistochemical results and indicated that the virus was likely to replicate especially in myocardial fibres. The isolated ALV failed to induce other neoplastic lesions in this line of chickens within the experimental period of 100 days, despite the broad tissue tropism throughout the body. These results confirmed that this virus was able to induce glioma in embryo-inoculated chickens.
• The effects of antipyretics on influenza virus encephalitis in mice and chicks

  Y Sunden, CH Park, K Matsuda, A Anagawa, T Kimura, K Ochiai, H Kida, T Umemura

  JOURNAL OF VETERINARY MEDICAL SCIENCE 65 11 1185 - 1188 2003年11月 [査読有り][通常論文]
   

  To investigate the effect of antipyretics on the murine and poultry models of influenzal encephalitis, we injected large quantities of antipyretics, acetylsalicylic acid (aspirin) and dicrofenac sodium (voltaren). The effect of antipyretic treatment on the murine encephalitis model was unremarkable histologically and immunohistochemically. Whereas in chicks, CNS lesions consisting of perivascular cuffing and gliosis appeared only in those animals treated with the antipyretics and viral antigen was detected mainly in the nuclei of glial cells or vascular endothelia of voltaren-treated animals. We here demonstrate that antipyretic treatment aggravated the hematogenous spread of the influenza virus to the CNS in chicks, but did not affect the transneural infection in mice.
• Involvement of the JNK-like protein of the Aedes albopictus mosquito cell line, C6/36, in phagocytosis, endocytosis and infection of West Nile virus

  T Mizutani, M Kobayashi, Y Eshita, K Shirato, T Kimura, Y Ako, H Miyoshi, T Takasaki, Kurane, I, H Kariwa, T Umemura, Takashima, I

  INSECT MOLECULAR BIOLOGY 12 5 491 - 499 2003年10月 [査読有り][通常論文]
   

  We recently cloned a c-Jun amino-terminal kinase (JNK) sequence from the C6/36 cell line, derived from the mosquito Aedes albopictus. We showed that SP600125, an inhibitor of JNK proteins, inhibits phagocytosis by C6/36 cells, suggesting that the JNK-like protein regulates phagocytosis. Here, we show that C6/36 cells constitutively express low levels of mRNA encoding the antibacterial peptides, cecropin and defensin, but that these mRNAs were up-regulated upon stimulation by lipopolysaccharide (LPS). Thus, the C6/36 cells have properties similar to those of mammalian macrophages. To characterize further the functional properties of C6/36 cells, we have assayed the role of the JNK-like protein in phagocytosis, endocytosis, and viral infection. C6/36 cells phagocytosed bacteria and artificial beads, and this was only slightly up-regulated following LPS stimulation, suggesting that newly stimulated JNK-like protein was not necessary for phagocytosis. SP600125 inhibited the acidification of intracellular compartments, including those involved in the endocytic pathway. Pretreatment of C6/36 cells with SP600125 or bafilomycin A1, but not cytochalasin D, inhibited the entry of West Nile virus (WNV), suggesting that WNV is internalized mainly by endocytosis, and that the JNK signalling pathway is important for endocytic entry. These findings indicate that the JNK-like protein regulates basic physiological functions, including phagocytosis and endocytosis and infection of WNV.
• Impaired myelinogenesis and early maturation of oligodendrocytes inblack tremor hamster with central nervous system myelin deficiency

  Kim HO, Matsuda K, Kimura T, Ochiai K, Yazawa H, Itakura C, Umemura T

  Journal of Toxicological Pathology 16 209 - 213 2003年10月 [査読有り][通常論文]
  
• Sustained hypomyelination and high serum thyroid hormone in aged black tremor hamster

  HO Kim, T Kimura, K Ochiai, H Yazawa, C Itakura, T Umemura

  JAPANESE JOURNAL OF VETERINARY RESEARCH 51 2 113 - 120 2003年08月 [査読有り][通常論文]
   

  Oligodendrocytes and myelin in the corpus callosum of black tremor and normal hamsters aged over 1.5 years were ultrastructurally examined to determine the myelination index (ratio of myelin thickness/diameter of axon), percentage of naked axons, and proportions of oligodendroglial subtypes (light, medium and dark). The mutant hamsters were remarkably hypomyelinated, with a low myelination index and a high proportion of naked axons, and high proportions of the dark subtypes. Serum concentrations of thyroid hormones (T-3 and T-4) in 6-week-old mutant hamsters were 2-fold (T-3) to 3-fold (T-4) higher than those of age-matched normal animals. However, in the aged animals (over 1.5 years old) only T4 levels of the mutant hamsters were higher in the mutant than normal hamsters. The black tremor hamsters were hypomyelinated throughout their life and high serum level of thyroid hormones might have played a role in the hypomyelination.
• Extensive immune-mediated hippocampal damage in mice surviving infection with neuroadapted Sindbis virus

  T Kimura, DE Griffin

  VIROLOGY 311 1 28 - 39 2003年06月 [査読有り][通常論文]
   

  Viral infections of the central nervous system and immune responses to these infections cause a variety of neurological diseases. Infection of weanling mice with Sindbis virus causes acute nonfatal encephalomyelitis followed by clearance of infectious virus, but persistence of viral RNA. Infection with a neuroadapted strain of Sindbis virus (NSV) causes fatal encephalomyelitis, but passive transfer of immune serum after infection protects from fatal disease and infectious virus is cleared. To determine whether persistent NSV RNA is associated with neurological damage, we examined the brains of recovered mice and found progressive loss of the hippocampal gyrus, adjacent white matter, and deep cerebral cortex associated with mononuclear cell infiltration. Mice deficient in CD4(+) T cells showed less tissue loss, while mice lacking CD8(+) T cells showed lesions comparable to those in immunocompetent mice. Mice deficient in both CD4(+) and CD8(+) T cells developed severe tissue loss similar to immunocompetent mice and this was associated with extensive infiltration of macrophages. The number of CD4(+) cells and macrophage/microglial cells, but not CD8(+) cells, infiltrating the hippocampal gyrus was correlated with the number of terminal deoxynucleotidyltransferase-mediated dUTP nick end-labeling positive pyramidal neurons. These results suggest that CD4(+) T cells can promote progressive neuronal death and tissue injury, despite clearance of infectious virus. (C) 2003 Elsevier Science (USA). All rights reserved.
• Passage of equine herpesvirus-1 in suckling mouse brain enhances extraneural virus growth and subsequent hematogenous neuroinvasion

  R Hasebe, T Kimura, K Nakamura, K Okazaki, K Ochiai, R Wada, T Umemura

  JOURNAL OF VETERINARY MEDICAL SCIENCE 64 10 907 - 912 2002年10月 [査読有り][通常論文]
   

  Intracerebral inoculation of field-isolates as well as established strains of equine herpesvirus-1 (EHV-1) in suckling mice results in viral replication in neurons and glial cells and induces encephalitis. By intraperitoneal (i.p.) inoculation, no histological lesion was observed in the central nervous system (CNS) in suckling mice with the EHV-1 HH1 strain (HH1), whereas a neuroadapted variant (NHH1) produced by serial passage of HH1 in the mouse brain caused severe encephalomyelitis after i.p. inoculation. The purpose of this study was to determine the route of neuroinvasion after i.p. inoculation of NHH1 and to clarify the effects of the brain passage on viral neuroinvasion. NHH1, but not HH1, targeted splenic and pulmonary macrophages and omental fat cells on days 1 and 2 post-inoculation (p.i.). From days 1 to 3 p.i., cell-associated viremia was occurred in NHH1-infected mice, but not in HH1-infected mice. On day 4 p.i., viral antigen was detected in a few endothelial cells, perivascular glial cells and neurons in the CNS in NHH1-infected mice. The number of viral antigen-positive cells increased markedly after day 5 p.i. In contrast, no viral antigen-positive cell was detected in the CNS in HH1-infected mice, except for a few nerve cells in the thoracic cord on day 4 p.i. These results suggest that NHH1 neuroinvasion is hematogenous and is correlated with enhanced extraneural virus growth.
• Steroid hormones do not reactivate Neospora caninum in ovariectomized mice

  A Kobayashi, S Katagiri, T Kimura, K Ochiai, T Umemura

  JOURNAL OF VETERINARY MEDICAL SCIENCE 64 9 773 - 777 2002年09月 [査読有り][通常論文]
   

  The direct effects of three steroid hormones (progesterone, estradiol-17beta and corticosterone) on the growth of Neospora caninum (N. caninum) tachyzoite were examined in Vero cells. Subsequently, ovariectomized BALB/c mice infected with N. caninum were treated with physiological concentrations of the steroid hormones for I or 2 weeks. These hormones had no direct effect on the parasite growth in vitro. In the infected mice, there was no significant difference in the parasite distribution and histopathological changes between the hormone-injected and control groups. No mice showed parasitemia at the time of autopsy. These results suggest that physiological levels of steroid hormones (progesterone, estradiol- 17beta and corticosterone) do not reactivate N. caninum in mice.
• Equine herpesvirus-1-induced encephalomyelitis in mice: a comparative study of neuroadapted virus and its parental strain

  R Hasebe, T Kimura, E Sato, K Okazaki, K Ochiai, R Wada, T Umemura

  JOURNAL OF COMPARATIVE PATHOLOGY 127 2-3 118 - 125 2002年08月 [査読有り][通常論文]
   

  Little is known about the neuropathogenicity of equine herpesvirus-1 (EHV-1) in mice. No neurological signs were observed in 6-day-old mice inoculated intracerebrally with the HH1 strain (HH1) of EHV-1. However, 6-day-old mice inoculated intracerebrally with a variant derived by serial passage of HH1 in mouse brain showed severe neurological symptoms and eventually died. Histological analyses were performed on 6-day-old mice inoculated with the neuroadapted HH1 (NHH1) and the parental HH1 strain by the intracerebral, intranasal or intraperitoneal route. All routes of inoculation with NHH1 caused encephalitis, but myelitis was observed only in mice inoculated intraperitoneally. Prominent histological findings were perivascular cuffing sometimes associated with small fibrin thrombi, neuronal and glial degeneration and necrosis, and intranuclear inclusion bodies in neurons, glial cells and ependymal cells. Intracerebral and intranasal inoculation, but not intraperitoneal inoculation, with HH1 induced central nervous system (CNS) lesions that were milder than those in mice inoculated with NHH1. The distribution of viral antigen was more widespread in mice inoculated with NHH1 than with HH1 No viral antigen was detected in the CNS of mice inoculated intraperitoneally with HH1. These results indicate that increased viral multiplication and spreading in the CNS were responsible for the enhanced neurovirulence of NHH1. Although EHV-1 has been considered to be primarily endotheliotropic in horses, both NHH1 and HH1 showed tropism for the parenchymal cells of the CNS in mice, namely neurons, glial cells and ependymal cells. (C) 2002 Elsevier Science Ltd. All rights reserved.
• The invasion routes of neurovirulent A Hong Kong 483/97 (H5N1) influenza virus into the central nervous system after respiratory infection in mice

  CH Park, M Ishinaka, A Takada, H Kida, T Kimura, K Ochiai, T Umemura

  ARCHIVES OF VIROLOGY 147 7 1425 - 1436 2002年 [査読有り][通常論文]
   

  A/Hong Kong/483/97 (H5N1) influenza virus (HK483) isolated from the third patient during the outbreak of chicken and human influenza in Hong Kong in 1997 was shown to be neurovirulent in mice. HK483 was inoculated intranasally to mice, and the invasion routes of the virus in the central nervous system (CNS) were investigated by immunohistochemical and in situ hybridization. The pathological changes consisted of bronchopneumonia, ganglionitis, and nonpurulent encephalomyelitis of the brain stem and the anterior part of the thoracic cord. Viral antigens and viral nucleic acids (RNA and mRNA) were demonstrated in the pterygopalatine, trigeminal and superior ganglions prior to or simultaneously with their detection in the CNS. The antigens and nucleic acids were also observed in the olfactory bulb from an early stage of the infection. In the spinal cord, virus-infected cells were first demonstrated in the grey matter of the thoracic cord. The virus, which primarily replicated in the lungs, was considered to invade the thoracic cord via cardiopulmonary splanchnic nerves and sympathetic nerves. These findings indicate that the virus reached the CNS through afferent fibers of the olfactory, vagal, trigeminal, and sympathetic nerves following replication in the respiratory mucosa.
• Distribution of rabbit haemorrhagic disease virus RNA in experimentally infected rabbits

  T Kimura, Mitsui, I, Y Okada, T Furuya, K Ochiai, T Umemura, C Itakura

  JOURNAL OF COMPARATIVE PATHOLOGY 124 2-3 134 - 141 2001年02月 [査読有り][通常論文]
   

  Adult rabbits were inoculated with liver homogenate from a rabbit haemorrhagic disease (RHD). All experimentally infected rabbits died with typical clinical, gross and histological findings of RHD. Distribution of RHD virus in tissues of the infected rabbits was studied by non-isotopic in-situ hybridization. Both viral plus- and minus-strand RNAs were detected within the cytoplasm of hepatocytes. Kupffer cells and splenic and alveolar macrophages. mainly in morphologically intact cells. Strand-specific reverse transcriptase polymerase chain reaction also demonstrated viral minus-strand RNA as well as plus-strand RNA in the liver, lung and spleen of infected rabbits. Those results suggest that viral replication occurs not only in hepatocytes but also in macrophages. The infected macrophages may contribute to viral dissemination in RHD. (C) 2001 Harcourt Publishers Ltd.
• Role of antibodies in controlling alphavirus infection of neurons

  DE Griffin, S Ubol, P Despres, T Kimura, A Byrnes

  ANTIBODIES IN VIRAL INFECTION 260 191 - 200 2001年 [査読有り][通常論文]
  
• Demonstration of equine herpesvirus-1 gene expression in the placental trophoblasts of naturally aborted equine fetuses

  R Mukaiya, T Kimura, K Ochiai, R Wada, T Umemura

  JOURNAL OF COMPARATIVE PATHOLOGY 123 2-3 119 - 125 2000年08月 [査読有り][通常論文]
   

  Equine herpesvirus-1 (EHV-1) infection was demonstrated in the lung tissue of seven aborted fetuses by immunohistochemical labelling and polymerase chain reaction. The placentas of the fetuses were also examined by non-isotopic in-situ hybridization for the EHV-1 glycoprotein B (gB) gene. Positive hybridization signals were observed in the cytoplasm of trophoblasts, especially in microcotyledons, of all seven placentas, and in villous epithelium of the allantochorion of six placentas. Despite the presence of EHV-1 RNA, EHV-1 antigens were not detected in placentas by immunohistochemical examination. The present study represents the first in-vivo demonstration of the EHV-1 gene in equine trophoblasts. The findings suggest direct cell-to-cell spread of EHV-1 from endometrial cells to trophoblasts. (C) 2000 Harcourt Publishers Ltd.
• The role of CD8(+) T cells and major histocompatibility complex class I expression in the central nervous system of mice infected with neurovirulent Sindbis virus

  T Kimura, DE Griffin

  JOURNAL OF VIROLOGY 74 13 6117 - 6125 2000年07月 [査読有り][通常論文]
   

  Little is known about the role of CD8(+) T cells infiltrating the neural parenchyma during encephalitis induced by neurovirulent Sindbis virus (NSV), NSV preferentially infects neurons in the mouse brain and spinal cord; however, it is generally accepted that neurons can express few if any major histocompatibility complex (MHC) class I molecules. We evaluated the possible roles and interactions of CD8(+) T cells during NSV encephalitis and demonstrated that MHC class I antigen (H2K/D) was expressed on endothelial cells, inflammatory cells, and ependymal cells after intracerebral inoculation of NSV, No immunoreactivity was observed in neurons. On the other hand, in situ hybridization with probes for MHC class I heavy chain, beta 2 microglobulin, and TAP1 and TAP2 mRNAs revealed increased expression in a majority of neurons, as well as in inflammatory cells, endothelial cells, and ependymal cells in the central nervous system of infected mice. NSV-infected neurons may fail to express MHC class I molecules due to a posttranscriptional block or may express only nonclassical MHC class I genes. To better understand the role CD8(+) T cells play during fatal encephalitis induced by NSV, mice lacking functional CD8(+) T cells were studied. The presence or absence of CD8 did not alter outcome, but absence of beta 2 microglobulin improved survival, Interestingly, the intracellular levels of viral RNA decreased more rapidly in immunocompetent mice than in mice without functional CD8(+) T cells, These observations suggest that CD8(+) T cells may act indirectly, possibly via cytokines, to contribute to the clearance of viral RNA in neurons.
• Differences between C57BL/6 and BALB/cBy mice in mortality and virus replication after intranasal infection with neuroadapted Sindbis virus

  DC Thach, T Kimura, DE Griffin

  JOURNAL OF VIROLOGY 74 13 6156 - 6161 2000年07月 [査読有り][通常論文]
   

  Neuroadapted Sindbis virus (NSV), given intranasally, caused fatal encephalitis in 100% of adult C57BL/6 mice and 0% of BALB/cBy mice. Most C57BL/6 mice developed severe kyphoscoliosis followed by hind-limb paralysis, while BALB/cBy mice did not. In situ hybridization for detecting NSV RNA and immunohistochemistry for detecting NSV antigen indicated that virus delivered by this route infected neurons of the olfactory region and spread caudally without infection of ependymal cells. Virus antigen was more abundant and infectious virus increased more rapidly and reached higher levels in C57BL/6 mice than in BALB/cBy mice. Surprisingly, infectious virus was cleared faster in C57BL/6 mice, and this was associated with more rapid production of neutralizing antibody. However, viral RNA was cleared more slowly in C57BL/6 mice. In both mouse strains, more infectious virus was present in the lumbar spinal cord than in the cervical spinal cord. These data suggest that genetic susceptibility to fatal NSV encephalomyelitis is determined at least Tn part by the efficiency of viral replication and spread in the central nervous system. The differences identified in this study provide possible phenotypes for mapping genetic loci involved in susceptibility.
• Suppressive effects of dominant negative ras mutant N116Y on transformed phenotypes of human bladder cancer cells

  T Watanabe, N Shinohara, A Sazawa, Y Kobayashi, Y Ogiso, T Kimura, M Takiguchi, J Yasuda, A Hashimoto, T Koyanagi, N Kuzumaki

  CANCER LETTERS 149 1-2 195 - 202 2000年02月 [査読有り][通常論文]
   

  To investigate the suppressive effect of dominant negative H-ras mutant N116Y on transformed phenotypes, we established two N116Y ras mutant stable transfectant clones (C5, C13) of human bladder cancer cell line, UMUC-2. These N116Y ras mutant transfectants, especially the C5 cells, showed a dramatic change of cellular morphology and significantly reduced growth in soft agar compared to their control. Furthermore, phosphorylation of the Jun NH2-terminal kinase (JNK) was significantly decreased in these transfectants compared to the control. These results suggest that the N116Y-induced suppression of transformed phenotypes in UMUC-2 cells is associated with inhibition of JNK phosphorylation, (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.
• Borna disease virus infection in domestic cats: Evaluation by RNA and antibody detection

  Y Nishino, M Funaba, R Fukushima, T Mizutani, T Kimura, R Iizuka, H Hirami, M Hara

  JOURNAL OF VETERINARY MEDICAL SCIENCE 61 10 1167 - 1170 1999年10月 [査読有り][通常論文]
   

  Borna disease virus (BDV) infection has been suggested to cause spontaneous neurological disease in cats referred to as staggering disease. However the evaluation of BDV infection in neurologically asymptomatic cats remained unclear. In the present study, BDV infected, asymptomatic cats in Tokyo were surveyed both by the presence of plasma antibodies against BDV-p24 and -p40 and by RNA detection in peripheral blood mononuclear cells. Seven of 32 domestic cats (21.9%) were serologically or genetically judged to be BDV-infected. Six cats were positive for anti-BDV antibody and two cats were positive for BDV RNA. Within the 2 RNA-positive cats, only one was positive for anti-BDV antibodies. Furthermore, the findings of anti-BDV-p40 and anti-BDV-p24 antibody-positive cats did not completely overlap. These results suggest that there are neurologically asymptomatic domestic cats infected with BDV present in the Tokyo area.
• Lead poisoning in wild waterfowl in Japan

  K Ochiai, T Kimura, K Uematsu, T Umemura, C Itakura

  JOURNAL OF WILDLIFE DISEASES 35 4 766 - 769 1999年10月 [査読有り][通常論文]
   

  We collected 430 harvested ducks (Anas sp. and Aythya sp.) from nine prefectures in Japan between 1994 and 1997. Fifteen (4%) of 363 birds harvested during and after hunting seasons had one lead pellet each in the proventriculus and gizzard. In addition, 32 (34%) of 93 swans (Cygnus sp.) and two of 14 geese (Anser sp.) found dead from various wetlands had lesions consistent with lead poisoning. One to nine swans suspected of having toxicosis from ingestion of lead shot were found dead each year. Twenty-seven (84%) of the 32 lead-exposured swans were found in Hokkaido Prefecture. We concluded that lead poisoning is still a serious threat to waterfowl in Japan and that there is considerable need for environmental improvement concerning this problem.
• Borna Disease Virus Infection in Domestic Cats: Evaluation by RNA and Antibody Detection

  Yoshii Nishino, Masayuki Funaba, Ryoko Fukushima, Tetsuya Mizutani, Takashi Kimura, Reiko Iizuka, Hiroshi Hirami, Motonobu Hara

  Journal of Veterinary Medical Science 61 10 1167 - 1170 1999年10月 [査読有り][通常論文]
   

  Borna disease virus (BDV) infection has been suggested to cause spontaneous neurological disease in cats referred to as staggering disease. However the evaluation of BDV infection in neurologically asymptomatic cats remained unclear. In the present study, BDV infected, asymptomatic cats in Tokyo were surveyed both by the presence of plasma antibodies against BDV-p24 and -p40 and by RNA detection in peripheral blood mononuclear cells. Seven of 32 domestic cats (21.9%) were serologically or genetically judged to be BDV-infected. Six cats were positive for anti-BDV antibody and two cats were positive for BDV RNA. Within the 2 RNA-positive cats, only one was positive for anti-BDV antibodies. Furthermore, the findings of anti-BDV-p40 and anti-BDV-p24 antibody-positive cats did not completely overlap. These results suggest that there are neurologically asymptomatic domestic cats infected with BDV present in the Tokyo area.
• Canine hemangiopericytoma: an evaluation of metastatic potential

  E Handharyani, K Ochiai, T Kadosawa, T Kimura, T Umemura

  JOURNAL OF VETERINARY DIAGNOSTIC INVESTIGATION 11 5 474 - 478 1999年09月 [査読有り][通常論文]
  
• Complementary DNA cloning, tissue distribution, and synthesis of canine brain natriuretic peptide

  K Asano, M Murakami, D Endo, T Kimura, T Fujinaga

  AMERICAN JOURNAL OF VETERINARY RESEARCH 60 7 860 - 864 1999年07月 [査読有り][通常論文]
   

  Objective-To determine complimentary DNA (cDNA) sequence and tissue distribution of canine brain natriuretic peptide (BNP), and to investigate whether synthesis of canine BNP increases in association with cardiovascular dysfunction. Animals-5 healthy adult mixed-breed dogs and 3 healthy adult Beagles. Procedure-Total RNA was extracted from normal canine hearts and was used in a reverse transcription-polymerase chain reaction IRT-PCR) procedure to isolate canine BNP cDNA. Sequence of the isolated cDNA was analyzed. Gene expression of canine BNP in various tissues from 2 mixed-breed dogs was investigated, using RT-PCR and northern blot analyses. Moreover, messenger RNA (mRNA) expression of canine BNP, using northern blot analysis, was compared between grossly normal hearts from 3 Beagles and hearts from 3 mixed-breed dogs with acute myocardial infarction created by surgical ligation. Results-The cDNA sequence and deduced amino acid residues of canine BNP precursor were 420 base pairs and 140 residues, respectively. Messenger RNA expression of canine BNP was delectable in the atria but not in the ventricles and the other tissues. Messenger RNA expression of canine BNP was, however, detectable in the infarcted portion of the ventricles. The amount of canine BNP mRNA in the infarcted ventricles was significantly increased, compared with that of noninfarcted ventricles. Conclusion-The cDNA sequence of canine BNP was determined. Expression of canine BNP mRNA was detected not only in the atria but also in infarcted ventricles. Synthesis of canine BNP increases in association with ischemic myocardial injury. Canine BNP may be used as an indicator of severity of ventricular myocardial injury.
• Arteriovenous malformation of the cervical spinal cord in a dog

  E Hayashida, K Ochiai, T Kadosawa, T Kimura, T Umemura

  JOURNAL OF COMPARATIVE PATHOLOGY 121 1 71 - 76 1999年07月 [査読有り][通常論文]
   

  An 8-year-old female German Shepherd dog showed first order Horner's syndrome associated with progressive right-sided hemiplegia and megaoesophagus. Intramedullary and leptomeningeal arteriovenous malformation (AVM) was identified in the cervical spinal cord. The morphological characteristics were arteriovenous shunting, intramedullary multiple thromboses and haemorrhage, non-inflammatory necrosis of white and grey matter around the shunt, and intervening neural gliosis with neovascularization. These findings suggested that the malformation induced a focal circulatory disturbance within the cervical spinal cord and that fatal thrombosis was responsible for the sudden onset of the nervous signs and progressive neurological deterioration. This is the first report of intramedullary spinal AVM in a dog. (C) 1999 W.B. Saunders Company Limited.
• Evidence of neoplastic nature and viral aetiology of so-called fowl glioma

  K Ochiai, K Ohashi, T Mukai, T Kimura, T Umemura, C Itakura

  VETERINARY RECORD 145 3 79 - 81 1999年07月 [査読有り][通常論文]
  
• Hepatic lesions in young rabbits experimentally infected with rabbit haemorrhagic disease virus

  D Mikami, JH Park, T Kimura, K Ochiai, C Itakura

  RESEARCH IN VETERINARY SCIENCE 66 3 237 - 242 1999年06月 [査読有り][通常論文]
   

  Twenty young rabbits (eleven 2-week-old and nine 4-week-old) were experimentally infected with rabbit haemorrhagic disease virus (RHDV) to clarify susceptibility. They were killed chronologically up to 96 hours post-inoculation (PI) and examined for lesions. All inoculated rabbits were clinically normal, but grossly minute white or grey spots were detected throughout the liver. Histologically, the lesions consisted of aggregates of lymphocytes, macrophages and heterophils, with or without acidophilic bodies and necrotic hepatocytes. Immunohistochemically, RHDV antigens were found in the degenerated hepatocytes and in macrophages. The cellular aggregates were considered to be a reaction to necrotic hepatocytes infected with RHDV. It was concluded that some hepatocytes are susceptible to RHDV in young rabbits.
• Neurotoxic effects of 2,5-hexanedione on normal and neurofilament-deficient quail

  T Hirai, M Mizutani, T Kimura, K Ochiai, T Umemura, C Itakura

  TOXICOLOGIC PATHOLOGY 27 3 348 - 353 1999年05月 [査読有り][通常論文]
   

  The neurotoxic effects of 2,5-hexanedione (2,5-HD) were investigated using neurofilament (NF)-deficient (Quv) Japanese quail in comparison with normal Japanese quail. Both Quv and normal Japanese quail were inoculated intraperitoneally with 350 mg/kg/day 2,5-HD for 6 consecutive wk. The results of 2,5-HD exposure differed substantially between the 2 strains of Japanese quail. The 2,5 HD-exposed normal quail showed leg paralysis about 4 wk after initiation of dosing. Some treated normal quail fell into dysstasia and died of nutritional disturbances. Histologically, 2,5-HD-treated normal quail had NF-rich axonal swellings and degeneration in the distal parts of the peripheral nerves, spinal cord, and cerebellar peduncles. In contrast, 2,5-HD-injected Quv quail showed tonic convulsion, ataxia gait, severe quivering, and excitation about 2-3 days after administration. Some treated Quv birds died immediately after systemic tonic convulsion, probably because of asphyxia. Although all treated Quv quail showed neurologic signs, there were no recognizable 2,S-HD-induced lesions in the nervous system. After about 4-6 wk of dosing, 2,5-HD induced distal axonopathy in normal quail and acute neurotoxicity in Quv quail.
• Single-step reverse transcriptase polymerase chain reaction for detection of Borna disease virus RNA in vitro and in vivo

  T Mizutani, M Ogino, Y Nishino, T Kimura, H Inagaki, D Hayasaka, H Kariwa, Takashima, I

  JAPANESE JOURNAL OF VETERINARY RESEARCH 46 4 165 - 169 1999年02月 [査読有り][通常論文]
   

  There are few copies of Borna disease virus (BDV) genome in peripheral blood mononuclear cells and no reliable standard reverse transcriptase-polymerase chain reaction (RT-PCR) method for the detection of BDV RNA, which is both highly sensitive and free of contamination. Single-step RT-PCR, in which both reverse transcription and amplification by Tag DNA polymerase work efficiently in a single buffer, was applied to detect the p24 region of BDV RNA in vitro and in vivo. Using in vitro synthesized RNA, it was demonstrated that at least 100 copies of BDV RNA could be detected and the sensitivity and specificity were nearly equal to those obtained by RT-nested PCR. We could detect BDV RNA from more than 1 pg of cellular RNA obtained from BDV-persistently infected MDCK cells. Furthermore, this method was successfully performed on brain specimens obtained from a BDV-infected rat at II weeks post-inoculation. This single-step RT-PCR method will be convenient for detecting limited amounts of BDV RNA in various cells and tissue samples.
• A single-tube RT-PCR method for the detection of Borna disease viral genomic RNA

  T Mizutani, M Ogino, Y Nishino, T Kimura, H Kariwa, K Tsujimura, H Inagaki, Takashima, I

  JAPANESE JOURNAL OF VETERINARY RESEARCH 46 2-3 73 - 81 1998年11月 [査読有り][通常論文]
   

  For detecting Borna disease virus (BDV) genomic stranded RNA, single-tube reverse transcription-polymerase chain reaction (St RT-PCR) was developed to equal the sensitivity of RT-nested PCR but with reduced risk of contamination. BDV-genomic stranded RNA was synthesized in vitro using plasmid cDNA of BDV p24 region as a template and RNA was also extracted from BDV-persistently infected MDCK (MDCK/BDV) cells. Both RNAs were amplified by St RT-PCR in which a single round of RT and a single round of PCR were performed in the same tube. Ten copies of synthesized RNA could be amplified by St RT-PCR, indicating that St RT-PCR method is as sensitive as the ordinary RT-nested PCR method. Furthermore, this method was applied to quantify the exact copy number of genomic RNA in MDCK/BDV cells. Signals were obtained from the samples containing more than 1 pg total cellular RNA. From the results, approximately 100 copies of BDV genomic RNA exist in one MDCK/BDV cell. BDV genomic RNA from the in viva RNA samples using St RT-PCR, indicating this method is applicable for the epidemiological study of BDV without contamination.
• イヌの脳性ナトリウム利尿ペプチド遺伝子のクローニング

  浅野 和之, 村上 正晃, 遠藤 大二, 木村 享史, 奥村 正裕, 廉澤 剛, 藤永 徹

  日本獣医学会学術集会講演要旨集 125回 267 - 267 (公社)日本獣医学会 1998年03月
• Gizzard adenocarcinoma in an aged Humboldt penguin (Spheniscus humboldti)

  KO Cho, T Kimura, K Ochiai, C Itakura

  AVIAN PATHOLOGY 27 1 100 - 102 1998年02月 [査読有り][通常論文]
   

  Spontaneous gizzard adenocarcinoma was found in a 15-year-old male Humboldt penguin (Spheniscus humboldti). The lesser curvature of the gizzard wall was extensively and markedly thickened with a hard white tumour mass. Histopathologically, the tumour in the gizzard consisted of two structures: small to large acini lined by cuboidal or flattened cells with poor stroma in the mucosa, and undifferentiated tumour cells arranged singly or in cords with acinar structures separated by dense fibrous tissue in the muscular layer. Small white metastatic tumours of the intestine and the pancreas had a similar histological appearance to that in the muscular layer of the gizzard. From the location of tumour in the gizzard and the stainability with combined alcian blue pH 2.5-periodic acid Schiff, it was considered that this tumour might originate from the lining cells of the tip of the gizzard mucosal folds.
• Bronchiolar-alveolar carcinoma in a cow

  Y Okada, K Ochiai, K Osaki, T Kimura, C Itakura

  JOURNAL OF COMPARATIVE PATHOLOGY 118 1 69 - 74 1998年01月 [査読有り][通常論文]
   

  Bronchiolar-alveolar carcinoma was observed in the lung of an 8-year-old Holstein cow. Grossly, the lung contained multiple tumour masses, which were solid, yellowish-white in colour, and firm in consistency. These tumours also occurred in the liver, pancreas, uterus and lymph nodes in the thoracic, abdominal and pelvic cavities. Histologically, the masses were composed of abundant fibrous stroma and proliferating atypical cuboidal epithelial cells, occasionally forming glandular structures. Electron microscopy revealed that the neoplastic cells had microvilli and lamellar bodies in the cytoplasm, suggesting that they originated from immature respiratory epithelial cells differentiating towards either Clara cells or type II pneumocytes. (C) 1998 W.B. Saunders Company Limited.
• Significance of Marek's disease virus serotype 1-specific phosphorylated proteins in Marek's disease skin lesions

  KO Cho, D Endoh, T Kimura, K Ochiai, C Itakura

  AVIAN PATHOLOGY 26 4 707 - 720 1997年12月 [査読有り][通常論文]
   

  Skin tissues were obtained from 12 chickens showing Marek's disease (MD) skin tumour induced by a very virulent MD/5 strain of Marek's disease virus serotype 1 (MDV1) and taken from 24 field broiler chickens (8 to 9-weeks-old) manifesting MD skin tumour. They were examined by the immunohistochemical method in order to examine the topographic expression of MDV1-specific phosphorylated proteins (PP) and by the reverse-transcription polymerase chain reaction following Southern hybridization to analyse their gene transcriptional activity. PP were massively detected and their gene transcripts were abundantly expressed only in the necrotic areas of some experimentally-induced MD cutaneous lymphoma lesions. Strong PP-positive cells were degenerate or necrotic lymphoblasts. The other field or experimentally-induced MD cutaneous lymphoma lesions, even those consisting almost completely of tumorous lymphoblasts, had only a few PP-positive lymphoid cells and few gene transcripts. Necrotic areas of feather pulp MD lymphoma lesions consisting mainly of degenerate or necrotic lymphoblasts and cytolycic feather follicle epithelium (FFE) were also strongly positive for PP. PPs and their gene transcripts were detectable only in the cytolytically infected cells such as FFE and tumorous lymphoblasts converting to the abortic state.
• Apocrine gland tumor of the eyelid in a dog

  T Hirai, M Mubarak, T Kimura, K Ochiai, C Itakura

  VETERINARY PATHOLOGY 34 3 232 - 234 1997年05月 [査読有り][通常論文]
   

  A 13-year-old male Shetland Sheepdog with progressive exophthalmos had a neoplastic mass in the ocular adnexa. Histologically, this neoplasm was composed of duct-forming epithelial cells with decapitation secretion. Tumor cells invaded the globe through the tunica conjunctiva and replaced the vitreous body. The cornea, iris, ciliary body, and retina were extensively destroyed. Both the epithelial and spindle-shaped myoepithelial cells showed nuclear atypia and mitotic activity in the globe. The primary tumor was diagnosed as adenocarcinoma, probably originating from apocrine sweat glands of the eyelid, and the infiltrating intraocular neoplasm was diagnosed as a malignant mixed tumor.
• 静岡県における兎出血病の発生．

  伊藤謙一, 川嶋和晴, 大庭芳和, 播谷亮, 木村享史, 板倉智敏

  日本獣医師会雑誌 50 9 523 - 526 日本獸医師会 1997年 [査読有り][通常論文]
   

  岡県の一観光牧場で, 9日間に成兎60羽中53羽 (88.3%) が沈うつ症状を示して発症後数時間で死亡し, 死亡3例について病理学的に検索したところ, 兎出血病 (Rabbit hemorrhagic disease: RHD) に特微的な病変が認められた. すなわち, 組織学的に壊死性肝炎, 肺・腎臓における播種性血管内凝固が認められ, 抗RHDウィルスIgGによる免疫染色で肝細胞に陽性反応が認められた. また, 電子顕微鏡学的には, 壊死肝細胞細胞質にカリシウイルス様粒子が観察された. 死亡例の肝臓乳剤を接種された2羽の兎で本病の再現がなされ, 肝臓からカリシウイルス粒子が精製された. 今回の例は1994年 (北海道) に続いてわが国における第2件目であった.
• Inherited muscular disorder in mutant Japanese quail (Coturnix coturnix japonica): An immunohistochemical study

  S Tanaka, IS Braga, T Kimura, K Ochiai, C Itakura, M Mizutani

  JOURNAL OF COMPARATIVE PATHOLOGY 115 2 139 - 150 1996年08月 [査読有り][通常論文]
   

  Cryostat sections of myofibres from the Musculus pectoralis thoracicus of a newly established mutant strain (LWC) of Japanese quail with a myotonic dystrophy-like myopathy were labelled with antibody against myosin heavy chain (MHC) isoforms and neural cell adhesion molecule (N-CAM). The characteristic lesions found in sections of muscle of LWC quail stained with haematoxylin and eosin were type 2B fibre atrophy, sarcoplasmic masses, and ring fibres. Immunohistochemical examination failed to distinguish type 2A and 2B fibres in the LWC quail. Antibody to adult fast MHC, which reacted only with type 2A fibres in normal quail, reacted in LWC quail with type 2B fibres, and to a limited degree with type 2A fibres. Sarcoplasmic masses reacted with both fast and slow MHC antibodies. Some masses also reacted with N-CAM antibody, but apparently independently of similar reactions in fibres. These findings suggest that the changes observed in the myofibres of the LWC quail were not neurogenic but represented defects in both the plasma membrane and intermediate filaments. (C) 1996 W.B. Saunders Company Limited
• Sequential skin lesions in chickens experimentally infected with Marek's disease virus

  KO Cho, M Mubarak, T Kimura, K Ochiai, C Itakura

  AVIAN PATHOLOGY 25 2 325 - 343 1996年06月 [査読有り][通常論文]
   

  Skin biopsies taken at weekly intervals from the same specific-pathogen free (SPF) chickens inoculated with Md/5 Marek's disease virus revealed two distinctive patterns of perifollicular cutaneous lesions, tumour-associated and non-tumour-associated. The tumour-associated pattern was subdivided into two types. The progressive type was manifested by a continuous increase of lymphoid cell aggregates (LCA) in the skin, resulting in the development of gross skin rumours with or without visceral rumours, and the regressive type showed initially increased and finally regressed LCA in the skin, associated with the development of visceral rumours. The non-tumour-associated pattern was characterized by initial transient small LCA in the skin without evidence of tumour formation. Birds with the tumour-associated pattern, regardless of type, had persistent nuclear inclusions (NI) and positive reactions against MDV1-specific phosphorylated polypeptides in the feather follicle epithelium (FFE) and initial R(1)-type (consisting mainly of small lymphocytes with a few lymphoblasts) to advanced T-type (consisting predominantly of lymphoblasts) feather pulp lesions (FPL). On the other hand, birds with the non-tumour-associated pattern formed transient NI and positive reactions against MDV1-specific phosphorylated polypeptides in the FFE and R(1)-type to R(2)-type (consisting mainly of plasma cells with oedema) FPL. Antigen-positive lymphoid cells against MDV1-specific phosphorylated polypeptides were detected in both inflammatory and tumourous lesions, especially in the necrotic tumour lesions in the skin of birds showing the progressive type.
• Inherited muscular disorder in mutant Japanese quail (Coturnix coturnix japonica): An ultrastructural study

  S Tanaka, IS Braga, T Kimura, K Ochiai, C Itakura, M Mizutani

  JOURNAL OF COMPARATIVE PATHOLOGY 114 3 325 - 337 1996年04月 [査読有り][通常論文]
   

  Ultrastructural study of muscles taken from a mutant (LWC) strain of Japanese quail with myotonia showed type 2 fibre atrophy, ring fibre formation, sarcoplasmic masses, and ''moth-eaten'' fibres. In these abnormal fibres, the most characteristic feature was the loss of interconnection among the myofibrils, mitochondria, and T tubules. Apparently normal muscle fibres often showed mild changes, such as proliferation of T tubules and enlarged sarcoplasmic areas with increased glycogen granules and ribosomes at the periphery of the fibres. The study suggested that one possible cause of these ultrastructural changes was a defect in cytoskeleton of muscle cells, especially in intermediate filaments. (C) 1996 W.B. Saunders Company Limited
• INHERITED MUSCULAR DISORDER IN MUTANT JAPANESE-QUAIL (COTURNIX-COTURNIX-JAPONICA) - RELATIONSHIP BETWEEN THE DEVELOPMENT OF MUSCLE LESIONS AND AGE

  IS BRAGA, S TANAKA, T KIMURA, C ITAKURA, M MIZUTANI

  JOURNAL OF COMPARATIVE PATHOLOGY 113 2 131 - 143 1995年08月 [査読有り][通常論文]
   

  The progression of the pathological changes that occur in the skeletal muscle was examined in 19 Japanese quail of the LWC strain, affected with an autosomal dominant inherited muscular disorder producing electrical myotonia. The muscle samples were obtained every 10 days from 20 to 70 days of age. Muscle samples from 18 age-matched commercial quail were used as normal controls. Characteristic histological lesions found in the skeletal muscles included sarcoplasmic masses, ringed fibres, internal migration of nuclei and fibre size variation. These lesions, which mainly occurred in the proximal muscles, appeared first in the pectoral region and later in the muscles of the thoracic and pelvic limbs. The most predominant lesion observed at all ages consisted of sarcoplasmic masses. The presence of histological changes did not affect muscle fibre typing by two staining methods, for myosin ATPase at pH 4.5, and by NADH-TR stain. The histological changes were observed in type 2A and less commonly in 2B fibres, but not in type 1. The pectoralis thoracicus muscle, in which lesions were particularly common, showed abnormally large type 2B muscle fibres at 20 days of age. These fibres began to decrease in size at 30 days of age, and at 70 days had become strikingly atrophic, their diameter being only about half that observed at 20 days. The atrophic type 2B muscle fibres were eventually replaced by lipocytes. Chronological staging of the histopathological changes in muscle was impossible since no inter-relationship was observed between the age of the quail, the severity of clinical signs and the extent of muscle lesions. This variability in the severity and age of onset may have been due to the variable expression or incomplete penetrance of the defective gene. Because the disorder is hereditary and progressive in nature, it can be classified as a type of progressive muscular dystrophy. (C) 1995 Academic Press Limited
• ANALYSIS OF VIRUS-CELL BINDING CHARACTERISTICS ON THE DETERMINATION OF JAPANESE ENCEPHALITIS-VIRUS SUSCEPTIBILITY

  T KIMURA, J KIMURAKURODA, K NAGASHIMA, K YASUI

  ARCHIVES OF VIROLOGY 139 3-4 239 - 251 1994年 [査読有り][通常論文]
   

  The susceptibility of fourteen established cell lines to infection with Japanese encephalitis virus (JEV) was assayed using an indirect fluorescent antibody technique. In kinetic studies, the degree of binding and internalization of JEV allowed the identification of high susceptibility and low-susceptibility cells. Scatchard analysis showed that JEV specifically bound to high-susceptibility Vero cells with greater affinity than to low-susceptibility NRK cells. Microinjection of viral genomic RNA into NRK cells induced highly efficient production of viral antigen and infectious virions. A hemagglutinin-inhibiting monoclonal antibody against JEV (MAb 301) inhibited the binding of JEV to the Vero and NRK cells. JEV was found to bind to a 74K molecule present in the membrane fraction of Vero cells and this binding was inhibited by MAb 301. Importantly, the 74K molecule was not detected in the membrane faction of NRK cells. These results suggest that early events in the JEV-cell interaction influence the susceptibility of cells to infection, and in particular suggests that the 74K molecule may be a possible candidate or component of the cellular receptor for JEV.
• キスカル酸レセプター刺激によるイノシトールリン脂質代謝応答の加齢ラット海馬での増大．

  斎藤巨志, 中川勇三, 石原高文, 木村享史, 長嶋和郎

  神経化学 29 472 - 473 1990年 [査読有り][通常論文]
  

MISC

• イヌ血管肉腫におけるヒストンラクチル化とグルタミン代謝の役割の解析

  鈴木玲海, 青島圭佑, 山崎淳平, 安井博宣, 平島一輝, 木村享史 がんと代謝研究会プログラム&抄録集 9th 2023年
• イヌ血管肉腫におけるDNAメチル化異常とDNAメチル化阻害による抗腫瘍効果

  鹿又崚, 青島圭佑, 青島圭佑, 鈴木玲海, 山崎淳平, 山崎淳平, 木村享史 日本獣医学会学術集会講演要旨集 166th 2023年
• イヌ血管肉腫におけるヒストンラクチル化およびグルタミン代謝の役割の解析

  鈴木玲海, 青島圭佑, 山崎淳平, 安井博宣, 平島一輝, 木村享史 日本癌学会学術総会抄録集(Web) 82nd 2023年
• Burkholderia thailandensis感染マクロファージ細胞株RAW264におけるパイロトーシスに対するCOX-2の影響

  市川世識, 青島圭佑, 小林篤史, 木村享史 日本獣医学会学術集会講演要旨集 164th (CD-ROM) 2021年
• ヒストン脱アセチル化酵素阻害剤およびブロモドメイン阻害剤はイヌ血管肉腫細胞株に細胞死を誘導する

  鈴木玲海, 青島圭佑, 小林篤史, 木村享史 日本獣医学会学術集会講演要旨集 164th (CD-ROM) 2021年
• 獣医病理学研修会出題標本 No.1248.コウジョウセンガゼルの肺(北海道大学)

  木村享史, ERDEMSURAKH Ochbayar, 青島圭佑, 小林篤史 日本獣医学会学術集会講演要旨集 163rd 2020年
• 鉛散弾の経口投与によるバルバリーガモの鉛の血中濃度の経時変化および体内分布の解析

  佐藤裕, 石井千尋, 中山翔太, 一瀬貴大, 齊藤慶輔, 渡邊有希子, 小笠原浩平, 鳥本亮太, 小林篤史, 木村享史, 池中良徳, 石塚真由美 日本獣医学会学術集会講演要旨集 162nd 504 2019年08月20日 [査読無し][通常論文]
  
• ラット由来細胞株Rn33Bの神経分化はEHV-1感受性を低下させる

  港江利奈, 青島圭佑, 小林篤史, 木村享史 日本ウイルス学会学術集会プログラム・予稿集(Web) 67th 2019年
• ドキソルビシン耐性イヌ血管肉腫細胞株の樹立と薬剤耐性機構の解析

  森田 敦也, 青島 圭佑, 小林 篤史, 木村 享史 日本獣医学会学術集会講演要旨集 161回 303 -303 2018年08月 [査読無し][通常論文]
  
• イヌ血管肉腫におけるCXCR7シグナル伝達機構の解析

  大西 慎一, 青島 圭佑, 小林 篤史, 木村 享史 日本獣医学会学術集会講演要旨集 160回 321 -321 2017年08月 [査読無し][通常論文]
  
• ウマ主要組織適合遺伝子複合体クラスI遺伝子導入マウスのウマヘルペスウイルス1型に対する感受性の検討

  港 江利奈, 青島 圭佑, 小林 篤史, 木村 享史 日本獣医学会学術集会講演要旨集 159回 319 -319 2016年08月 [査読無し][通常論文]
  
• 北大農場で発生したリステリア症罹患羊の飼育環境からのリステリア属菌の分離とゲノム解析

  長谷部 理絵, 青島 圭佑, 棟居 佳子, 三谷 朋弘, 平井 佑治, 山崎 剛士, 木村 享史, 堀田 基広 日本獣医学会学術集会講演要旨集 159回 418 -418 2016年08月 [査読無し][通常論文]
  
• 2010年に稚内で分離された2株の高病原性鳥インフルエンザウイルスの病原性比較

  直亨則, 梶原将大, 丸山隼輝, 木村享史, MANZOOR Rashid, 村松美笑子, 岡松正敏, 宮本洋子, 吉田玲子, 迫田義博, 喜田宏, 高田礼人 日本ウイルス学会学術集会プログラム・抄録集 61st 150 2013年10月29日 [査読無し][通常論文]
  
• Erratum: Immunohistochemical molecular expression profile of metastatic brain tumor for potent personalized medicine (Brain Tumor Pathol DOI: 10.1007/s10014-012-0124-y)

  Kato Y, Nishihara H, Yuzawa S, Mohri H, Kanno H, Hatanaka Y, Kimura T, Tanino M, Tanaka S Brain Tumor Pathology 30 (4) 266 -267 2013年10月 [査読無し][通常論文]
  
• 超音波検査により診断が可能であった子宮捻転の犬の1例

  中村 健介, 山崎 真大, 大田 寛, 森下 啓太郎, 木村 享史, 滝口 満喜 日本獣医画像診断学会抄録 54回 28 -28 2013年09月 [査読無し][通常論文]
  
• オートファジーはウエストナイルウイルスの増殖を抑制する

  小林進太郎, 大場靖子, 山口宏樹, 佐々木道仁, 長谷部理絵, 木村享史, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 61st 2013年
• 霊長類動物からの新規ポリオーマウイルスの検出

  山口宏樹, 小林進太郎, 石井秋宏, 小川寛人, 木村享史, 澤洋文, 大場靖子 日本分子生物学会年会プログラム・要旨集(Web) 36th 2013年
• インフルエンザウイルス感染におけるマウス肺の病理学的変化の観察

  澤洋文, MUTO Mton‐Akio, 寸田祐嗣, 服部ともえ, 藤倉大輔, 中山洋佑, 宮崎忠昭, 丸山光生, 木村享史 日本ウイルス学会学術集会プログラム・抄録集 60th 334 2012年10月31日 [査読無し][通常論文]
  
• ザンビアの野生動物におけるオルソポックスウイルス感染の疫学調査

  大場靖子, 石井秋宏, トーマス 由佳, 小川寛人, 中村一郎, 木村享史, 森川茂, 西條政幸, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 60th 443 2012年10月31日 [査読無し][通常論文]
  
• 両側性子宮分節性無形成から片側性子宮角捻転に至った犬の1例

  横山望, 中村健介, 木村享史, 大田寛, 山崎真大, 滝口満喜 北海道獣医師会雑誌 56 (8) 418 2012年08月10日 [査読無し][通常論文]
  
• ウエストナイルウイルス感染神経細胞におけるユビキチン化タンパク質の蓄積

  小林進太郎, 小林進太郎, 大場靖子, 山口宏樹, 木村享史, 澤洋文, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• 霊長類動物におけるパラインフルエンザウイルスの疫学調査

  佐々木道仁, 石井秋宏, 大場靖子, トーマス由佳, 小川寛人, 中村一郎, 木村享史, 澤洋文, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• ザンビアにおける霊長類動物からの新規ポリオーマウイルスの検出

  山口宏樹, 山口宏樹, 大場靖子, 小林進太郎, 小林進太郎, 石井秋宏, 小川寛人, 中村一郎, THOMAS由佳, 木村享史, 澤洋文, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 60th 2012年
• 霊長類動物におけるパラインフルエンザウイルス3型の疫学調査

  佐々木道仁, 石井秋宏, 大場靖子, THOMAS Yuka, 小川寛人, 中村一郎, 木村享史, 澤洋文, 澤洋文 日本分子生物学会年会プログラム・要旨集(Web) 35th 2012年
• ウマMHC class Iが示すウマヘルペスウイルス1型レセプター機能の解析

  佐々木道仁, 五十嵐学, 澤洋文, 伊藤公人, 福士秀人, 木村享史 日本獣医学会学術集会講演要旨集 152nd 239 2011年08月31日 [査読無し][通常論文]
  
• ザンビア共和国の霊長類動物におけるポリオーマウイルスの疫学調査

  山口宏樹, 小林進太郎, 大場靖子, 石井秋宏, 小川寛人, Thomas由佳, 木村享史, 澤洋文 日本獣医学会学術集会講演要旨集 152nd 2011年
• 野生齧歯動物からの新規ポリオーマウイルスの検出

  大場靖子, 小林進太郎, 中村一郎, 石井秋宏, 木村享史, 澤洋文 日本ウイルス学会学術集会プログラム・抄録集 58th 445 2010年10月15日 [査読無し][通常論文]
  
• Disruption of Intracellular Vesicular Trafficking by Agnoprotein is Essential for Viroporin Activity and JC Virus Replication

  Tadaki Suzuki, Yasuko Orba, Yoshinori Malcino, Yuki Okada, Yuji Sunden, Takashi Kimura, Hideki Hasegawa, Tetsutaro Sata, William W. Hall, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 16 84 -84 2010年10月 [査読無し][通常論文]
  
• Large T antigen promotes JC virus replication in G2 arrest by inducing G2 checkpoint signaling

  Yasuko Orba, Tadaki Suzuki, Takashi Kimura, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 16 64 -65 2010年10月 [査読無し][通常論文]
  
• Cys80 of JC Virus Capsid Protein, VP1 is Essential for Intrapentamer Disulfide Bond and Pentamer Formation

  Shintaro Kobayashi, Tadaki Suzuki, Manabu Igarashi, Noriko Ohtake, Keita Nagakawa, Kimihito Ito, Kenichi Niikura, Takashi Kimura, Harumi Kasamatsu, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 16 45 -46 2010年10月 [査読無し][通常論文]
  
• 新規エントリーレセプターを介したウマヘルペスウイルス1型の細胞内侵入機構の解析

  佐々木道仁, 長谷部理絵, 福士秀人, 谷山弘行, 澤洋文, 木村享史 日本獣医学会学術集会講演要旨集 150th 2010年
• Inhibition of the SDF-1F?-CXCR4 Axis by the CXCR4 Antagonist AMD3100 Suppresses the Migration of Human ATLL Cells and Murine Lymphoblastoid Cells from HTLV-I Tax Transgenic Mice

  A. Kawaguichi, Hidekatsu, I, H. Hasegawa, H. Sawa, M. Ogata, T. Tsuji, T. Kimura, T. Sata, W. W. Hall, Y. Orba AIDS RESEARCH AND HUMAN RETROVIRUSES 25 (11) 1212 -1212 2009年11月 [査読無し][通常論文]
  
• CXCR4アンタゴニスト(AMD3100)を用いたSCIDマウスにおけるTaxトランスジェニックマウス由来腫瘍細胞の組織浸潤抑制

  川口晶, 辻隆裕, 大場靖子, 木村享史, 相内章, 伊波英克, 緒方正男, 佐多徹太郎, 澤洋文, 長谷川秀樹 日本ウイルス学会学術集会プログラム・抄録集 57th 445 2009年10月01日 [査読無し][通常論文]
  
• CNS lymphomatoid granulomatosis with lymph node and bone marrow involvements. (vol 28, pg 640, 2008)

  A. Takiyama, H. Nishihara, U. Tateishi, T. Kimura, W. Lei, K. Marukawa, T. Itoh, S. Hashino, K. Nagashima, S. Tanaka NEUROPATHOLOGY 29 (4) 520 -520 2009年08月 [査読無し][通常論文]
  
• Involvement of Adaptor Protein Crk in Malignant Features of Human Mesothelioma.

  M. A. Tanino, L. Wang, S. Kohsaka, T. Kimura, K. Sasai, H. Nishihara, M. Tsuda, S. Yano, S. Tanaka AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE 179 2009年 [査読無し][通常論文]
  
• Viroporin activity of JCV agnoprotein

  Tadaki Suzuki, Yasuko Orba, Yuji Sunden, Takashi Kimura, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 15 96 -97 2009年 [査読無し][通常論文]
  
• ヒトATL細胞及びTaxトランスジェニックマウス由来腫瘍細胞のSDF‐1αに対する走化性

  川口晶, 大場靖子, 木村享史, 伊波英克, 緒方正男, 辻隆裕, 佐多徹太郎, 澤洋文, 長谷川秀樹 日本ウイルス学会学術集会プログラム・抄録集 56th 247 2008年10月01日 [査読無し][通常論文]
  
• 気胸を併発した先天性肺葉性肺気腫の犬の1例

  上條弘美, 高木哲, 大田寛, 山崎真大, 木村享史, 滝口満喜 北海道獣医師会雑誌 52 (8) 420 2008年08月01日 [査読無し][通常論文]
  
• ATL腫瘍細胞の走化性におけるCXCR4を介したシグナル経路の解析

  川口晶, 大場靖子, 木村享史, 伊波英克, 緒方正男, 佐多徹太郎, 澤洋文, 長谷川秀樹 補体シンポジウム講演集 45th 179 2008年07月 [査読無し][通常論文]
  
• JC virus agnoprotein modifies plasma membrane permeability and facilitates

  Tadaki Suzuki, Yuki Okada, Yasuko Orba, Yuji Sunden, Takashi Kimura, Kazuo Nagashima, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 13 128 -128 2007年 [査読無し][通常論文]
  
• Pharmacological cdk inhibitor suppresses JC virus proliferation

  Yasuko Orba, Yuji Sunden, Tadaki Suzuki, Takashi Kimura, Shinya Tanaka, Kazuo Nagashima, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 13 109 -110 2007年 [査読無し][通常論文]
  
• Biological effects of Natalizurnab on JC virus infectivity in its permissive human neural cell lines in vitro

  Tadaki Suzuki, Satoko Yamanouchi, Yuji Sunden, Yasuko Orba, Shinya Tanaka, Takashi Kimura, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 13 127 -127 2007年 [査読無し][通常論文]
  
• Visualization of West Nile virus particles for examination on the cellular entry mechanism

  Yoshinori Makino, Tadaki Suzuki, Rie Hasebe, Akihiko Maeda, Hidehiro Takahashi, Takashi Kimura, Hirofumi Sawa JOURNAL OF NEUROVIROLOGY 13 100 -100 2007年 [査読無し][通常論文]
  
• Efficacy of intracerebral vaccination against pseudorabies virus in mice

  Jae-Ho Shin, Yoshihiro Sakoda, Jae Hoon Kim, Tomohisa Tanaka, Hiroshi Kida, Takashi Kimura, Kenji Ochiai, Takashi Umemura JOURNAL OF NEUROIMMUNOLOGY 178 114 -114 2006年09月 [査読無し][通常論文]
  
• スリランカ津波被災地ならびにその近郊に生息する野生げっ歯類の病理学的検査

  木村享史, 沢洋文, 片倉賢, 中村一郎, 有川二郎, 松本芳嗣, RAJAPAKSE R.P.V.Jayanthe, 高島郁夫, 梅村孝司 日本獣医学会学術集会講演要旨集 140th 72 -72 2005年08月31日 [査読無し][通常論文]
  
• ホジキン型リンパ腫が疑われた猫の1例

  岡崎和子, 滝口満喜, 木村享史, 松田英一郎, 稲葉睦 北海道獣医師会雑誌 48 (8) 319 2004年08月01日 [査読無し][通常論文]
  
• 超音波検査によりしん出性収縮性心膜炎と診断された犬の1例

  浜本耕平, 滝口満喜, 伊藤啓, 木村享史, 廉沢剛, 稲葉睦 北海道獣医師会雑誌 47 (8) 315 2003年08月01日 [査読無し][通常論文]
  
• 神経症状を呈するネコにおけるボルナ病ウイルス感染の診断と病体の調査

  西野 佳以, 吉田 倫子, 田所 真弓, 栗田 吾郎, 辻本 元, 奥村 正裕, 木村 享史, 水谷 哲也 日本獣医学会学術集会講演要旨集 129回 81 -81 2000年03月 [査読無し][通常論文]
  
• 日本で発生した兎出血病由来ウイルスを用いての実験病理学的研究

  三井 一鬼 日本獣医学会学術集会講演要旨集 123回 96 -96 1997年03月 [査読無し][通常論文]
  

書籍等出版物

• 動物病理学各論第3版

  木村享史 (担当:共編者(共編著者)範囲:第1章心臓P1-16; 第3章体腔P78-81)
  文永堂出版 2021年03月 (ISBN: 9784830032806) xv, 509p
• 動物病理カラーアトラス第2版

  木村享史 (担当:共編者(共編著者)範囲:第1編Ⅲ心内膜の病変P4；心筋の病変P10-11；第5編Ⅱ肝臓の病変P142)
  文永堂出版 2018年01月 (ISBN: 9784830032684) xvii, 320p
• 動物病理学総論第3版．日本獣医病理学会編

  木村 享史 (担当:分担執筆範囲:第7章第4節 炎症のメディエーター p.129-135，第7章第6節第3項 肉芽腫性炎症に分類される疾患p.148-152)
  文永堂出版 2013年04月
• 動物病理学各論第2版．日本獣医病理学会編．

  木村 享史 (担当:分担執筆範囲:第1章第2節第4項 リンパ管 p.26-27，第1章第2節第5項 血管およびリンパ管の腫瘍p.28-29，第3章第2節 腹腔，腹膜 p.77-81)
  文永堂出版 2010年03月
• 新獣医学辞典．新獣医学辞典編集委員会編．

  木村 享史 (担当:分担執筆範囲:壊死後性肝硬変 p.122，間質性肺炎 p.238，奇異性塞栓症 p.259，クロイツフェルト‐ヤコブ病 p.340)
  チクサン出版社 2008年01月
• 動物病理カラーアトラス．日本獣医病理学会編．

  木村 享史 (担当:分担執筆範囲:第5編 消化器系Ⅱ 5-8. 兎出血病 p.115)
  文永堂出版 2007年02月

講演・口頭発表等

• ウイルス感染と免疫  [招待講演]

  木村 享史

  日本獣医病理学会スライドセミナー“炎症と免疫” 2013年03月 公開講演，セミナー，チュートリアル，講習，講義等 東京大学
• Seminar Public Awareness “Surveillance of Potential Pathogens in Fruitbats in Indonesia”  [招待講演]

  Takashi Kimura

  Joint Seminar between National Committee on Zoonosis and Bogor Agricultural University 2013年03月 公開講演，セミナー，チュートリアル，講習，講義等 IPB International Convention Center, Bogor, Indonesia
• MHC class I を介したウマヘルペスウイルス1型の細胞内侵入機構  [招待講演]

  木村 享史

  第６回獣医学科特別セミナー 2011年08月 公開講演，セミナー，チュートリアル，講習，講義等 山口大学
• Immune responses to Sindbis virus infection determine the outcome of acute encephalitis in mice.  [通常講演]

  Takashi Kimura

  Joint Symposium between Hokkaido University Graduate School of Veterinary Medicine and Seoul National University College of Veterinary Medicine-Second COE International Symposium for Zoonosis Control- 2003年12月 シンポジウム・ワークショップパネル（指名） Seoul National University, Korea
• 中枢神経系におけるウイルス感染の免疫学的制御  [招待講演]

  木村 享史

  2003年度第5回生物研セミナー 2003年11月 公開講演，セミナー，チュートリアル，講習，講義等 麻布大学

担当経験のある科目（授業）

一般教養演習（フレッシュマンセミナー） 病気を考える北海道大学
神経・運動器病理学北海道大学獣医学部・帯広畜産大学畜産学部共同獣医学課程
泌尿器病理学北海道大学獣医学部・帯広畜産大学畜産学部共同獣医学課程
先端獣医科学科目 獣医病理学特論北海道大学
病理学総論北海道大学
人獣共通感染症制御学特論北海道大学
環境と人間「感染症と免疫」北海道大学
獣医科学特論演習北海道大学
獣医科学特別研究北海道大学
卒業論文北海道大学
研究方法論北海道大学
獣医学総合演習北海道大学
獣医病理組織学実習北海道大学
獣医病理学実習北海道大学
獣医学総合実習北海道大学

所属学協会

• 日本獣医病理学専門家協会   日本ウイルス学会   American Society for Virology   日本獣医学会   

共同研究・競争的資金等の研究課題

• ウマヘルペスウイルス1型感染神経細胞におけるウイルス遺伝子発現制御機構の解明

  日本学術振興会：科学研究費助成事業

  研究期間 : 2022年04月 -2026年03月 

  代表者 : 木村 享史, 青島 圭佑
• 結核と鼻疽の制圧プロジェクト

  国立研究開発法人日本医療研究開発機構：医療分野国際科学技術共同研究開発推進事業 地球規模課題対応国際科学技術協力プログラム

  研究期間 : 2020年04月 -2025年03月
• レセプター遺伝子導入モデルを用いたウマヘルペスウイルス1型潜伏感染機構の解明

  日本学術振興会：科学研究費助成事業 基盤研究(B)

  研究期間 : 2016年04月 -2020年03月 

  代表者 : 木村 享史, 青島 圭佑

   

  ウマヘルペスウイルス1型（EHV-1）は馬に呼吸器症状、流産および脊髄脳症を引き起す。EHV-1は神経細胞とリンパ球に潜伏感染することが報告されているが、その機序については不明な点が多く、これは小動物モデルおよびin vitroモデルが存在しないことによる。本研究では、EHV-1に感受性を有するin vitroモデルとマウスモデルをEHV-1エントリーレセプターの遺伝子導入によって作製、解析し、潜伏感染モデルとしての有用性を検証した。分化依存性にEHV-1感染に抵抗性を示すラット由来神経細胞株は、EHV-1の神経病原性と潜伏感染に関与する分子メカニズムの解明に有用であると考えられた。
• ウシの黄色ブドウ球菌性乳房炎の慢性化におけるCXCL8の持続的放出の役割

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2015年04月 -2018年03月 

  代表者 : 渡部 淳, 木村 和弘, 木村 享史, 秦 英司, 中島 恵一, 林 智人

   

  黄色ブドウ球菌性乳房炎が慢性化する過程で観察されるケモカインCXCL8の持続的放出について、ウシ乳腺上皮細胞における CXCL8の発現は主に黄色ブドウ球菌リポテイコ酸等によって誘発され、ラクトフェリンのエラスターゼ分解産物である催炎性ラクトフェリン由来ペプチドもCXCL8発現に部分的に関与することが示唆された。また同乳房炎においてCXCL8同様に持続的に放出される好中球エラスターゼは好中球の細胞死に伴なって放出され、その細胞死には壊死や好中球細胞外トラップ形成が関わることが示唆された。
• 神経細胞のフラビウイルス感受性を規定する宿主因子：細胞分化からのアプローチ

  日本学術振興会：科学研究費助成事業 挑戦的萌芽研究

  研究期間 : 2014年04月 -2016年03月 

  代表者 : 木村 享史

   

  ラット中脳由来神経細胞株CSM14.1は分化依存的に日本脳炎ウイルス（JEV）感受性の低下を示す。分化状態によって発現が変動する遺伝子群をマイクロアレイ法にて解析し、これらのうちから神経細胞のウイルス感受性に関与する未知の宿主遺伝子を同定する目的で、未分化CSM14.1細胞での発現が分化CSM14.1に比較して高い遺伝子群より2個、分化CSM14.1細胞での発現が未分化CSM14.1細胞に比較して高い遺伝子群より2個の遺伝子を選別した。これら遺伝子を未分化CSM14.1細胞に過剰発現させ、JEV感受性の変動を解析した。
• レセプター遺伝子導入マウスを利用した抗ウマヘルペスウイルス1型戦略

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2012年 -2015年 

  代表者 : 木村 享史

   

  ウマ主要組織適合遺伝子複合体（MHC）クラスⅠ分子はウマヘルペスウイルス1型（EHV-1）レセプターのエントリーレセプターとして機能する。全身諸臓器にウマMHCクラスⅠ遺伝子を発現するトランスジェニックマウス（Tgマウス）を作製し、EHV-1を経鼻感染させた。TgマウスではWTマウスと比較してより重度の肺炎を発症し、また、肺組織中により多数のウイルス感染細胞が認められた。以上の結果から、外来性に導入したウマMHCクラスIがマウスのEHV-1感受性の増強に関与することが示唆された。また、EHV-1の主要標的細胞であるT細胞に特異的にMHCクラスIを発現するTgマウスを作製した。
• ウシの黄色ブドウ球菌性乳房炎における好中球介在性炎症増幅機構の役割

  日本学術振興会：科学研究費助成事業 基盤研究(C)

  研究期間 : 2010年 -2012年 

  代表者 : 渡部 淳, 秦 英司, 木村 和弘, 平井 綱雄, 木村 享史

   

  牛の黄色ブドウ球菌性乾乳期乳房炎における炎症が持続的に進行する特徴的病態に、連鎖的に乳汁中に放出されるインターロイキン-8(IL-8)(CXCL8ともいう)、好中球エラスターゼ、催炎性ラクトフェリン由来ペプチド(ラクトフェリンのエラスターゼによる分解産物)が関与することを明らかにした。また慢性乳房炎において、IL-8の分泌を抑制するトランスフォーミング成長因子-β2は炎症の鎮静化に関与することが示唆された。
• ウマヘルペスウイルス1型レセプターの機能解析とそれを基盤とした感染予防法の検討

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2008年 -2011年 

  代表者 : 木村 享史, 澤 洋文

   

  ウマヘルペスウイルス1型(EHV-1)はアルファヘルペスウイルス亜科に属し、ウマに呼吸器症状、流産、脳脊髄炎を惹き起こす。臨床上特に問題となる流産と脳脊髄炎では、妊娠子宮、中枢神経系特異的に血管内皮細胞へのウイルス感染が生じ、それに続発する血管炎、血栓形成、循環障害が病態発生に大きな役割を果たしている。従って、血管内皮細胞へのウイルス感染は流産、脳脊髄炎の発症において最も重要なステップであるにも関わらず、その感受性を規定するウイルスレセプターに関しては不明な点が多い。本研究では、ウマ脳血管内皮細胞からクローニングしたEHV-1レセプターの機能解析を行い、レセプター遺伝子導入によるマウスモデルの作製とウイルスエンベロープ蛋白-レセプター相互作用を利用した感染予防法の検討を行う。
• フラビウイルス脳炎の新側面-ウイルス感染に起因する精神機能異常の病態解明-

  文部科学省：科学研究費補助金(挑戦的萌芽研究)

  研究期間 : 2009年 -2010年 

  代表者 : 木村 享史

   

  日本脳炎ウイルス(JEV)感染ラットの脳内では低レベルのウイルスゲノムRNA複製が持続している。この感染状態に類似したin vitroシステムを確立する目的で、以下の実験を行った。ラット神経細胞由来株化細胞として、温度感受性SV40T抗原で不死化されたCSM14.1を使用した。JEVの構造蛋白遺伝子を組み込んだ発現プラスミドを作製し、ウエストナイルウイルス(WNV)非構造蛋白遺伝子とEGFP発現カセットを有するWNVレプリコン(ペンシルベニア大学より分与)と共に293T細胞へ導入することにより、上清中に産生されるVLPを回収した。CSM14.1細胞にVLPを感染させた場合、31℃にて維持した細胞では13.6%がGFP陽性を示した。一方、37℃下で72時間培養し神経系分化マーカーが発現した細胞では、VLPの感染により51%がGFP陽性を示し、37℃下での培養によってVLP感染率が低下することが明らかとなった。また、37℃にて培養した細胞では、31℃で維持した細胞と比較して、エンドサイトーシスによるJEV particleの取り込み効率に差異があることが示唆された。従って、レプリコンRNAの導入には31℃にて維持した細胞を使用することが適切であることが確認された。JEV感染ラットの脳内ではセロトニン産生酵素であるトリプトファン水解酵素(TPH)の遺伝子発現が上昇している。F344...
• 血液脳関門モデルを用いたウエストナイルウイルス神経侵襲メカニズムの解明

  文部科学省：科学研究費補助金(基盤研究(C))

  研究期間 : 2006年 -2007年 

  代表者 : 木村 享史, 好井 健太朗, 澤 洋文

   

  1.ウエストナイルウイルス(WNV)が血液脳関門(Blood-brain barrier; BBB)を通過する機序には不明な点が多い。本研究では、ヒト臍静脈内皮細胞(HUVEC)をTranswell filterの上面に接着、単層培養を行い、BBBのin vitroモデルとして使用した。2.pCMVベクターにWNV NY99 6LP株およびEg101株の構造遺伝子(C、PrM、E)を組み込んだ発現プラスミドを作製した。これとWNVレプリコン(WNV非構造蛋白遺伝子全長と緑色蛍光蛋白EGFP発現カセットを有する)をBHK細胞に共導入し、ウイルス様粒子(Virus-like particles; VLPs)を作製した。3.WNV強毒株である 6LP株のVLPs(6LP-VLPs)および弱毒株であるg101株のVLPs(Eg-VLPs)をin vitro BBBモデルに感染させ、BBBを透過したVLPsの量を24時間後に測定した。Transwell内に接種した6LP-VLPsの約1/10量が下方のチャンバーから検出されたが、同様に接種したEg-VLPsは下方のチャンバーからほとんど検出されなかった。このように両者のin vitroBBB透過性には有意な差が認められ、ウイルスの毒力とBBB透過性との関連性が示唆された。次にHUVECに6LP-VLPsを感染させ、タイトジャンクション(...
• 野生のニホンジカは人あるいは他の動物種の感染源になり得るか?

  文部科学省：科学研究費補助金(基盤研究(B))

  研究期間 : 2004年 -2006年 

  代表者 : 鈴木 正嗣, 梶 光一, 山内 貴義, 有川 二郎, 横山 真弓, 堀内 基広, 木村 享史

   

  1.ニホンジカ(976頭)とニホンイノシシ(87頭)から得た血清を用い,ELISA法とウエスタンブロット法によりE型肝炎に対する血清学的診断を行った.その結果,それぞれの種で2.6%および2.3%が抗体陽性と判断された.イノシシにおいては,RT-PCR法により3頭でHEV-RNAが確認されたが,捕獲状況からこれらは同腹子と考えられた2.野生のニホンイノシシ(88頭)から得た血清を用い,ELISA法と顕微鏡下凝集試験(MAT)により,レプトスピフに対する抗体の検出を行った.その結果,12検体(13.6%)が抗体陽性と判断され,さらにMATでは血清型copenhageni, australis, bratislavaの抗体が検出された.そのため,捕獲ならびに解体にともなう人や猟犬に対する感染リスクが示唆された.3.野生のニホンジカ173頭を材料に,糞便培養法,LAMP法,nested PCR法によりヨーネ菌の検出を試みた.その結果,糞便培養法では菌は確認されなかった.また,LAMP法とnested PCR法でもヨーネ菌DNAは検出されなかった.4.北海道産の野生ニホンジカ(エゾシカ)においては,末梢血よりRickettsia helveticaとEhrlichiamurisの遺伝子が確認された.5.岩手県で捕獲された野生ニホンジカからは,槍形吸虫Dicrocoelium chin...
• マウス脳継代により獲得されたウマヘルペスウイルス1型神経病原性の分子基盤

  文部科学省：科学研究費補助金(若手研究(B))

  研究期間 : 2002年 -2003年 

  代表者 : 木村 享史

   

  ウマヘルペスウイルス1型(EHV-1)の神経向性は他のヘルペスウイルスに比較して非常に弱いことが知られているが、脳継代によって作出したEHV-1神経馴化株(NHH1)は親株(HH1)に比べて強い神経病原性を示す。脳継代による変異領域を同定する目的で、両ウイルス株のDNAを比較解析した。LA-PCR法によって13Kbに至るウイルスDNA断片を複数増幅し、RFLP解析を行ったが、両ウイルス間に差異は検出されなかった。平成14年度の解析により、NHH1はHH1に比べてより広範囲な組織向性を示すことが明らかとなっているため、細胞への吸着、侵入に関与するエンベロープ蛋白に変異が生じている可能性が考えられた。そこで、NHH1、HH1のgB遺伝子、gD遺伝子をそれぞれシークエンスしたが、両ウイルス株間でコードされるアミノ酸配列に相違は認められなかった。平成14年度に行った解析により、NHH1、HH1の示す神経侵襲性の差異が脳血管内皮細胞への感染性によって一部規定される可能性が考えられた。また、馬のEHV-1脳脊髄炎においても血行性にCNSに到達したウイルスが脳血管内皮細胞に感染することが知られている。そこで、馬、マウスの大脳皮質より脳微小血管内皮細胞(BMEC)を分離、培養し、HH1株に対する感受性を検討した。その結果、馬BMECはEHV-1感受性であり、エンドサイトーシスによってウイルス...
• アルファウイルス脳炎の病態を制御する細胞性免疫因子の解明

  文部科学省：科学研究費補助金(特定領域研究(C))

  研究期間 : 2001年 -2001年 

  代表者 : 木村 享史

   

  神経強毒性シンドビスウイルス(NSV)は成マウスに致死性(死亡率70〜100%)の急性ウイルス性脳炎を引き起こすが、耐過マウスには海馬組織欠損を特徴とする水頭症様病変が頻繁に認められる。本研究はこの海馬組織傷害に関与する細胞性免疫因子の同定を目的として行われた。C57BL/6マウス(B6)および同系のCD4遺伝子欠損マウス(CD4 -/-)、CD8α遺伝子欠損マウス(CD8 -/-)、recombination activating gene 1遺伝子欠損マウス(RAG -/-)にNSVを脳内接種し、接種後24時間目に抗NSV免疫血清を腹腔内投与することによってマウスを人為的に耐過させた。B6マウスにはマクロファージ、リンパ球浸潤を伴った海馬組織欠損が認められ、同病変は脳内から感染性ウイルスが消失した後に形成された。CD4 -/-マウスの海馬組織欠損は軽度であったが、CD8 -/-マウスはB6マウスと同様の水頭症病変を形成した。一部のRAG -/-マウスにおいてもマクロファージ浸潤を伴った重度の水頭症病変が形成された。TUNEL法陽性海馬分子層ニューロン数は海馬組織中に浸潤するCD4陽性細胞数およびマクロファージ数と比例したが、CD8陽性細胞数との相関は認められなかった。なお、抗CD4および抗CD8モノクローナル抗体投与によって作成したCD4陽性細胞欠損マウス、CD8陽性細胞...
• インターロイキン-2遺伝子導入マウスにおける小脳病変の形成機序に関する実験的研究

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1996年 -1996年 

  代表者 : 木村 享史

   

  ヒトIL-2 (hIL-2)ゲノムDNAを導入して作製したトランスジェニックマウス(hIL-2マウス)を免疫組織化学的、分子生物学的に解析し、以下の知見を得た。1.hIL-2マウスの小脳病変の免疫組織化学的検索5週齢のhIL-2マウスの小脳凍結切片を抗Thy-1抗体を用いた酵素抗体法によって検索したところ、クモ膜下腔に集簇するリンパ球のうち多くのものはThy-1陽性を呈した。2.hIL-2発現細胞の同定9日齢のhIL-2マウスの小脳パラフィン包埋切片に対し、hIL-2遺伝子を特異的に増幅するプライマー対を用いたRT-in situPCR法を施行した。hIL-2mRNAの5'末端を増幅するプライマー対と、第2エクソンから第3エクソンにわたる領域を増幅するプライマー対の2種類を用いて解析を行った結果、いずれのプライマー対を用いた場合においても、プルキンエ細胞層に限局して弱いシグナルが観察された。クモ膜下腔に浸潤している少数の炎症性細胞にシグナルは認められなかった。3.小脳において発現しているhIL-2mRNAの解析hIL-2マウス小脳mRNAからRT-PCR法によって第1イントロンを含んだhIL-2primary transcriptを増幅した。増幅したcDNA (293bp)をpGEM-4Zベクターにクローニングし、ジデオキシ法による塩基配列の解読を行った。その結果、第1イント...
• 神経細胞の細胞骨格ニューロフィラメントの生物学的並びに病理学的意義の究明

  文部科学省：科学研究費補助金(一般研究(B), 基盤研究(B))

  研究期間 : 1995年 -1996年 

  代表者 : 板倉 智敏, 木村 亨史, 落合 謙爾

   

  1.ニューロフィラメント(NF)欠損ウズラ(Quv)の本態についてQuvの末梢神経線維における軸索と髄鞘の変化,およびその相関関係を調べた.無髄神経線維では,NF欠損を代償してマイクロチューブ(MT)数が増加しているようであった.有髄神経線維では,軸索の大きさが髄鞘層数の規定因子ではないと判断された.次に,NFの神経線維再生への関与を調べた.このため,Quv坐骨神経を鉗子で押して砕き,2週間後にこれより末梢部位を形態学的に調べた.この結果,Quvでは正常対照例より再生神経線維が少なく,NF欠損はその再生を遅延させることが明らかとなった.さらに,NFの欠損が三量体型GTP結合(G)蛋白質に影響を及ぼすか否かを検討した.G蛋白質はQuvでは正常ウズラよりも増量していたが,これがQuvの振戦症状発現に関与しているか否かは明らかではない.2.NF欠損ウズラ(Quv)に対する神経毒の影響Quvに対して,神経毒であるアクリルアミドに続いてtri-ortho-cresyl-phosphate(TOCP)の影響を調べた.TOCPの連続投与は,正常ウズラの脊髄白質の神経路に遠位軸索症を引き起こしたが,Quvにはその変化は生じなかった.よってNFの存在がその病変発現に関与していることは明らかであった.一方,TOCPの正常およびQuvウズラへの投与は,軸索終末変性を両者に引き起こし,この変性病変発...
• インターロイキン-2遺伝子導入マウスにおける神経病変の発症機構に関する実験的研究

  文部科学省：科学研究費補助金(奨励研究(A))

  研究期間 : 1995年 -1995年 

  代表者 : 木村 享史

   

  ヒトIL-2ゲノムDNAを導入して作製したトランスジェニックマウス(hIL-2マウス)を解析し、以下の知見を得た。1.hIL-2マウスの組織学的検索9日令、5週令および12週令の雄マウスについて病理組織学的検索を行ったところ、小脳、皮膚に限局して病変が認められた。9日令の小脳では、クモ膜下腔にリンパ球、マクロファージ、好中球の集簇が認められた。5週令、12週令ではクモ膜下腔のリンパ球浸潤に加え脂肪顆粒細胞が小脳実質内に浸潤し、これに伴って小脳皮質の菲薄化、小脳小葉の縮小が認められた。9日令の皮膚には変化が認められなかったが、5週令、12週令の皮膚では、真皮における毛包および血管を中心としたリンパ球、マクロファージ浸潤ならびに毛包、皮脂腺の消失が生じていた(第120回日本獣医学会発表、1995年11月)。2.hIL-2発現組織の同定9日令、5週令、12週令のhIL-2マウスの前進諸臓器ならびに末梢血白血球からtotal RNAを抽出し、ヒトhIL-2を特異的に増幅するプライマーペアを用いてRT-PCRを行ったところ、9日令、5週令の小脳、皮膚12週令の皮膚、体表リンパ節に特異的に導入遺伝子の発現が確認された(第120回日本獣医学会発表、1995年11月)。3.抗神経組織抗体の存在の有無の検討C57BL/6マウス小脳ホモジネートをhIL-2マウス血清を抗体として用いたImmuno...