Researcher Database

Junya Yamagishi
International Institute for Zoonosis Control Division of Collaboration and Education
Associate Professor

Researcher Profile and Settings

Affiliation

  • International Institute for Zoonosis Control Division of Collaboration and Education

Job Title

  • Associate Professor

URL

J-Global ID

Research Interests

  • トランスクリプトーム   転写制御   転写開始点   バイオインフォマティクス   TSS-seq   原虫   次世代シーケンサー   トキソプラズマ   遺伝子診断   ステージ変換   血清学診断   マダニ媒介   Theileria   ブラディゾイト   Neospora caninum   マダニ   組換えワクチン   Babesia   弱毒化   Toxoplasma   TgSAG1   予防対策   生活環   タキゾイト   Toxoplasma gondii   疫学調査   東南アジア   病原性   発現ベクター   

Research Areas

  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine
  • Life sciences / Veterinary medicine

Academic & Professional Experience

  • 2015 Hokkaido University

Research Activities

Published Papers

  • Lavel C Moonga, Kyoko Hayashida, Namwiinga R Mulunda, Yukiko Nakamura, James Chipeta, Hawela B Moonga, Boniface Namangala, Chihiro Sugimoto, Zephaniah Mtonga, Mable Mutengo, Junya Yamagishi
    Emerging infectious diseases 27 (8) 2237 - 2239 2021/08 
    Rickettsia asembonensis is a flea-related Rickettsia with unknown pathogenicity to humans. We detected R. asembonensis DNA in 2 of 1,153 human blood samples in Zambia. Our findings suggest the possibility of R. asembonensis infection in humans despite its unknown pathogenicity.
  • Jumpei Yamazaki, Yuki Matsumoto, Jaroslav Jelinek, Teita Ishizaki, Shingo Maeda, Kei Watanabe, Genki Ishihara, Junya Yamagishi, Mitsuyoshi Takiguchi
    Scientific reports 11 (1) 10005 - 10005 2021/05/11 
    DNA methylation plays important functions in gene expression regulation that is involved in individual development and various diseases. DNA methylation has been well studied in human and model organisms, but only limited data exist in companion animals like dog. Using methylation-sensitive restriction enzyme-based next generation sequencing (Canine DREAM), we obtained canine DNA methylation maps of 16 somatic tissues from two dogs. In total, we evaluated 130,861 CpG sites. The majority of CpG sites were either highly methylated (> 70%, 52.5-64.6% of all CpG sites analyzed) or unmethylated (< 30%, 22.5-28.0% of all CpG sites analyzed) which are methylation patterns similar to other species. The overall methylation status of CpG sites across the 32 methylomes were remarkably similar. However, the tissue types were clearly defined by principle component analysis and hierarchical clustering analysis with DNA methylome. We found 6416 CpG sites located closely at promoter region of genes and inverse correlation between DNA methylation and gene expression of these genes. Our study provides basic dataset for DNA methylation profiles in dogs.
  • Mulenga Mwenda, Ngonda Saasa, Nyambe Sinyange, George Busby, Peter J Chipimo, Jason Hendry, Otridah Kapona, Samuel Yingst, Jonas Z Hines, Peter Minchella, Edgar Simulundu, Katendi Changula, King Shimumbo Nalubamba, Hirofumi Sawa, Masahiro Kajihara, Junya Yamagishi, Muzala Kapin'a, Nathan Kapata, Sombo Fwoloshi, Paul Zulu, Lloyd B Mulenga, Simon Agolory, Victor Mukonka, Daniel J Bridges
    MMWR. Morbidity and mortality weekly report 70 (8) 280 - 282 2021/02/26 
    The first laboratory-confirmed cases of coronavirus disease 2019 (COVID-19), the illness caused by SARS-CoV-2, in Zambia were detected in March 2020 (1). Beginning in July, the number of confirmed cases began to increase rapidly, first peaking during July-August, and then declining in September and October (Figure). After 3 months of relatively low case counts, COVID-19 cases began rapidly rising throughout the country in mid-December. On December 18, 2020, South Africa published the genome of a SARS-CoV-2 variant strain with several mutations that affect the spike protein (2). The variant included a mutation (N501Y) associated with increased transmissibility.†,§ SARS-CoV-2 lineages with this mutation have rapidly expanded geographically.¶,** The variant strain (PANGO [Phylogenetic Assignment of Named Global Outbreak] lineage B.1.351††) was first detected in the Eastern Cape Province of South Africa from specimens collected in early August, spread within South Africa, and appears to have displaced the majority of other SARS-CoV-2 lineages circulating in that country (2). As of January 10, 2021, eight countries had reported cases with the B.1.351 variant. In Zambia, the average number of daily confirmed COVID-19 cases increased 16-fold, from 44 cases during December 1-10 to 700 during January 1-10, after detection of the B.1.351 variant in specimens collected during December 16-23. Zambia is a southern African country that shares substantial commerce and tourism linkages with South Africa, which might have contributed to the transmission of the B.1.351 variant between the two countries.
  • Yukiko Nakamura, Kyoko Hayashida, Victoire Delesalle, Yongjin Qiu, Ryosuke Omori, Martin Simuunza, Chihiro Sugimoto, Boniface Namangala, Junya Yamagishi
    Frontiers in veterinary science 8 599815 - 599815 2021 
    We clarified the genetic diversity of Trypanosoma spp. within the Kafue ecosystem, using PCR targeting the internal transcribed spacer 1 and the cathepsin L-like cysteine protease (CatL) sequences. The overall prevalence of Trypanosoma spp. in cattle and tsetse flies was 12.65 and 26.85%, respectively. Cattle positive for Trypanosoma vivax had a significantly lower packed cell volume, suggesting that T. vivax is the dominant Trypanosoma spp. causing anemia in this area. Among the 12 operational taxonomic units (OTUs) of T. vivax CatL sequences detected, one was from a known T. vivax lineage, two OTUs were from known T. vivax-like lineages, and nine OTUs were considered novel T. vivax-like lineages. These findings support previous reports that indicated the extensive diversity of T. vivax-like lineages. The findings also indicate that combining CatL PCR with next generation sequencing is useful in assessing Trypanosoma spp. diversity, especially for T. vivax and T. vivax-like lineages. In addition, the 5.42% prevalence of Trypanosoma brucei rhodesiense found in cattle raises concern in the community and requires careful monitoring of human African trypanosomiasis.
  • David Squarre, Yukiko Nakamura, Kyoko Hayashida, Naoko Kawai, Herman Chambaro, Boniface Namangala, Chihiro Sugimoto, Junya Yamagishi
    Parasites & vectors 13 (1) 599 - 599 2020/11/30 
    BACKGROUND: Piroplasms are vector-borne intracellular hemoprotozoan parasites that infect wildlife and livestock. Wildlife species are reservoir hosts to a diversity of piroplasms and play an important role in the circulation, maintenance and evolution of these parasites. The potential for likely spillover of both pathogenic and non-pathogenic piroplasm parasites from wildlife to livestock is underlined when a common ecological niche is shared in the presence of a competent vector. METHOD: To investigate piroplasm diversity in wildlife and the cattle population of the greater Kafue ecosystem, we utilized PCR to amplify the 18S rRNA V4 hyper-variable region and meta-barcoding strategy using the Illumina MiSeq sequencing platform and amplicon sequence variant (ASV)-based bioinformatics pipeline to generate high-resolution data that discriminate sequences down to a single nucleotide difference. RESULTS: A parasite community of 45 ASVs corresponding to 23 species consisting of 4 genera of Babesia, Theileria, Hepatozoon and Colpodella, were identified in wildlife and the cattle population from the study area. Theileria species were detected in buffalo, impala, hartebeest, sable antelope, sitatunga, wild dog and cattle. In contrast, Babesia species were only observed in cattle and wild dog. Our results demonstrate possible spillover of these hemoprotozoan parasites from wildlife, especially buffalo, to the cattle population in the wildlife-livestock interface. CONCLUSION: We demonstrated that the deep amplicon sequencing of the 18S rRNA V4 hyper-variable region for wildlife was informative. Our results illustrated the diversity of piroplasma and the specificity of their hosts. They led us to speculate a possible ecological cycle including transmission from wildlife to domestic animals in the greater Kafue ecosystem. Thus, this approach may contribute to the establishment of appropriate disease control strategies in wildlife-livestock interface areas.
  • Lavel Chinyama Moonga, Kyoko Hayashida, Naoko Kawai, Ryo Nakao, Chihiro Sugimoto, Boniface Namangala, Junya Yamagishi
    Diagnostics (Basel, Switzerland) 10 (11) 2020/11/02 
    Spotted fever group (SFG) rickettsiae causes febrile illness in humans worldwide. Since SFG rickettsiosis's clinical presentation is nonspecific, it is frequently misdiagnosed as other febrile diseases, especially malaria, and complicates proper treatment. Aiming at rapid, simple, and simultaneous detection of SFG Rickettsia spp. and Plasmodium spp., we developed a novel multiple pathogen detection system by combining a loop-mediated isothermal amplification (LAMP) method and dipstick DNA chromatography technology. Two primer sets detecting SFG Rickettsia spp. and Plasmodium spp. were mixed, and amplified products were visualized by hybridizing to dipstick DNA chromatography. The multiplex LAMP with dipstick DNA chromatography distinguished amplified Rickettsia and Plasmodium targeted genes simultaneously. The determined sensitivity using synthetic nucleotides was 1000 copies per reaction for mixed Rickettsia and Plasmodium genes. When genomic DNA from in vitro cultured organisms was used, the sensitivity was 100 and 10 genome equivalents per reaction for Rickettsia monacensis and Plasmodium falciparum, respectively. Although further improvement will be required for more sensitive detection, our developed simultaneous diagnosis technique will contribute to the differential diagnosis of undifferentiated febrile illness caused by either SFG Rickettsia spp. or Plasmodium spp. in resource-limited endemic areas. Importantly, this scheme is potentially versatile for the simultaneous detection of diverse infectious diseases.
  • Lucky R Runtuwene, Shuichi Kawashima, Victor D Pijoh, Josef S B Tuda, Kyoko Hayashida, Junya Yamagishi, Chihiro Sugimoto, Shoko Nishiyama, Michihito Sasaki, Yasuko Orba, Hirofumi Sawa, Tomohiko Takasaki, Anthony A James, Takashi Kobayashi, Yuki Eshita
    International journal of molecular sciences 21 (20) 2020/10/12 
    Efforts to determine the mosquito genes that affect dengue virus replication have identified a number of candidates that positively or negatively modify amplification in the invertebrate host. We used deep sequencing to compare the differential transcript abundances in Aedes aegypti 14 days post dengue infection to those of uninfected A. aegypti. The gene lethal(2)-essential-for-life [l(2)efl], which encodes a member of the heat shock 20 protein (HSP20) family, was upregulated following dengue virus type 2 (DENV-2) infection in vivo. The transcripts of this gene did not exhibit differential accumulation in mosquitoes exposed to insecticides or pollutants. The induction and overexpression of l(2)efl gene products using poly(I:C) resulted in decreased DENV-2 replication in the cell line. In contrast, the RNAi-mediated suppression of l(2)efl gene products resulted in enhanced DENV-2 replication, but this enhancement occurred only if multiple l(2)efl genes were suppressed. l(2)efl homologs induce the phosphorylation of eukaryotic initiation factor 2α (eIF2α) in the fruit fly Drosophila melanogaster, and we confirmed this finding in the cell line. However, the mechanism by which l(2)efl phosphorylates eIF2α remains unclear. We conclude that l(2)efl encodes a potential anti-dengue protein in the vector mosquito.
  • Hassan Hakimi, Thomas J Templeton, Miako Sakaguchi, Junya Yamagishi, Shinya Miyazaki, Kazuhide Yahata, Takayuki Uchihashi, Shin-Ichiro Kawazu, Osamu Kaneko, Masahito Asada
    PLoS pathogens 16 (10) e1008917  2020/10 
    Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.
  • Kyoko Hayashida, Peter Nambala, Nick Van Reet, Philippe Büscher, Naoko Kawai, Mable Mwale Mutengo, Janelisa Musaya, Boniface Namangala, Chihiro Sugimoto, Junya Yamagishi
    PLoS neglected tropical diseases 14 (10) e0008753  2020/10 
    Human African trypanosomiasis (HAT) is one of the neglected tropical diseases in sub-Saharan Africa. Early diagnosis and treatment prior to disease progression are crucial for the survival of HAT patients. We had previously established a loop-mediated isothermal amplification (LAMP) method for HAT diagnosis in which the reagents were dried for field-use purposes. In this study, we used a semi-automated process to produce the test tubes using a bio-inkjet printer to achieve an accurate production. The performance of the inkjet printer-produced dried LAMP test (CZC-LAMP) was found to be stable after storage for up to 180 days at 30 °C. The diagnostic accuracy of CZC-LAMP HAT was evaluated using DNA samples that were extracted from 116 Trypanosoma brucei gambiense patients and 66 T. b. rhodesiense patients. The sensitivity was 72% for T. b. gambiense (95%CI: 63%-80%) and 80% for T. b. rhodesiense (95%CI: 69%-89%). The specificity determined using DNA from 116 endemic control DNA samples was 95% (95%CI: 89%-98%). The performance of the CZC-LAMP HAT and CZC-LAMP rHAT were also evaluated using 14 crude blood lysate samples obtained from T. b. rhodesiense patients and endemic control samples collected from Rumphi District in Malawi. The sensitivity and specificity were both 100% (95%CI: 77%-100%). As the developed CZC-LAMP test does not require a cold chain or a sophisticated laboratory, it holds promise for use as a routine simple molecular tool for point-of-care HAT diagnosis in endemic areas.
  • David Squarre, Kyoko Hayashida, Alex Gaithuma, Herman Chambaro, Naoko Kawai, Ladslav Moonga, Boniface Namangala, Chihiro Sugimoto, Junya Yamagishi
    International journal for parasitology. Parasites and wildlife 12 34 - 41 2020/08 
    The Kafue ecosystem is a vast conservation protected area comprising the Kafue National Park (KNP) and the Game Management Areas (GMA) that act as a buffer around the national park. The KNP has been neglected as a potential foci for rhodesiense sleeping sickness despite the widespread presence of the tsetse vector and abundant wildlife reservoirs. The aim of this study was to generate information on circulating trypanosomes and their eminent threat/risk to public health and livestock production of a steadily growing human and livestock population surrounding the park. We detected various trypanosomes circulating in different mammalian wildlife species in KNP in Zambia by applying a high throughput ITS1-polymerase chain reaction (PCR)/nanopore sequencing method in combination with serum resistant associated-PCR/Sanger sequencing method. The prevalence rates of trypanosomes in hartebeest, sable antelope, buffalo, warthog, impala and lechwe were 6.4%, 37.2%, 13.2%, 11.8%, 2.8% and 11.1%, respectively. A total of six trypanosomes species or subspecies were detected in the wildlife examined, including Trypanosoma brucei brucei, T. godfreyi, T. congolense, T. simiae and T. theileri. Importantly we detected human infective T. b. rhodesiense in buffalo and sable antelope with a prevalence of 9.4% and 12.5%, respectively. In addition, T. b. rhodesiense was found in the only vervet monkey analyzed. The study thus reaffirmed that the Kafue ecosystem is a genuine neglected and re-emerging foci for human African trypanosomiasis. This is the first assessment of the trypanosome diversity circulating in free-ranging wildlife of the KNP.
  • Makoto Matsubayashi, Tomoyuki Shibahara, Tomohide Matsuo, Toshimitsu Hatabu, Junya Yamagishi, Kazumi Sasai, Takashi Isobe
    The Journal of veterinary medical science 82 (5) 516 - 519 2020/05/30 [Refereed][Not invited]
     
    There have been no reports of the prevalence of Eimeria spp. in poultry breeding farms in Japan unlike those of broiler farms. From 2017 to 2018, we examined the prevalence of Eimeria spp. on breeding farms in Japan by oocyst morphology and PCR analyses. A total of 143 samples was collected from 37 breeding farms in 21 prefectures of Japan. We detected oocysts of seven species at 34 of 37 breeding farms by PCR, and we identified E. brunetti at 51.5% of farms found to be positive for Eimeria. The differences in the identification of Eimeria spp. between the morphology and PCR assay methods of oocysts were pronounced for E. maxima and E. necatrix. We confirmed that molecular tools were more suitable for accurately estimating prevalence of Eimeria spp., and these findings suggest that E. brunetti could be widespread in Japan.
  • Yasuyuki Goto, Akihiro Kuroki, Kengo Suzuki, Junya Yamagishi
    Microbiology resource announcements 9 (21) 2020/05/21 
    Leishmania tarentolae is a protozoan parasite of lizards and is nonpathogenic to mammals. Genome information for the nonpathogenic species will facilitate an understanding of the pathology caused by species pathogenic to mammals. Here, we report resequencing of the L. tarentolae genome with a single-molecule real-time (SMRT) sequencer to provide a more complete genome assembly.
  • Alex Gaithuma, Junya Yamagishi, Kyoko Hayashida, Naoko Kawai, Boniface Namangala, Chihiro Sugimoto
    Scientific reports 10 (1) 5005 - 5005 2020/03/19 
    Tsetse flies are the vectors of African trypanosomiasis affecting 36 sub-Saharan countries. Both wild and domestic animals play a crucial role in maintaining the disease-causing parasites (trypanosomes). Thus, the identification of animal reservoirs of trypanosomes is vital for the effective control of African trypanosomiasis. Additionally, the biotic and abiotic factors that drive gut microbiome diversity in tsetse flies are primarily unresolved, especially under natural, field conditions. In this study, we present a comprehensive DNA metabarcoding approach for individual tsetse fly analysis in the identification of mammalian blood meal sources and fly bacterial microbiome composition. We analyzed samples from two endemic foci, Kafue, Zambia collected in June 2017, and Hurungwe, Zimbabwe sampled in April 2014 (pilot study) and detected DNA of various mammals including humans, wild animals, domestic animals and small mammals (rat and bat). The bacterial diversity was relatively similar in flies with different mammalian species DNA, trypanosome infected and uninfected flies, and female and male flies. This study is the first report on bat DNA detection in wild tsetse flies. This study reveals that small mammals such as bats and rats are among the opportunistic blood meal sources for tsetse flies in the wild, and the implication on tsetse biology and ecology needs to be studied.
  • Hayato Harima, Masahiro Kajihara, Edgar Simulundu, Eugene Bwalya, Yongjin Qiu, Mao Isono, Kosuke Okuya, Gabriel Gonzalez, Junya Yamagishi, Bernard M Hang'ombe, Hirofumi Sawa, Aaron S Mweene, Ayato Takada
    Viruses 12 (2) 2020/02/05 [Refereed][Not invited]
     
    Porcine sapelovirus (PSV) has been detected worldwide in pig populations. Although PSV causes various symptoms such as encephalomyelitis, diarrhea, and pneumonia in pigs, the economic impact of PSV infection remains to be determined. However, information on the distribution and genetic diversity of PSV is quite limited, particularly in Africa. In this study, we investigated the prevalence of PSV infection in Zambia and characterized the isolated PSVs genetically and biologically. We screened 147 fecal samples collected in 2018 and found that the prevalences of PSV infection in suckling pigs and fattening pigs were high (36.2% and 94.0%, respectively). Phylogenetic analyses revealed that the Zambian PSVs were divided into three different lineages (Lineages 1-3) in the clade consisting of Chinese strains. The Zambian PSVs belonging to Lineages 2 and 3 replicated more efficiently than those belonging to Lineage 1 in Vero E6 and BHK cells. Bioinformatic analyses revealed that genetic recombination events had occurred and the recombination breakpoints were located in the L and 2A genes. Our results indicated that at least two biologically distinct PSVs could be circulating in the Zambian pig population and that genetic recombination played a role in the evolution of PSVs.
  • Megasari Marsela, Kyoko Hayashida, Ryo Nakao, Elisha Chatanga, Alex Kiarie Gaithuma, Kawai Naoko, Janelisa Musaya, Chihiro Sugimoto, Junya Yamagishi
    Parasite (Paris, France) 27 46 - 46 2020 
    This study aimed to identify trypanosomes infecting cattle in Malawi in order to understand the importance of cattle in the transmission dynamics of Human African Trypanosomiasis (HAT) and Animal African Trypanosomosis (AAT). A total of 446 DNA samples from cattle blood from three regions of Malawi were screened for African trypanosomes by ITS1 PCR. The obtained amplicons were sequenced using a portable next-generation sequencer, MinION, for validation. Comparison of the results from ITS1 PCR and MinION sequencing showed that combining the two methods provided more accurate species identification than ITS1 PCR alone. Further PCR screening targeting the serum resistance-associated (SRA) gene was conducted to detect Trypanosoma brucei rhodesiense. Trypanosoma congolense was the most prevalent Trypanosoma sp., which was found in Nkhotakota (10.8%; 20 of 185), followed by Kasungu (2.5%; 5 of 199). Of note, the prevalence of T. b. rhodesiense detected by SRA PCR was high in Kasungu and Nkhotakota showing 9.5% (19 of 199) and 2.7% (5 of 185), respectively. We report the presence of animal African trypanosomes and T. b. rhodesiense from cattle at the human-livestock-wildlife interface for the first time in Malawi. Our results confirmed that animal trypanosomes are important causes of anemia in cattle and that cattle are potential reservoirs for human African trypanosomiasis in Malawi.
  • Pengfei Li, Dane Vassiliadis, Sze Ying Ong, Vicki Bennett-Wood, Chihiro Sugimoto, Junya Yamagishi, Elizabeth L Hartland, Shivani Pasricha
    Frontiers in cellular and infection microbiology 10 428 - 428 2020 
    Legionella pneumophila is an environmental bacterium that has evolved to survive predation by soil and water amoebae such as Acanthamoeba castellanii, and this has inadvertently led to the ability of L. pneumophila to survive and replicate in human cells. L. pneumophila causes Legionnaire's Disease, with human exposure occurring via the inhalation of water aerosols containing both amoebae and the bacteria. These aerosols originate from aquatic biofilms found in artifical water sources, such as air-conditioning cooling towers and humidifiers. In these man-made environments, A. castellanii supports L. pneumophila intracellular replication, thereby promoting persistence and dissemination of the bacteria and providing protection from external stress. Despite this close evolutionary relationship, very little is known about how A. castellanii responds to L. pneumophila infection. In this study, we examined the global transcriptional response of A. castellanii to L. pneumophila infection. We compared A. castellanii infected with wild type L. pneumophila to A. castellanii infected with an isogenic ΔdotA mutant strain, which is unable to replicate intracellularly. We showed that A. castellanii underwent clear morphological and transcriptional rewiring over the course of L. pneumophila infection. Through improved annotation of the A. castellanii genome, we determined that these transcriptional changes primarily involved biological processes utilizing small GTPases, including cellular transport, signaling, metabolism and replication. In addition, a number of sirtuin-encoding genes in A. castellanii were found to be conserved and upregulated during L. pneumophila infection. Silencing of sirtuin gene, sir6f (ACA1_153540) resulted in the inhibition of A. castellanii cell proliferation during infection and reduced L. pneumophila replication. Overall our findings identified several biological pathways in amoebae that may support L. pneumophila replication and A. castellanii proliferation in environmental conditions.
  • Namwiinga Rozaria Mulunda, Kyoko Hayashida, Junya Yamagishi, Sandie Sianongo, Gilbert Munsaka, Chihiro Sugimoto, Mable Mwale Mutengo
    Parasite (Paris, France) 27 53 - 53 2020 
    Cryptosporidium is a major etiological agent of diarrhoeal diseases among children and immune-compromised individuals in sub-Saharan African countries. We conducted a study to determine the prevalence and genetic characteristics of Cryptosporidium spp. in stool samples from patients with diarrhoea who presented at the University Teaching Hospital in Lusaka, Zambia. Cryptosporidium species and subtypes from 71 microscopically confirmed cryptosporidiosis stool samples collected between 2017 and 2019 were determined by polymerase chain reaction followed by partial sequencing of the small subunit rRNA and 60-kDa glycoprotein (gp60) gene. Additionally, data for the period between 2014 and 2019 were reviewed and analysed for cryptosporidiosis seasonal and age distribution. Cryptosporidium was more prevalent in the rainy season. The highest number of cases was reported among the 1-4 year age group. By sequence analysis of the 71 positive isolates, Cryptosporidium hominis (n = 42; 59.2%), C. parvum (n = 27; 38%), C. felis (n = 1; 1.4%), and C. meleagridis (n = 1; 1.4%) were identified. Four C. hominis subtype families (Ia, Ib, Id, and Ie) and three C. parvum subtype families (IIc, IIe, and IIs) were identified. The most frequent subtypes were IeA11G3T3 (n = 20; 28.2%), IIcA5G3 (n = 12; 16.9%), IIeA12G1 (n = 11; 15.5%) and IaA30R3 (n = 10; 14.1%). The observed species/subtypes of C. hominis and C. parvum indicated that the infection was mainly transmitted through the anthroponotic route. The identification of C. felis and C. meleagridis suggests that an atypical zoonotic transmission cycle also exists.
  • Yamagishi J, Hayashida K, Matsuo J, Okubo T, Kuroda M, Nagai H, Sekizuka T, Yamaguchi H, Sugimoto C
    Journal of human genetics 1434-5161 2019/11 [Refereed][Not invited]
  • Nakagawa S, Inoue S, Kryukov K, Yamagishi J, Ohno A, Hayashida K, Nakazwe R, Kalumbi M, Mwenya D, Asami N, Sugimoto C, Mutengo MM, Imanishi T
    Clinical & Translational Immunology 8 (11) e01087  2019/11 [Refereed][Not invited]
  • Kobayashi K, Umeda K, Ihara F, Tanaka S, Yamagishi J, Suzuki Y, Nishikawa Y
    BMC genomics 20 (1) 705  2019/09 [Refereed][Not invited]
  • Thu MJ, Qiu Y, Yamagishi J, Kusakisako K, Ogata S, Moustafa MAM, Isoda N, Sugimoto C, Katakura K, Nonaka N, Nakao R
    Microbiology resource announcements 8 (37) 2019/09 [Refereed][Not invited]
  • Matsubayashi M, Yamaguchi H, Hatta T, Kawahara F, Hatabu T, Iseki H, Yamagishi J, Isobe T, Teramoto I, Kaneko A, Kita K, Tsuji N, Sasai K
    Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases 75 103993  1567-1348 2019/08 [Refereed][Not invited]
  • Nguyen TT, Dang-Trinh MA, Higuchi L, Mosqueda J, Hakimi H, Asada M, Yamagishi J, Umemiya-Shirafuji R, Kawazu SI
    Pathogens (Basel, Switzerland) 8 (3) 2019/07 [Refereed][Not invited]
  • Nakamura Y, Yamagishi J, Hayashida K, Osada N, Chatanga E, Mweempwa C, Chilongo K, Chisi J, Musaya J, Inoue N, Namangala B, Sugimoto C
    PLoS neglected tropical diseases 13 (7) e0007568  1935-2727 2019/07 [Refereed][Not invited]
  • Hayashida K, Orba Y, Sequeira PC, Sugimoto C, Hall WW, Eshita Y, Suzuki Y, Runtuwene L, Brasil P, Calvet G, Rodrigues CDS, Santos CCD, Mares-Guia MAM, Yamagishi J, Filippis AMB, Sawa H
    PLoS neglected tropical diseases 13 (6) e0007480  1935-2727 2019/06 [Refereed][Not invited]
  • Moonga LC, Hayashida K, Nakao R, Lisulo M, Kaneko C, Nakamura I, Eshita Y, Mweene AS, Namangala B, Sugimoto C, Yamagishi J
    Parasites & vectors 12 (1) 168  2019/04 [Refereed][Not invited]
  • Gaithuma AK, Yamagishi J, Martinelli A, Hayashida K, Kawai N, Marsela M, Sugimoto C
    PLoS neglected tropical diseases 13 (2) e0006842  1935-2727 2019/02 [Refereed][Not invited]
  • Batdorj Davaasuren, Junya Yamagishi, Daiki Mizushima, Sandagdorj Narantsatsral, Davaajav Otgonsuren, Punsantsogvoo Myagmarsuren, Badgar Battsetseg, Banzragch Battur, Noboru Inoue, Keisuke Suganuma
    Microbiology resource announcements 8 (9) 2576-098X 2019/02 [Refereed][Not invited]
     
    Trypanosoma equiperdum primarily parasitizes the genital organs and causes dourine in equidae. We isolated a new T. equiperdum strain, T. equiperdum IVM-t1, from the urogenital tract of a horse definitively diagnosed as having dourine in Mongolia. Here, we report the whole-genome sequence, the predicted gene models, and their annotations.
  • Nao N, Sato K, Yamagishi J, Tahara M, Nakatsu Y, Seki F, Katoh H, Ohnuma A, Shirogane Y, Hayashi M, Suzuki T, Kikuta H, Nishimura H, Takeda M
    PloS one 14 (4) e0215822  2019 [Refereed][Not invited]
  • Novel Characteristics of Mitochondrial Electron Transport Chain from Eimeria tenella.
    Matsubayashi M, Inaoka DK, Komatsuya K, Hatta T, Kawahara F, Sakamoto K, Hikosaka K, Yamagishi J, Sasai K, Shiba T, Harada S, Tsuji N, Kita K
    Genes 10 (1) 2019/01 [Refereed][Not invited]
  • Kyoko Hayashida, Rika Umemiya-Shirafuji, Thillaiampalam Sivakumar, Junya Yamagishi, Yutaka Suzuki, Chihiro Sugimoto, Naoaki Yokoyama
    International journal for parasitology 48 (12) 915 - 924 0020-7519 2018/10 [Refereed][Not invited]
     
    Oriental theileriosis caused by Theileria orientalis is an economically significant disease in cattle farming. The lack of laboratory animal models and in vitro culture systems is a major obstacle in the drive to better understand the biology of this parasite. Notably, research on the sporozoite stage of T. orientalis has rarely been undertaken, although such investigations are of paramount importance for vaccine development based on blocking sporozoite invasion of its host animals. In the present study, we established a mouse-tick infection model for propagating T. orientalis in mice and for producing the sporozoite stage in tick salivary glands. Splenectomized severe combined immunodeficient mice transfused with bovine erythrocytes were infected with T. orientalis. The larval ticks of Haemaphysalis longicornis were then fed on the T. orientalis-infected mice. The piroplasm and sporozoite stages were microscopically observed in the mouse blood and nymphal salivary glands, respectively. The transcriptomics data generated from the piroplasm and sporozoite stages revealed a stage-specific expression pattern for the parasite genes. The mouse-tick infection model and the transcriptomics data it has provided will contribute to a better understanding of T. orientalis biology and will also provide much needed information for the design of effective control measures targeting oriental theileriosis.
  • Schuelein R, Spencer H, Dagley LF, Li PF, Luo L, Stow JL, Abraham G, Naderer T, Gomez-Valero L, Buchrieser C, Sugimoto C, Yamagishi J, Webb AI, Pasricha S, Hartland EL
    Cellular microbiology 20 (9) e12852  1462-5814 2018/09 [Refereed][Not invited]
  • Sivakumar T, Tuvshintulga B, Zhyldyz A, Kothalawala H, Yapa PR, Kanagaratnam R, Vimalakumar SC, Abeysekera TS, Weerasingha AS, Yamagishi J, Igarashi I, Silva SSP, Yokoyama N
    Journal of clinical microbiology 0095-1137 2018/08 [Refereed][Not invited]
  • Salim B, Hayashida K, Mossaad E, Nakao R, Yamagishi J, Sugimoto C
    Veterinary parasitology 260 53 - 57 0304-4017 2018/08 [Refereed][Not invited]
  • Kazuo Imai, Norihito Tarumoto, Lucky Ronald Runtuwene, Jun Sakai, Kyoko Hayashida, Yuki Eshita, Ryuichiro Maeda, Josef Tuda, Hideaki Ohno, Takashi Murakami, Shigefumi Maesaki, Yutaka Suzuki, Junya Yamagishi, Takuya Maeda
    Malaria journal 17 (1) 217 - 217 2018/05/29 [Refereed][Not invited]
     
    BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.
  • Lucky R Runtuwene, Josef S B Tuda, Arthur E Mongan, Wojciech Makalowski, Martin C Frith, Mallika Imwong, Suttipat Srisutham, Lan Anh Nguyen Thi, Nghia Nguyen Tuan, Yuki Eshita, Ryuichiro Maeda, Junya Yamagishi, Yutaka Suzuki
    Scientific reports 8 (1) 8286 - 8286 2018/05/29 [Refereed][Not invited]
     
    Here, we report the application of a portable sequencer, MinION, for genotyping the malaria parasite Plasmodium falciparum. In the present study, an amplicon mixture of nine representative genes causing resistance to anti-malaria drugs is diagnosed. First, we developed the procedure for four laboratory strains (3D7, Dd2, 7G8, and K1), and then applied the developed procedure to ten clinical samples. We sequenced and re-sequenced the samples using the obsolete flow cell R7.3 and the most recent flow cell R9.4. Although the average base-call accuracy of the MinION sequencer was 74.3%, performing >50 reads at a given position improves the accuracy of the SNP call, yielding a precision and recall rate of 0.92 and 0.8, respectively, with flow cell R7.3. These numbers increased significantly with flow cell R9.4, in which the precision and recall are 1 and 0.97, respectively. Based on the SNP information, the drug resistance status in ten clinical samples was inferred. We also analyzed K13 gene mutations from 54 additional clinical samples as a proof of concept. We found that a novel amino-acid changing variation is dominant in this area. In addition, we performed a small population-based analysis using 3 and 5 cases (K13) and 10 and 5 cases (PfCRT) from Thailand and Vietnam, respectively. We identified distinct genotypes from the respective regions. This approach will change the standard methodology for the sequencing diagnosis of malaria parasites, especially in developing countries.
  • Mingming Liu, Paul Franck Adjou Moumouni, Masahito Asada, Hassan Hakimi, Tatsunori Masatani, Patrick Vudriko, Seung-Hun Lee, Shin-Ichiro Kawazu, Junya Yamagishi, Xuenan Xuan
    Parasites and Vectors 11 (1) 260  1756-3305 2018/04/23 [Refereed][Not invited]
     
    Background: Genetic manipulation techniques, such as transfection, have been previously reported in many protozoan parasites. In Babesia, stable transfection systems have only been established for bovine Babesia parasites. We recently reported a transient transfection system and the selection of promoter candidates for Babesia gibsoni. The establishment of a stable transfection system for B. gibsoni is considered to be urgent to improve our understanding of the basic biology of canine Babesia parasites for a better control of babesiosis. Results: GFP-expressing parasites were observed by fluorescence microscopy as early as two weeks after drug selection, and consistently expressed GFP for more than 3 months without drug pressure. Genome integration was confirmed by PCR, sequencing and Southern blot analysis. Conclusions: We present the first successful establishment of a stable transfection system for B. gibsoni. This finding will facilitate functional analysis of Babesia genomes using genetic manipulation and will serve as a foundation for the development of tick-Babesia and host-Babesia infection models.
  • Mamoru Niikura, Shin Ichi Inoue, Toshiyuki Fukutomi, Junya Yamagishi, Hiroko Asahi, Fumie Kobayashi
    Experimental Parasitology 185 1 - 9 0014-4894 2018/02 [Refereed][Not invited]
     
    © 2018 Elsevier Inc. Plasmodium berghei (Pb) XAT, a rodent malaria parasite, is an irradiation-attenuated variant derived from the lethal strain Pb NK65. Differences in genome sequence, protein structure and function between Pb XAT and Pb NK65 are currently unknown. In this study, to investigate genetic alterations in Pb XAT, we performed comparative genomics and proteomics analyses of nonlethal and lethal strains of Pb. We found mutations, such as a deletion mutation in rhoptry-associated protein (rap) 1, and deletion of rap2/3 and skeleton-binding protein 1 (sbp1), in Pb XAT. RAP1 is required for targeting of RAP2 to the rhoptries. However, the contribution of RAP2/3 to the lethality of Plasmodium is unclear. Therefore, we generated RAP1- and RAP2/3-deficient mutants of Pb ANKA, a reference strain of P. berghei. Furthermore, we investigated the effect of RAP1 and RAP2/3 deficiency on the outcome of infection. The parasitemia in mice infected with RAP1-deficient parasites was increased compared to that in control parasite-infected mice during the early phase of infection. However, mice infected with RAP1-deficient parasites survived longer than did control parasite-infected mice. Moreover, mice infected with RAP2/3-deficient parasites showed low levels of parasitemia and ultimately recovered from the infection The aim of this study was to investigate the effect of RAP2/3 expression on the outcome of infection with Pb XAT using a RAP2/3-expressing Pb XAT. Results showed that complementation of RAP2/3 expression in Pb XAT partially restored virulence. Our findings suggest that RAP1 and RAP2/3 contribute to virulence and a decrease in their expression explains the loss of virulence of the Pb XAT strain.
  • Kousuke Umeda, Sachi Tanaka, Fumiaki Ihara, Junya Yamagishi, Yutaka Suzuki, Yoshifumi Nishikawa
    PLOS ONE 12 (11) e0187703  1932-6203 2017/11 [Refereed][Not invited]
     
    Background Toxoplasma gondii is capable of persisting in the brain, although it is efficiently eliminated by cellular immune responses in most other sites. While Toll-like receptor 2 (TLR2) reportedly plays important roles in protective immunity against the parasite, the relationship between neurological disorders induced by T. gondii infection and TLR2 function in the brain remains controversial with many unknowns. In this study, primary cultured astrocytes, microglia, neurons, and peritoneal macrophages obtained from wild-type and TLR2-deficient mice were exposed to T. gondii tachyzoites. To characterize TLR2-dependent functional pathways activated in response to T. gondii infection, gene expression of different cell types was profiled by RNA sequencing. Results During T. gondii infection, a total of 611, 777, 385, and 1105 genes were upregulated in astrocytes, microglia, neurons, and macrophages, respectively, while 163, 1207, 158, and 1274 genes were downregulated, respectively, in a TLR2-dependent manner. Overrepresented Gene Ontology (GO) terms for TLR2-dependently upregulated genes were associated with immune and stress responses in astrocytes, immune responses and developmental processes in microglia, metabolic processes and immune responses in neurons, and metabolic processes and gene expression in macrophages. Overrepresented GO terms for downregulated genes included ion transport and behavior in astrocytes, cell cycle and cell division in microglia, metabolic processes in neurons, and response to stimulus, signaling and cell motility in macrophages. Conclusions To our knowledge, this is the first transcriptomic study of TLR2 function across different cell types during T. gondii infection. Results of RNA-sequencing demonstrated roles for TLR2 varied by cell type during T. gondii infection. Our findings facilitate understanding of the detailed relationship between TLR2 and T. gondii infection, and elucidate mechanisms underlying neurological changes during infection.
  • Liu M, Adjou Moumouni PF, Cao S, Asada M, Wang G, Gao Y, Guo H, Li J, Vudriko P, Efstratiou A, Ringo AE, Lee SH, Hakimi H, Masatani T, Sunaga F, Kawazu SI, Yamagishi J, Jia L, Inoue N, Xuan X
    Ticks and tick-borne diseases 1877-959X 2017/11 [Refereed][Not invited]
  • Junya Yamagishi, Masahito Asada, Hassan Hakimi, Takeshi Q. Tanaka, Chihiro Sugimoto, Shin-ichiro Kawazu
    BMC GENOMICS 18 (1) 832  1471-2164 2017/10 [Refereed][Not invited]
     
    Background: Babesia ovata, belonging to the phylum Apicomplexa, is an infectious parasite of bovids. It is not associated with the manifestation of severe symptoms, in contrast to other types of bovine babesiosis caused by B. bovis and B. bigemina; however, upon co-infection with Theileria orientalis, it occasionally induces exacerbated symptoms. Asymptomatic chronic infection in bovines is usually observed only for B. ovata. Comparative genomic analysis could potentially reveal factors involved in these distinguishing characteristics; however, the genomic and molecular basis of these phenotypes remains elusive, especially in B. ovata. From a technical perspective, the current development of a very long read sequencer, MinION, will facilitate the obtainment of highly integrated genome sequences. Therefore, we applied next-generation sequencing to acquire a high-quality genome of the parasite, which provides fundamental information for understanding apicomplexans. Results: The genome was assembled into 14,453,397 bp in size with 5031 protein-coding sequences (91 contigs and N50 = 2,090,503 bp). Gene family analysis revealed that ves1 alpha and beta, which belong to multigene families in B. bovis, were absent from B. ovata, the same as in B. bigemina. Instead, ves1a and ves1b, which were originally specified in B. bigemina, were present. The B. ovata and B. bigemina ves1a configure one cluster together even though they divided into two sub-clusters according to the spp. In contrast, the ves1b cluster was more dispersed and the overlap among B. ovata and B. bigemina was limited. The observed redundancy and rapid evolution in sequence might reflect the adaptive history of these parasites. Moreover, same candidate genes which potentially involved in the distinct phenotypes were specified by functional analysis. An anamorsin homolog is one of them. The human anamorsin is involved in hematopoiesis and the homolog was present in B. ovata but absent in B. bigemina which causes severe anemia. Conclusions: Taking these findings together, the differences demonstrated by comparative genomics potentially explain the evolutionary history of these parasites and the differences in their phenotypes. Besides, the draft genome provides fundamental information for further characterization and understanding of these parasites.
  • Mingming Liu, Masahito Asada, Shinuo Cao, Paul Franck Adjou Moumouni, Patrick Vudriko, Artemis Efstratiou, Hassan Hakimi, Tatsunori Masatani, Fujiko Sunaga, Shin-ichiro Kawazu, Junya Yamagishi, Xuenan Xuan
    MOLECULAR AND BIOCHEMICAL PARASITOLOGY 216 56 - 59 0166-6851 2017/09 [Refereed][Not invited]
     
    The development of gene manipulation techniques has been reported in many protozoan parasites over the past few years. However, these techniques have not yet been established for Babesia gibsoni. Here, we report for the first time, the successful transient transfection of B. gibsoni. The plasmid containing the firefly luciferase reporter gene (pBS-ELA) was transfected into B. gibsoni by an AMAXA 4D Nudeofector (TM) device. Transfection using program FA113 and Lonza buffer SF showed the highest luciferase expression. Twenty micrograms of plasmid produced the highest relative transfection efficiency. The fluorescent protein-expressing parasites were determined by GFP-containing plasmid (pBS-EGA) at 48 and 72 h post transfection. This finding is the first step towards a stable transfection method for B. gibsoni, which may contribute to a better understanding of the biology of the parasite.
  • Kazuo Imai, Norihito Tarumoto, Kazuhisa Misawa, Lucky Ronald Runtuwene, Jun Sakai, Kyoko Hayashida, Yuki Eshita, Ryuichiro Maeda, Josef Tuda, Takashi Murakami, Shigefumi Maesaki, Yutaka Suzuki, Junya Yamagishi, Takuya Maeda
    BMC INFECTIOUS DISEASES 17 (1) 621  1471-2334 2017/09 [Refereed][Not invited]
     
    Background: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION (TM) nanopore sequencer. Methods: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION (TM) nanopore sequencer to identify each Plasmodium species. Results: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION (TM) was consistent with the reference plasmid sequences and the results of nested PCR. Conclusions: Our diagnostic method combined with LAMP and MinION (TM) could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.
  • Toshiki Sekiya, Junya Yamagishi, John Henry V. Gray, Paul G. Whitney, Axel Martinelli, Weiguang Zeng, Chinn Yi Wong, Chihiro Sugimoto, David C. Jackson, Brendon Y. Chua
    BIOMATERIALS 137 61 - 72 0142-9612 2017/08 [Refereed][Not invited]
     
    The lipopeptide R4Pam2Cys is an agonist for toll-like receptor-2 (TLR2), a key pathogen-associated molecular pattern receptor expressed on many antigen-presenting cells such as dendritic cells (DCs). Electrostatic association of R4Pam2Cys with soluble protein antigens significantly enhances their immunogenicity and there is evidence to suggest that reducing the size of suitably adjuvanted-antigen complexes in solution may further improve their immunostimulatory capabilities. In this study, we investigated how incorporation of polyethylene glycol (PEG) into R4Pam2Cys affects the size, activity and efficacy of formed antigen-lipopeptide complexes. The presence of PEG was shown to increase solubility with a concomitant reduction in the particle size of vaccine formulations that was dependent on the length of PEG used. When compared to non-PEGylated R4Pam2Cys, vaccination of animals with antigen-complexed PEGylated R4Pam2Cys resulted not only in improvements in antibody production but significantly higher antigen-specific CD8(+) T cell responses. Both lipopeptides exhibited similar in vitro capabilities to induce DC maturation, facilitate antigen uptake and presentation to T cells. Moreover, analyses of the transcriptomes obtained from DCs treated with either lipopeptide revealed a large number of commonly induced genes with similar transcript expression levels, suggesting that common signalling pathways and processes were engaged following activation by either lipopeptide. In vivo analysis however revealed that vaccination with antigen-complexed PEGylated R4Pam2Cys resulted in improved antigen presentation to T cells. These heightened responses were not attributed to prolonged antigen persistence but rather due to more rapid transportation of antigen from the injection site into the draining lymph nodes over a short period of time. Our results indicate that reducing the size of formed antigen-TLR2-agonist complexes by PEGylation does not compromise the activity of the agonist but in fact enhances its trafficking in vivo ultimately leading to improved humoral and cell-mediated immune responses. (C) 2017 Elsevier Ltd. All rights reserved.
  • Use of the Oxford Nanopore MinION sequencer for MLST genotyping of vancomycin-resistant enterococci.
    Tarumoto N, Sakai J, Sujino K, Yamaguchi T, Ohta M, Yamagishi J, Runtuwene LR, Murakami T, Suzuki Y, Maeda T, Maesaki S
    J Hosp Infect 96 (3) 296 - 298 2017/06 [Refereed][Not invited]
  • Junya Yamagishi, Lucky R. Runtuwene, Kyoko Hayashida, Arthur E. Mongan, Lan Anh Nguyen Thi, Linh Nguyen Thuy, Cam Nguyen Nhat, Kriengsak Limkittikul, Chukiat Sirivichayakul, Nuankanya Sathirapongsasuti, Martin Frith, Wojciech Makalowski, Yuki Eshita, Sumio Sugano, Yutaka Suzuki
    SCIENTIFIC REPORTS 7 (1) 3510  2045-2322 2017/06 [Refereed][Not invited]
     
    The recent development of a nanopore-type portable DNA sequencer has changed the way we think about DNA sequencing. We can perform sequencing directly in the field, where we collect the samples. Here, we report the development of a novel method to detect and genotype tropical disease pathogens, using dengue fever as a model. By combining the sequencer with isothermal amplification that only requires a water bath, we were able to amplify and sequence target viral genomes with ease. Starting from a serum sample, the entire procedure could be finished in a single day. The analysis of blood samples collected from 141 Indonesian patients demonstrated that this method enables the clinical identification and serotyping of the dengue virus with high sensitivity and specificity. The overall successful detection rate was 79%, and a total of 58 SNVs were detected. Similar analyses were conducted on 80 Vietnamese and 12 Thai samples with similar performance. Based on the obtained sequence information, we demonstrated that this approach is able to produce indispensable information for etiologically analyzing annual or regional diversifications of the pathogens.
  • Yohei Takeda, Keisuke Kataoka, Junya Yamagishi, Seishi Ogawa, Tsukasa Seya, Misako Matsumoto
    CELL REPORTS 19 (9) 1874 - 1887 2211-1247 2017/05 [Refereed][Not invited]
     
    Cancer patients having anti-programmed cell death-1 (PD-1)/PD ligand 1 (L1)-unresponsive tumors may benefit from advanced immunotherapy. Double-stranded RNA triggers dendritic cell (DC) maturation to cross-prime antigen-specific cytotoxic T lymphocytes (CTLs) via Toll-like receptor 3 (TLR3). The TLR3-specific RNA agonist, ARNAX, can induce anti-tumor CTLs without systemic cytokine/interferon (IFN) production. Here, we have developed a safe vaccine adjuvant for cancer that effectively implements anti-PD-L1 therapy. Co-administration of ARNAX with a tumor-associated antigen facilitated tumor regression in mouse models, and in combination with anti-PD-L1 antibody, activated tumor-specific CTLs in lymphoid tissues, enhanced CTL infiltration, and overcame anti-PD-1 resistance without cytokinemia. The TLR3-TICAM-1-interferon regulatory factor (IRF) 3-IFN-beta axis in DCs exclusively participated in CD8(+) T cell cross-priming. ARNAX therapy established Th1 immunity in the tumor microenvironment, upregulating genes involved in DC/T cell/natural killer (NK) cell recruitment and functionality. Human ex vivo studies disclosed that ARNAX+ antigen induced antigen-specific CTL priming and proliferation in peripheral blood mononuclear cells (PBMCs), supporting the feasibility of ARNAX for potentiating anti-PD-1/PD-L1 therapy in human vaccine immunotherapy.
  • Patrick Reteng, Visia Vrisca, Inka Sukarno, Ilham Habib Djarkoni, Jane Angela Kalangi, George Eduardo Jacobs, Lucky Ronald Runtuwene, Yuki Eshita, Ryuichiro Maeda, Yutaka Suzuki, Arthur Elia Mongan, Sarah Maria Warouw, Junya Yamagishi, Josef Tuda
    BMC Research Notes 10 (1) 147  1756-0500 2017/04/04 [Refereed][Not invited]
     
    Background: Malaria still poses one of the major threats to human health. Development of effective antimalarial drugs has decreased this threat however, the emergence of drug-resistant Plasmodium falciparum, a cause of Malaria, is disconcerting. The antimalarial drug chloroquine has been effectively used, but resistant parasites have spread worldwide. Interestingly, the withdrawal of the drug reportedly leads to an increased population of susceptible parasites in some cases. We examined the prevalence of genomic polymorphisms in a malaria parasite P. falciparum, associated with resistance to an antimalarial drug chloroquine, after the withdrawal of the drug from Indonesia. Results: Blood samples were collected from 95 malaria patients in North Sulawesi, Indonesia, in 2010. Parasite DNA was extracted and analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for pfcrt and pfmdr1. In parallel, multiplex amplicon sequencing for the same genes was carried out with Illumina MiSeq. Of the 59 cases diagnosed as P. falciparum infection by microscopy, PCR-RFLP analysis clearly identified the genotype 76T in pfcrt in 44 cases. Sequencing analysis validated the identified genotypes in the 44 cases and demonstrated that the haplotype in the surrounding genomic region was exclusively SVMNT. Results of pfmdr1 were successfully obtained for 51 samples, where the genotyping results obtained by the two methods were completely consistent. In pfmdr1, the 86Y mutant genotype was observed in 45 cases (88.2%). Conclusions: Our results suggest that the prevalence of the mutated genotypes remained dominant even 6 years after the withdrawal of chloroquine from this region. Diversified haplotype of the resistance-related locus, potentially involved in fitness costs, unauthorized usage of chloroquine, and/or a short post-withdrawal period may account for the observed high persistence of prevalence.
  • Naganori Nao, Junya Yamagishi, Hiroko Miyamoto, Manabu Igarashi, Rashid Manzoor, Aiko Ohnuma, Yoshimi Tsuda, Wakako Furuyama, Asako Shigeno, Masahiro Kajihara, Noriko Kishida, Reiko Yoshida, Ayato Takada
    MBIO 8 (1) 2150-7511 2017/01 [Refereed][Not invited]
     
    Highly pathogenic avian influenza viruses with H5 and H7 hemagglutinin (HA) subtypes evolve from low-pathogenic precursors through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been observed to occur naturally only in these HA subtypes, little is known about the genetic basis for the acquisition of the polybasic HA cleavage site. Here we show that consecutive adenine residues and a stem-loop structure, which are frequently found in the viral RNA region encoding amino acids around the cleavage site of low-pathogenic H5 and H7 viruses isolated from waterfowl reservoirs, are important for nucleotide insertions into this RNA region. A reporter assay to detect nontemplated nucleotide insertions and deep-sequencing analysis of viral RNAs revealed that an increased number of adenine residues and enlarged stem-loop structure in the RNA region accelerated the multiple adenine and/or guanine insertions required to create codons for basic amino acids. Interestingly, nucleotide insertions associated with the HA cleavage site motif were not observed principally in the viral RNA of other subtypes tested (H1, H2, H3, and H4). Our findings suggest that the RNA editing-like activity is the key mechanism for nucleotide insertions, providing a clue as to why the acquisition of the polybasic HA cleavage site is restricted to the particular HA subtypes. IMPORTANCE Influenza A viruses are divided into subtypes based on the antigenicity of the viral surface glycoproteins hemagglutinin (HA) and neuraminidase. Of the 16 HA subtypes (H1 to -16) maintained in waterfowl reservoirs of influenza A viruses, H5 and H7 viruses often become highly pathogenic through the acquisition of multiple basic amino acid residues at the HA cleavage site. Although this mechanism has been known since the 1980s, the genetic basis for nucleotide insertions has remained unclear. This study shows the potential role of the viral RNA secondary structure for nucleotide insertions and demonstrates a key mechanism explaining why the acquisition of the polybasic HA cleavage site is restricted to particular HA subtypes in nature. Our findings will contribute to better understanding of the ecology of influenza A viruses and will also be useful for the development of genetically modified vaccines against H5 and H7 influenza A viruses with increased stability.
  • Tatsunori Masatani, Masahito Asada, Hassan Hakimi, Kei Hayashi, Junya Yamagishi, Shin-ichiro Kawazu, Xuenan Xuan
    PARASITOLOGY RESEARCH 115 (8) 3139 - 3145 0932-0113 2016/08 [Refereed][Not invited]
     
    Cysteine-based peroxidases, known as peroxiredoxins (Prx) or thioredoxin peroxidases (TPx), are important antioxidant enzymes that prevent oxidative damage caused by reactive oxygen species (ROS). In this study, we identified a novel mitochondrial 2-Cys Prx, BbTPx-2, from a bovine Babesia parasite, B. bovis. BbTPx-2 complementary DNA (cDNA) encodes a polypeptide of 254 amino acid residues. This protein has a mitochondrial targeting peptide at the N-terminus and two conserved cysteine residues of the typical 2-Cys Prx. By using a thiol mixed-function oxidation assay, the antioxidant activity of recombinant BbTPx-2 was revealed, and its antioxidant activity was comparable to that of a cytosolic 2-Cys Prx from B. bovis, BbTPx-1. Notably, we confirmed that BbTPx-2 was expressed in the mitochondrion of B. bovis merozoites. Taken together, the results suggest that the mitochondrial BbTPx-2 is an antioxidative enzyme for scavenging ROS in B. bovis.
  • Keisuke Suganuma, Albertus Eka Yudistira Sarwono, Shinya Mitsuhashi, Marcin Jakalski, Tadashi Okada, Molefe Nthatisi, Junya Yamagishi, Makoto Ubukata, Noboru Inoue
    ANTIMICROBIAL AGENTS AND CHEMOTHERAPY 60 (7) 4391 - 4393 0066-4804 2016/07 [Refereed][Not invited]
     
    This study aimed to evaluate the trypanocidal activity of mycophenolic acid (MPA) and its derivatives for Trypanosoma congolense. The proliferation of T. congolense was completely inhibited by adding <1 mu M MPA and its derivatives. In addition, the IMP dehydrogenase in T. congolense was molecularly characterized as the target of these compounds. The results suggest that MPA and its derivatives have the potential to be new candidates as novel trypanocidal drugs.
  • Makoto Matsubayashi, Fumiya Kawahara, Takeshi Hatta, Junya Yamagishi, Takeharu Miyoshi, Anisuzzaman, Kazumi Sasai, Takashi Isobe, Kiyoshi Kita, Naotoshi Tsuji
    INFECTION GENETICS AND EVOLUTION 40 54 - 62 1567-1348 2016/06 [Refereed][Not invited]
     
    Chicken coccidiosis is caused by Eimeria spp., particularly Eimeria tenella, and is characterized by watery or hemorrhagic diarrhea, resulting in death in severe cases. Precociously attenuated live vaccines are widely used to control the disease, and these are produced by serially passaging virulent strains through chickens, and the collection of oocysts from feces at progressively earlier time points during oocyst shedding. Sporozoites of the precocious strain rapidly enter the intestinal mucosa, and their subsequent asexual development reduces their growth. However, there have been few detailed genetic or transcriptional analyses of the strains. Here, we used RNA sequencing to gain novel biological insight into the pathogenicity and precocity of E. tenella. We compared the differential transcription in the sporozoites (the initial stage of endogenous development) of virulent and precocious strains by mapping the sequence reads onto the draft genome of E. tenella. About 90% of the reads from both strains were mapped to the genome, and 16,630 estimated transcript regions were identified. Using Gene Ontology slim and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses and the annotation of the estimated transcripts with Blastx, we found that the expression of some genes involved in carbohydrate metabolism were expressed two-fold more strongly in the virulent strain than in the precocious strain. Characteristically, genes related to proteins secreted from the apical complex, proteases, cell attachment proteins, mitochondrial proteins, and transporters were most strongly upregulated in the virulent strain. Interestingly, the expression of genes associated with cell survival, development, or proliferation was strongly upregulated in the precocious strain. These findings suggest that virulent strains survive long before invasion and invade actively/successfully into host cells, whereas proliferative processes appear to affect precocity. (c) 2016 Elsevier B.V. All rights reserved.
  • Shino Yamasaki, Keisuke Suganuma, Junya Yamagishi, Masahito Asada, Naoaki Yokoyama, Shin-ichiro Kawazu, Noboru Inoue
    PARASITES & VECTORS 9 (1) 299  1756-3305 2016/05 [Refereed][Not invited]
     
    Background: Since Trypanosorna spp. lack a complete heme synthesis pathway, the parasites are totally dependent on their host for heme throughout all of the stages of their life -cycle. We herein report the identification and characterization of a T. congolense epimastigote form (EMF)-specific hemoglobin (Hb) receptor. The gene was initially reported to encode a T. congolense haptoglobin (Hp)-Hb complex receptor (TcHpHbR) based on its similarity to a gene encoding a T brucei Hp-Hb complex receptor (TbHpHbR).Methods: Trypanosorna congolense IL3000 was used in this study. A TcHpHbR gene was PCR amplified from the parasite genome. The recombinant protein was used as an immunogen to raise antibodies for immunofluorescence assay and immunoblotting. Hemoglobin uptake by the parasite was examined by using Alexa 488 labelled Hb and visualized by confocal laser scanning microscopy. The qualitative and quantitative interaction between TcHpHbR and its ligand were measured using a surface plasmon resonance assay.Results: We found that, unlike TbHpHbR, TcHpHbR was exclusively expressed in the EMF stage at RNA and protein levels. The recombinant TcHpHbR (rTcHpHbR) was co-precipitated with free-Hb in a GST-pull down assay. Surface plasmon resonance revealed that rTcHpHbR binds free-Hb with high affinity (dissociation constant (K,A) =2.1x10(-8) M) but free-Hp with low affinity (Kd = 2.2x10(-7) M). Furthermore, Alexa 488-labelled-Hb was only taken up by the EMF and co-localized with tomato lectin, which is a marker of endocytic compartments (flagellar pocket and lysosome).Conclusion: We conclude that the T. congolense EMF takes up free-Hb via TcHpHbR, a receptor which is specific to this developmental stage. We therefore propose renaming TcHpHbR as T congolense EMF-specific Hb receptor (TcEpHbR).
  • Junya Yamagishi, Yukuto Sato, Natsuko Shinozaki, Bin Ye, Akito Tsuboi, Masao Nagasaki, Riu Yamashita
    PLOS ONE 11 (4) e0154389  1932-6203 2016/04 [Refereed][Not invited]
     
    The rapid improvement of next-generation sequencing performance now enables us to analyze huge sample sets with more than ten thousand specimens. However, DNA extraction can still be a limiting step in such metagenomic approaches. In this study, we analyzed human oral microbes to compare the performance of three DNA extraction methods: PowerSoil (a method widely used in this field), QIAsymphony (a robotics method), and a simple boiling method. Dental plaque was initially collected from three volunteers in the pilot study and then expanded to 12 volunteers in the follow-up study. Bacterial flora was estimated by sequencing the V4 region of 16S rRNA following species-level profiling. Our results indicate that the efficiency of PowerSoil and QIAsymphony was comparable to the boiling method. Therefore, the boiling method may be a promising alternative because of its simplicity, cost effectiveness, and short handling time. Moreover, this method was reliable for estimating bacterial species and could be used in the future to examine the correlation between oral flora and health status. Despite this, differences in the efficiency of DNA extraction for various bacterial species were observed among the three methods. Based on these findings, there is no "gold standard" for DNA extraction. In future, we suggest that the DNA extraction method should be selected on a case-by-case basis considering the aims and specimens of the study.
  • Hassan Hakimi, Junya Yamagishi, Yuto Kegawa, Osamu Kaneko, Shin-ichiro Kawazu, Masahito Asada
    PARASITES & VECTORS 9 171  1756-3305 2016/03 [Refereed][Not invited]
     
    Background: Bovine babesiosis is a tick-borne disease caused by several species of Babesia which produce acute and fatal disease in cattle and affect livestock industry worldwide. Babesia ovata is a benign species widespread in east Asian countries and causes anemia, particularly in cattle which are co-infected with Theileria orientalis. The development of genetic manipulation methods is necessary to improve our understanding of the basic biology of protozoan pathogens toward a better control of disease. Such tools have not been developed for B. ovata, and are the aim of this study. Methods: In this study we transfected constructs that were designed to evaluate the ability of several B. ovata promoter candidates to drive expression of a reporter luciferase. We found that the elongation factor-1 alpha intergenic region (ef-1 alpha IG) and the actin 5' non-coding region (NR) had highest promoter activities. To establish a stable transfection system, we generated a plasmid construct in which the ef-1 alpha IG promoter drives gfp expression, and the actin 5' NR mediates expression of the selectable marker hdhfr. The plasmid was designed for episomal transfection, as well as to integrate by double cross-over homologous recombination into the ef-1 alpha locus. Circular or linearized plasmid was transfected by electroporation into in vitro cultured B. ovata and retention of the plasmid was facilitated by drug selection with 5 nM WR99210 initiated 48 h after transfection. Results: After one-week cultivation with WR99210, GFP-expressing parasites were observed by fluorescence microscopy. Integration of the plasmid construct into the ef-1 alpha locus was confirmed by PCR, Southern blot analysis, and sequencing of recombination sites. These results confirm successful development of a stable transfection system for B. ovata. Conclusion: The current study provides a fundamental molecular tool to aid in molecular and cellular studies of B. ovata.
  • Youn-Kyoung Goo, Junya Yamagishi, Akio Ueno, Mohamad Alaa Terkawi, Gabriel Oluga Aboge, Dongmi Kwak, Yeonchul Hong, Dong-Il Chung, Makoto Igarashi, Yoshifumi Nishikawa, Xuenan Xuan
    PARASITES & VECTORS 8 654  1756-3305 2015/12 [Refereed][Not invited]
     
    Background: The glyoxalase pathway, which includes two enzymes, glyoxalase 1 and 2 (Glo1 and Glo2), is a ubiquitous cellular system responsible for the removal of cytotoxic methylglyoxal produced during glycolysis. Protozoan parasites, including Toxoplasma gondii (T. gondii) tachyzoites, produce methylglyoxal because of increased glycolytic fluxes. A Glo1 inhibitor such as curcumin could be considered a drug candidate for anti-protozoan, anti-inflammatory, and anti-cancer therapy. Methods: The T. gondii Glo1 gene (TgGlo1) was cloned and the recombinant protein was produced. Enzyme kinetics of TgGlo1 and five mutants were evaluated by adding methylglyoxal and glutathione to a reaction mixture. Finally, the inhibitory effects of various concentrations of curcumin on recombinant TgGlo1 were evaluated using in vitro cultures of T. gondii. Results: Active recombinant TgGlo1 was successfully produced and the active sites (E166 and E251) of TgGlo1 were verified by point mutagenesis. Curcumin at the tested doses inhibited the enzymatic activity of recombinant TgGlo1 as well as the parasitic propagation of in vitro-cultured T. gondii. The K-i and IC50 were 12.9 +/- 0.5 mu M and 38.3 +/- 0.9 mu M, respectively. Conclusion: The inhibitory effect of curcumin on the enzymatic activity of TgGlo1 and parasitic propagation of T. gondii could be explored in the potential development of a potent drug for the treatment of toxoplasmosis. However, considering the fact that curcumin is known to have many effects on other molecules in the micromolar range, further elucidation of curcumin's direct inhibition of the glyoxalase system of T. gondii will be needed.
  • Yukuto Sato, Junya Yamagishi, Riu Yamashita, Natsuko Shinozaki, Bin Ye, Takuji Yamada, Masayuki Yamamoto, Masao Nagasaki, Akito Tsuboi
    PLOS ONE 10 (6) e0131607  1932-6203 2015/06 [Refereed][Not invited]
     
    Given the advent of massively parallel DNA sequencing, human microbiome is analyzed comprehensively by metagenomic approaches. However, the inter-and intra-individual variability and stability of the human microbiome remain poorly characterized, particularly at the intra-day level. This issue is of crucial importance for studies examining the effects of microbiome on human health. Here, we focused on bacteriome of oral plaques, for which repeated, time-controlled sampling is feasible. Eighty-one supragingival plaque subjects were collected from healthy individuals, examining multiple sites within the mouth at three time points (forenoon, evening, and night) over the course of 3 days. Bacterial composition was estimated by 16S rRNA sequencing and species-level profiling, resulting in identification of a total of 162 known bacterial species. We found that species compositions and their relative abundances were similar within individuals, and not between sampling time or tooth type. This suggests that species-level oral bacterial composition differs significantly between individuals, although the number of subjects is limited and the intra-individual variation also occurs. The majority of detected bacterial species (98.2%; 159/162), however, did not fluctuate over the course of the day, implying a largely stable oral microbiome on an intra-day time scale. In fact, the stability of this data set enabled us to estimate potential interactions between rare bacteria, with 40 co-occurrences supported by the existing literature. In summary, the present study provides a valuable basis for studies of the human microbiome, with significant implications in terms of biological and clinical outcomes.
  • Marcin Jakalski, Hiroyuki Wakaguri, Tabea G. Kischka, Yoshifumi Nishikawa, Shin-ichiro Kawazu, Makoto Matsubayashi, Fumiya Kawahara, Naotoshi Tsuji, Shinuo Cao, Fujiko Sunaga, Xuenan Xuan, Kazuhiro Okubo, Ikuo Igarashi, Josef Tuda, Arthur E. Mongan, Yuki Eshita, Ryuichiro Maeda, Wojciech Makalowski, Yutaka Suzuki, Junya Yamagishi
    NUCLEIC ACIDS RESEARCH 43 (D1) D631 - D636 0305-1048 2015/01 [Refereed][Not invited]
     
    The previous release of our Full-parasites database ( ext-link-type="uri" xlink:href="http://fullmal.hgc.jp/" xlink:type="simple">http://fullmal.hgc.jp/) brought enhanced functionality, an expanded full-length cDNA content, and new RNA-Seq datasets from several important apicomplexan parasites. The 2015 update witnesses the major shift in the databases content with focus on diverse transcriptomes of the apicomplexan parasites. The content of the database was substantially enriched with transcriptome information for new apicomplexan parasites. The latest version covers a total of 17 species, with addition of our newly generated RNA-Seq data of a total of 909 150 388 tags. Moreover, we have generated and included two novel and unique datasets, which represent diverse nature of transcriptomes in individual parasites in vivo and in vitro. One is the data collected from 116 Indonesian patients infected with Plasmodium falciparum. The other is a series of transcriptome data collected from a total of 38 single cells of P. falciparum cultured in vitro. We believe that with the recent advances our database becomes an even better resource and a unique platform in the analysis of apicomplexan parasites and their interaction with their hosts. To adequately reflect the recent modifications and the current content we have changed the database name to DB-AT-DataBase of Apicomplexa Transcriptomes.
  • Maki Nishimura, Sachi Tanaka, Fumiaki Ihara, Yoshikage Muroi, Junya Yamagishi, Hidefumi Furuoka, Yutaka Suzuki, Yoshifumi Nishikawa
    SCIENTIFIC REPORTS 5 7936  2045-2322 2015/01 [Refereed][Not invited]
     
    Neospora caninum is a protozoan parasite that causes neurological disorders in dogs and cattle. It can cause nonsuppurative meningoencephalitis and a variety of neuronal symptoms are observed, particularly in dogs. However, the pathogenic mechanism, including the relationship between the parasite distribution and the clinical signs, is unclear. In this study, to understand the pathogenic mechanism of neosporosis, parasite distribution and lesions were assessed in the brain of mice infected with N. caninum (strain Nc-1). Host gene expression was also analyzed with RNA sequencing (RNA-Seq). The histopathological lesions in the frontal lobe and the medulla oblongata were significantly more severe in symptomatic mice than in asymptomatic mice, although no association between the severity of the lesions and parasite numbers was found. In infected mice, the expression of 772 mouse brain genes was upregulated. A GOstat analysis predicted that the upregulated genes were involved in the host immune response. Genes whose expression correlated positively and negatively with parasite numbers were involved in the host immune response, and neuronal morphogenesis and lipid metabolic processes, respectively. These results suggest that changes in the gene expression profile associated with neuronal functions as well as immune responses can contribute to the pathogenesis in N. caninum-infected animals.
  • Cloning and characterization of aspartic protease 3 of Toxoplasma gondii.
    Kamyingkird K, Goo YK, Cao S, Adjou Moumouni PF, Aboge GO, Yamagishi J, Terkawi MA, Masatani T, Yu L, Nishikawa Y, Xuan X
    Journal of Protozoology Research 24 18 - 25 2014/12 [Not refereed][Not invited]
  • Nadia Sokal, Yingchao Nie, Leslie G. Willis, Junya Yamagishi, Gary W. Blissard, Mark R. Rheault, David A. Theilmann
    VIROLOGY 468 160 - 171 0042-6822 2014/11 [Refereed][Not invited]
     
    IE0 and IE1 of the baculovirus Autographa californica multiple nucleopolyhedrovirus are essential transregulatory proteins required for both viral DNA replication and transcriptional transactivation. IE0 is identical to IE1 except for 54 amino acids at the N-terminus but the functional differences between these two proteins remain unclear. The purpose of this study was to determine the separate roles of these critical proteins in the virus life cycle. Unlike prior studies, IE0 and IE1 were analyzed using viruses that expressed ie0 and ie1 from an identical promoter so that the timing and levels of expression were comparable. IE0 and IE1 were found to equally support viral DNA replication and budded virus (BV) production. However, specific viral promoters were selectively transactivated by IE0 relative to IE1 but only when expressed at low levels. These results indicate that IE0 preferentially transactivates specific viral genes at very early times post-infection enabling accelerated replication and BV production. Crown Copyright (C) 2014 Published by Elsevier Inc. All rights reserved.
  • Junya Yamagishi, Anna Natori, Mohammed E. M. Tolba, Arthur E. Mongan, Chihiro Sugimoto, Toshiaki Katayama, Shuichi Kawashima, Wojciech Makalowski, Ryuichiro Maeda, Yuki Eshita, Josef Tuda, Yutaka Suzuki
    GENOME RESEARCH 24 (9) 1433 - 1444 1088-9051 2014/09 [Refereed][Not invited]
     
    To understand the molecular mechanisms of parasitism in vivo, it is essential to elucidate how the transcriptomes of the human hosts and the infecting parasites affect one another. Here we report the RNA-seq analysis of 116 Indonesian patients infected with the malaria parasite Plasmodium falciparum(Pf). We extracted RNAs from their peripheral blood as a mixture of host and parasite transcripts and mapped the RNA-seq tags to the human and Pf reference genomes to separate the respective tags. We were thus able to simultaneously analyze expression patterns in both humans and parasites. We identified human and parasite genes and pathways that correlated with various clinical data, which may serve as primary targets for drug developments. Of particular importance, we revealed characteristic expression changes in the human innate immune response pathway genes including TLR2 and T1CAM2 that correlated with the severity of the malaria infection. We also found a group of transcription regulatory factors, JUND, for example, and signaling molecules, TNFAIP3, for example, that were strongly correlated in the expression patterns of humans and parasites. We also identified several genetic variations in important anti-malaria drug resistance-related genes. Furthermore, we identified the genetic variations which are potentially associated with severe malaria symptoms both in humans and parasites. The newly generated data should collectively lay a unique foundation for understanding variable behaviors of the field malaria parasites, which are far more complex than those observed under laboratory conditions.
  • Junya Yamagishi, Hiroyuki Wakaguri, Naoaki Yokoyama, Riu Yamashita, Yutaka Suzuki, Xuenan Xuan, Ikuo Igarashi
    BMC GENOMICS 15 678  1471-2164 2014/08 [Refereed][Not invited]
     
    Background: Babesia bovis is an apicomplexan parasite that causes babesiosis in infected cattle. Genomes of pathogens contain promising information that can facilitate the development of methods for controlling infections. Although the genome of B. bovis is publically available, annotated gene models are not highly reliable prior to experimental validation. Therefore, we validated a preproposed gene model of B. bovis and extended the associated annotations on the basis of experimentally obtained full-length expressed sequence tags (ESTs). Results: From in vitro cultured merozoites, 12,286 clones harboring full-length cDNAs were sequenced from both ends using the Sanger method, and 6,787 full-length cDNAs were assembled. These were then clustered, and a nonredundant referential data set of 2,115 full-length cDNA sequences was constructed. The comparison of the preproposed gene model with our data set identified 310 identical genes, 342 almost identical genes, 1,054 genes with potential structural inconsistencies, and 409 novel genes. The median length of 5' untranslated regions (UTRs) was 152 nt. Subsequently, we identified 4,086 transcription start sites (TSSs) and 2,023 transcriptionally active regions (TARs) by examining 5' ESTs. We identified ATGGGG and CCCCAT sites as consensus motifs in TARs that were distributed around -50 bp from TSSs. In addition, we found ACACA, TGTGT, and TATAT sites, which were distributed periodically around TSSs in cycles of approximately 150 bp. Moreover, related periodical distributions were not observed in mammalian promoter regions. Conclusions: The observations in this study indicate the utility of integrated bioinformatics and experimental data for improving genome annotations. In particular, full-length cDNAs with one-base resolution for TSSs enabled the identification of consensus motifs in promoter sequences and demonstrated clear distributions of identified motifs. These observations allowed the illustration of a model promoter composition, which supports the differences in transcriptional regulation frameworks between apicomplexan parasites and mammals.
  • Sachi Tanaka, Maki Nishimura, Fumiaki Ihara, Junya Yamagishi, Yutaka Suzuki, Yoshifumi Nishikawa
    INFECTION AND IMMUNITY 81 (10) 3609 - 3619 0019-9567 2013/10 [Refereed][Not invited]
     
    Toxoplasma gondii is an obligate intracellular parasite that invades a wide range of vertebrate host cells. Chronic infections with T. gondii become established in the tissues of the central nervous system, where the parasites may directly or indirectly modulate neuronal function. However, the mechanisms underlying parasite-induced neuronal disorder in the brain remain unclear. This study evaluated host gene expression in mouse brain following infection with T. gondii. BALB/c mice were infected with the PLK strain, and after 32 days of infection, histopathological lesions in the frontal lobe were found to be more severe than in other areas of the brain. Total RNA extracted from infected and uninfected mouse brain samples was subjected to transcriptome analysis using RNA sequencing (RNA-seq). In the T. gondii-infected mice, 935 mouse brain genes were upregulated, whereas 12 genes were downregulated. GOstat analysis predicted that the upregulated genes were primarily involved in host immune responses and cell activation. Positive correlations were found between the numbers of parasites in the infected mouse brains and the expression levels of genes involved in host immune responses. In contrast, genes that had a negative correlation with parasite numbers were predicted to be involved in neurological functions, such as small-GTPase-mediated signal transduction and vesicle-mediated transport. Furthermore, differential gene expression was observed between mice exhibiting the clinical signs of toxoplasmosis and those that did not. Our findings may provide insights into the mechanisms underlying neurological changes during T. gondii infection.
  • Youn-Kyoung Goo, Akio Ueno, Mohamad Alaa Terkawi, G. Oluga Aboge, Yamagishi Junya, Makoto Igarashi, Jung-Yeon Kim, Yeon-Chul Hong, Dong-Il Chung, Yoshifumi Nishikawa, Xuenan Xuan
    EXPERIMENTAL PARASITOLOGY 135 (1) 42 - 49 0014-4894 2013/09 [Refereed][Not invited]
     
    Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species. (C) 2013 Elsevier Inc. All rights reserved.
  • Keisuke Suganuma, Kennedy Miyoro Mochabo, Hassan Hakimi, Shino Yamasaki, Junya Yamagishi, Masahito Asada, Shin-ichiro Kawazu, Noboru Inoue
    MOLECULAR AND BIOCHEMICAL PARASITOLOGY 191 (1) 36 - 43 0166-6851 2013/09 [Refereed][Not invited]
     
    It is known that gene expression in kinetoplastida is regulated post-transcriptionally. Several previous studies have shown that stage-specific gene expression in trypanosomes is regulated by cis-elements located in the 3' untranslated region (UTR) of each mRNA and also by RNA binding proteins. Our previous study revealed that gene expression of congolense epimastigote specific protein (cesp) was regulated by cis-elements located in the 3'UTR In the present study, we identified the adenosine and uridine rich region in the cesp 3'UTR. Using transgenic trypanosome cell lines with different egfp expression cassettes, we showed that this adenosine and uridine rich region is one of the regulatory elements for epimastigote form (EMF) stage-specific gene expression via the regulatory cis-element of the eukaryotic AU rich element (ARE). Therefore this required element within the cesp 3'UTR was designated as T. congolense ARE. This required cis-element might selectively stabilize mRNA in the EMF stage and destabilize mRNA in other stages. By RNA electro mobility shift assay, unknown stage-specific RNA binding proteins (RBPs) whose sequences specifically interacted with the required cis-element were found. These results indicate that EMF stage specific cis-element and REP complexes might specifically stabilize cesp mRNA in EMF. (C) 2013 Elsevier B.V. All rights reserved.
  • Tatsunori Masatani, Tomohide Matsuo, Tetsuya Tanaka, Mohamad Alaa Terkawi, Eung-Goo Lee, Youn-Kyoung Goo, Gabriel Oluga Aboge, Junya Yamagishi, Kei Hayashi, Kyohko Kameyama, Shinuo Cao, Yoshifumi Nishikawa, Xuenan Xuan
    PARASITOLOGY INTERNATIONAL 62 (4) 372 - 379 1383-5769 2013/08 [Refereed][Not invited]
     
    Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV. (C) 2013 Elsevier Ireland Ltd. All rights reserved.
  • Longzheng Yu, Mohmad Alaa Terkawi, Mary Jane Cruz-Flores, Florencia G. Claveria, Gabriel Oluga Aboge, Junya Yamagishi, Youn-Kyoung Goo, Shinuo Cao, Tatsunori Masatani, Yoshifumi Nishikawa, Xuenan Xuan
    Journal of Veterinary Medical Science 75 (7) 995 - 998 0916-7250 2013 [Refereed][Not invited]
     
    A total of 250 blood samples were collected from clinically healthy cattle in five provinces of Philippines. DNA was extracted from the samples and analyzed by nested PCR assays for an epidemiological survey of Babesia bovis and Babesia bigemina infections. Out of the 250 samples, 27 (10.8%) and 16 (6.4%) were positive for B. bovis infection and B. bigemina infection, respectively. Mixed infections were detected in a total of 4 samples (1.6%). Our data provide baseline information regarding the epidemiology of B. bovis and B. bigemina infections in cattle in Philippines, which can be utilized in developing proper strategies for disease control and management. © 2013 The Japanese Society of Veterinary Science.
  • Longzheng Yu, Junya Yamagishi, Shoufa Zhang, Chunmei Jin, Gabriel Oluga Aboge, Houshuang Zhang, Guohong Zhang, Tetsuya Tanaka, Kozo Fujisaki, Yoshifumi Nishikawa, Xuenan Xuan
    PARASITOLOGY INTERNATIONAL 61 (3) 481 - 486 1383-5769 2012/09 [Refereed][Not invited]
     
    A heterologous prime-boost strategy with priming plasmid DNA followed by recombinant virus expressing relevant antigens is known to stimulate protective immunity against intracellular parasites. In this study, we have evaluated a heterologous prime-boost strategy for immunizing mice against Toxoplasma gondii infection. Our results revealed that the prime-boost strategy using both plasmid DNA and adenoviral vector encoding TgAMA1 may stimulate both humoral and Th1/Th2 cellular immune responses specific for TgAMA1. Moreover, C57BL/6 mice immunized with the pAMA1/Ad5Null, pNull/Ad5AMA1, and pAMA1/Ad5AMA1 constructs showed survival rates of 12.5%, 37.5%, and 50%, respectively. In contrast, all the pNull/Ad5Null immunized mice died after infection with the PLK-GFP strain of T. gondii. Brain cyst burden was reduced by 23% in mice immunized with pAMA1/Ad5AMA1 compared with the pNull/Ad5AMA1 immunized mice. These results demonstrate that the heterologous DNA priming and recombinant adenovirus boost strategy may provide protective immunity against T. gondii infection. (C) 2012 Elsevier Ireland Ltd. All rights reserved.
  • Shinuo Cao, Gabriel Oluga Aboge, Mohamad Alaa Terkawi, Longzheng Yu, Ketsarin Kamyingkird, Yuzi Luo, Yan Li, Youn-Kyoung Goo, Junya Yamagishi, Yoshifumi Nishikawa, Naoaki Yokoyama, Hiroshi Suzuki, Ikuo Igarashi, Ryuichiro Maeda, Tawin Inpankaew, Sathaporn Jittapalapong, Xuenan Xuan
    PARASITOLOGY RESEARCH 111 (3) 1259 - 1266 0932-0113 2012/09 [Refereed][Not invited]
     
    Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.
  • Youn-Kyoung Goo, Gabriel Oluga Aboge, M. Alaa Terkawi, Honglin Jia, Junya Yamagishi, Fujiko Sunaga, Kazuhiko Namikawa, Se-Yeoun Cha, Hyung-Kwan Jang, Suk Kim, Yoshifumi Nishikawa, Xuenan Xuan
    PARASITOLOGY INTERNATIONAL 61 (2) 364 - 368 1383-5769 2012/06 [Refereed][Not invited]
     
    We determined the molecular characteristics of four proteins, BgP32, BgP45, BgP47, and BgP50, of Babesia gibsoni. Localization by subcellular fractionations followed by Western blotting revealed that the corresponding native proteins belong to merozoite surface protein family of B. gibsoni (BgMSP). Moreover, antisera against either rBgP45 or rBgP47 cross-reacted with all the proteins of the BgMSP family on ELISA and IFAT analyses. Of the four candidate antigens, ELISA with rBgP45 yielded high sensitivity, and ELISA with rBgP32 resulted in high specificity and in concordance with IFAT results. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Yamagishi J, Watanabe J, Goo YK, Masatani T, Suzuki Y, Xuan X
    The Journal of Parasitology 98 (2) 445 - 447 2012/02 [Refereed][Not invited]
  • Yan Li, Mohamad Alaa Terkawi, Yoshifumi Nishikawa, Gabriel Oluga Aboge, Yuzi Luo, Hideo Ooka, Youn-Kyoung Goo, Longzheng Yu, Shinuo Cao, Yongfeng Sun, Junya Yamagishi, Tatsunori Masatani, Naoaki Yokoyama, Ikuo Igarashi, Xuenan Xuan
    INFECTION AND IMMUNITY 80 (1) 311 - 320 0019-9567 2012/01 [Refereed][Not invited]
     
    Although primary infection of mice with Babesia microti has been shown to protect mice against subsequent lethal infection by Babesia rodhaini, the mechanism behind the cross-protection is unknown. To unravel this mechanism, we investigated the influence of primary infection of mice with nonlethal B. microti using different time courses on the outcome of subsequent lethal B. rodhaini infection. Simultaneous infections of mice with these parasites resulted in rapid increases in parasitemia, with 100% mortality in BALB/c mice, as observed with control mice infected with B. rodhaini alone. In contrast, mice with acute, resolving, and chronic-phase B. microti infections were completely protected against B. rodhaini, resulting in low parasitemia and no mortalities. Mice immunized with dead B. microti were not protected from B. rodhaini infection, although high antibody responses were induced. Interestingly, the protected mice had significantly decreased levels of antibody response, cytokines (including gamma interferon [IFN-gamma], interleukin-2 [IL-2], IL-8, IL-10, and IL-12), and nitric oxide levels after infection with B. rodhaini. SCID mice and IFN-gamma-deficient mice with chronic B. microti infections demonstrated protective responses comparable to those of immunocompetent mice. Likewise, in vivo NK cell depletion did not significantly impair the protective responses. Conversely, macrophage depletion resulted in increased susceptibility to B. rodhaini infection associated with changes in their antibody and cytokines profiles, indicating that macrophages contribute to the protection against this challenge infection. We conclude that future development of vaccines against Babesia should include a strategy that enhances the appropriate activation of macrophages.
  • Longzheng Yu, Shoufa Zhang, Wanfeng Liang, Chunmei Jin, Lijun Ji, Yuzi Luo, Yan Li, Shinuo Cao, Junya Yamagishi, Yoshifumi Nishikawa, Suguru Kawano, Kozo Fujisaki, Xuenan Xuan
    JOURNAL OF VETERINARY MEDICAL SCIENCE 73 (11) 1509 - 1512 0916-7250 2011/11 [Refereed][Not invited]
     
    An epidemiological survey on a Theileria parasite infection of cattle in Northeast China was carried out using allele-specific PCR and DNA sequence analysis of the major piroplasm surface protein (MPSP) gene. The results showed that 14 of 104 blood samples were positive for Theileria by PCR. Among the positive cases, co-infection with various combinations of C- and I-type parasites was detected in 12 samples; no B- and Thai-type parasites were detected by allele-specific PCR. Phylogenetic analysis based on the MPSP gene sequences revealed that Theileria parasites with the MPSP types 1, 2, and 4 were distributed in Northeast China.
  • Yuzi Luo, Honglin Jia, M. Alaa Terkawi, Youn-Kyoung Goo, Suguru Kawano, Hideo Ooka, Yan Li, Longzheng Yu, Shinuo Cao, Junya Yamagishi, Kozo Fujisaki, Yoshifumi Nishikawa, Atsuko Saito-Ito, Ikuo Igarashi, Xuenan Xuan
    PARASITOLOGY INTERNATIONAL 60 (2) 119 - 125 1383-5769 2011/06 [Refereed][Not invited]
     
    Here, we identified a novel secreted antigen designated as Babesia microti secreted antigen 1 (BmSA1) by immunoscreening a B. microti cDNA expression library using the sera from hamsters immunized with plasma, putatively containing secreted antigens, from B. microti-infected hamsters. Antibodies raised in mice immunized with recombinant BmSA1 (rBmSA1) recognized a native 33-kDa parasite protein. An enzyme-linked immunosorbent assay (ELISA) of rBmSA1 detected specific antibodies as early as 6 and 4 days post-infection in sera from a hamster experimentally infected with B. microti Gray strain (US type) and a mouse experimentally infected with B. microti Munich strain (rodent isolate), respectively. Moreover, a rapid immunochromatographic test (ICT) using rBmSA1 detected specific antibodies in a hamster experimentally infected with B. microti from day 6 to at least day 270 post-infection, which was quite consistent with the results of the ELISA. In addition, analysis of the sera involved in the first case of human babesiosis in Japan (Kobe type) showed that specific antibodies were detectable in the patient and the positive donor by ELISA using rBmSA1, and the ICT result was identical to the ELISA data. Taken together, these results indicated that BmSA1 could be a promising and universal target for developing both ELISA and ICT for the serodiagnosis of human babesiosis and for an epidemiological survey of its rodent reservoir. (C) 2010 Elsevier Ireland Ltd. All rights reserved.
  • Junya Yamagishi, Hiroyuki Wakaguri, Sumio Sugano, Suguru Kawano, Kozo Fujisaki, Chihiro Sugimoto, Junichi Watanabe, Yutaka Suzuki, Isao Kimata, Xuenan Xuan
    PARASITOLOGY INTERNATIONAL 60 (2) 199 - 202 1383-5769 2011/06 [Refereed][Not invited]
     
    A full-length cDNA library was constructed from the sporozoite of Cryptosporidium parvum. Normalized clones were subjected to Solexa shotgun sequencing, and then complete sequences for 1066 clones were reconfigured. Detailed analyses of the sequences revealed that 13.5% of the transcripts were spliced; the average and median 5' UTR lengths were 213.5 and 122 nucleotides, respectively. There were 148 inconsistencies out of 562 examined genes between the experimentally described cDNA sequence and the predicted sequence from its genome. In addition, we identified 118 sequences that had little homology against annotated genes of C. parvum as prospective candidates for addable genes. These observations should improve the reliability of C. parvum transcriptome and provide a versatile resource for further studies. (C) 2011 Elsevier Ireland Ltd. All rights reserved.
  • Hideo Ooka, Mohamad Alaa Terkawi, Youn-Kyoung Goo, Yuzi Luo, Yan Li, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Xuenan Xuan
    EXPERIMENTAL PARASITOLOGY 127 (1) 287 - 293 0014-4894 2011/01 [Refereed][Not invited]
     
    A novel gene. BmP94, encoding 94-kDa protein of Babesia micron was identified by immunoscreening of the cDNA expression library. The full-length of BmP94 was expressed in Escherichia coli (rBmP94), which resulted in insoluble form with low yield, and the truncated hydrophilic C-terminus region of the gene was expressed as a soluble protein (rBmP94/CT) with improved productivity. Antiserum raised against rBmP94/CT recognized the 94-kDa native protein in the parasite extract by Western blot analysis. Next, an ELISA using rBmP94/CT was evaluated for diagnostic use, and it demonstrated high sensitivity and specificity when tested with the sera from mice experimentally infected with B. microti and closely related parasites. Moreover, the immunoprotective property of rBmP94/CT as a subunit vaccine was evaluated in BALB/c mice against a B. microti challenge, but no significant protection was observed. Our data suggest that the immunodominant antigen BmP94 could be a promising candidate for diagnostic use for human babesiosis. (C) 2010 Elsevier Inc. All rights reserved.
  • Josef Tuda, Arthur E. Mongan, Mohammed E. M. Tolba, Mihoko Imada, Junya Yamagishi, Xuenan Xuan, Hiroyuki Wakaguri, Sumio Sugano, Chihiro Sugimoto, Yutaka Suzuki
    NUCLEIC ACIDS RESEARCH 39 (Database issue) D625 - D631 0305-1048 2011/01 [Refereed][Not invited]
     
    Full-Parasites (http://fullmal.hgc.jp/) is a transcriptome database of apicomplexa parasites, which include Plasmodium and Toxoplasma species. The latest version of Full-Parasites contains a total of 105 786 EST sequences from 12 parasites, of which 5925 full-length cDNAs have been completely sequenced. Full-Parasites also contain more than 30 million transcription start sites (TSS) for Plasmodium falciparum (Pf) and Toxoplasma gondii (Tg), which were identified using our novel oligo-capping-based protocol. Various types of cDNA data resources were interconnected with our original database functionalities. Specifically, in this update, we have included two unique RNA-Seq data sets consisting of 730 million mapped RNA-Seq tags. One is a dataset of 16 time-lapse experiments of cultured bradyzoite differentiation for Tg. The other dataset includes 31 clinical samples of Pf. Parasite RNA was extracted together with host human RNA, and the extracted mixed RNA was used for RNA sequencing, with the expectation that gene expression information from the host and parasite would be simultaneously represented. By providing the largest unique full-length cDNA and dynamic transcriptome data, Full-Parasites is useful for understanding host-parasite interactions and will help to eventually elucidate how monophyletic organisms have evolved to become parasites by adopting complex life cycles.
  • Guohong Zhang, Xiaohong Huang, Damdinsuren Boldbaatar, Banzragch Battur, Badgar Battsetseg, Houshuang Zhang, Longzheng Yu, Yan Li, Yuzi Luo, Shinuo Cao, Youn-Kyong Goo, Junya Yamagishi, Jinlin Zhou, Shoufa Zhang, Hiroshi Suzuki, Ikuo Igarashi, Takeshi Mikami, Yoshifumi Nishikawa, Xuenan Xuan
    VACCINE 28 (45) 7243 - 7247 0264-410X 2010/10 [Refereed][Not invited]
     
    Toxoplasma gondii and Neospora caninum are closely related apicomplexan parasites. The surface antigen 1 of T. gondii (TgSAG1) is a major immunodominant antigen and, therefore, is considered to be a good candidate for the development of an effective recombinant vaccine against toxoplasmosis. In this study, N. caninum stably expressing the TgSAG1 gene (Nc/TgSAG1) was constructed using pyrimethamine-resistant DHFR-TS and GFP genes as double-selection markers. The expression level, molecular weight, and antigenic property of recombinant TgSAG1 expressed by the Nc/TgSAG1 were similar to those of the native TgSAG1. The mice immunized with Nc/TgSAG1 induced TgSAG1-specific Th1-dominant immune responses and protected the mice from a lethal challenge infection with T. gondii. These results indicate that N. caninum may provide a new tool for the production of a live recombinant vector vaccine against toxoplasmosis in animals. To our knowledge, this is the first report to evaluate the usefulness of N. caninum-based live vaccine. (C) 2010 Elsevier Ltd. All rights reserved.
  • Junya Yamagishi, Hiroyuki Wakaguri, Akio Ueno, Youn-Kyoung Goo, Mohammed Tolba, Makoto Igarashi, Yoshifumi Nishikawa, Chihiro Sugimoto, Sumio Sugano, Yutaka Suzuki, Junichi Watanabe, Xuenan Xuan
    DNA RESEARCH 17 (4) 233 - 243 1340-2838 2010/08 [Refereed][Not invited]
     
    For the last couple of years, a method that permits the collection of precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high-throughput manner was established. We applied this novel method, 'tss-seq', to elucidate the transcriptome of tachyzoites of the Toxoplasma gondii, which resulted in the identification of 124 000 TSSs, and they were clustered into 10 000 transcription regions (TRs) with a statistics-based analysis. The TRs and annotated ORFs were paired, resulting in the identification of 30% of the TRs and 40% of the ORFs without their counterparts, which predicted undiscovered genes and stage-specific transcriptions, respectively. The massive data for TSSs make it possible to execute the first systematic analysis of the T. gondii core promoter structure, and the information showed that T. gondii utilized an initiator-like motif for their transcription in the major and novel motif, the downstream thymidine cluster, which was similar to the Y patch observed in plants. This encyclopaedic analysis also suggested that the TATA box, and the other well-known core promoter elements were hardly utilized.
  • Buyannemekh Tumurjav, Mohamad Alaa Terkawi, Houshuang Zhang, Guohong Zhang, Honglin Jia, Youn-Kyoung Goo, Junya Yamagishi, Yoshifumi Nishikawa, Ikuo Igarashi, Chihiro Sugimoto, Xuenan Xuan
    JAPANESE JOURNAL OF VETERINARY RESEARCH 58 (2) 111 - 119 0047-1917 2010/08 [Refereed][Not invited]
     
    Toxoplasma gondii matrix antigen 1 (TgMAG1), known as the 65-kDa protein, which is abundantly expressed in both bradyzoites and tachyzoites, was evaluated as a candidate for the development of a diagnostic reagent for ovine toxoplasmosis. The TgMAG1 gene was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST), and the recombinant TgMAG1 (rTgMAG1) was tested in an enzyme-linked immunosorbent assay (ELISA). The ELISA with rTgMAG1 showed a highly specific reaction with sera from mice experimentally infected with T. gondii but not with the closely related Neospora caninum. The antibodies to TgMAG1 were detectable from the acute to the chronic infectious stages in a mouse model. A total of 175 serum samples collected from sheep in 7 provinces of Mongolia were examined for the serodiagnosis of T. gondii infection by the ELISA with rTgMAG1, and the results were compared with those from the commercialized latex agglutination test (LAT). Of 175 serum samples analyzed, 42 (24.00%) and 29 (16.57%) samples were positive by the ELISA and LAT, respectively. Of 29 LAT-positive samples, 27 (93.10%) were positive by the ELISA. These results suggest that rTgMAG1 could be used as a reliable antigen for the detection of T. gondii infection in sheep.
  • Houshuang Zhang, Yoshifumi Nishikawa, Junya Yamagishi, Jinlin Zhou, Yuzuru Ikehara, Naoya Kojima, Naoaki Yokoyama, Xuenan Xuan
    EXPERIMENTAL PARASITOLOGY 125 (2) 130 - 136 0014-4894 2010/06 [Refereed][Not invited]
     
    Liposomes coated with neoglycolipids constructed with mannopentaose and dipalmitoylphosphatidy-lethanolamine (M3-DPPE), referred to as M3-DPPE liposomes, have been shown to induce cellular immunity against antigens encapsulated therein. To evaluate whether these M3-DPPE liposomes have an adjuvant capacity against Neospora caninum infection, a novel immunization method utilizing soluble N. caninum apical membrane antigen 1 (NcAMA1) encapsulated in the M3-DPPE liposomes (M3-NcAMA1) was employed. The results revealed that a significant amount of interferon (IFN)-gamma production was detected in culture supernatants of NcAMA1 protein- or N. caninum lysate-stimulated spleen cells obtained from the mice one week after the third immunization with M3-NcAMA1. The parasite burden in the dams' brain tissue was decreased and the survival rate of offspring increased significantly in M3-NcAMA1-immunized mice. Thus, a parasite-specific Th1 immune response was successfully induced in the pregnant mice immunized with M3-NcAMA1, and an effective reduction of offspring mortality from N. caninum infection was triggered. (C) 2010 Elsevier Inc. All rights reserved.
  • Honglin Jia, Yoshifumi Nishikawa, Yuzi Luo, Junya Yamagishi, Chihiro Sugimoto, Xuenan Xuan
    MOLECULAR AND BIOCHEMICAL PARASITOLOGY 170 (1) 1 - 6 0166-6851 2010/03 [Refereed][Not invited]
     
    The M17 family leucine aminopeptidase (LAP) hydrolyzes amino acids from the N-terminus of peptides. Many LAPS from parasitic protozoa, including Plasmodium, Trypanosoma, and Leishmania, have been intensely investigated because of their crucial roles in parasite biology. In this study, the functional recombinant Toxoplasma gondii LAP (rTgLAP) was expressed in Escherichia coli, and its enzymatic activity against synthetic substrates for aminopeptidase, as well as cellular localization, was determined. The activity was strongly dependent on metal divalent cations, and was inhibited by bestatin, which is an inhibitor for metalloprotease. Our results indicated that TgLAP is a functional aminopeptidase in the cytoplasm of T. gondii. (C) 2009 Elsevier B.V. All rights reserved.
  • Molecular characterization and expression of a 47-kDa merozoite surface protein of Babesia gibsoni for serodiagnosis by enzyme-linked immunosorbent assay.
    Aboge, G. O, Batbaatar, V, Goo, Y-K, Yamagishi, J, Nishikawa, Y, Sunaga, F, Namikawa, K, Igarashi, I, Fujisaki, K, Suzuki, H, Xuan, X
    J. Protozool. Res. 20 59 - 69 2010 [Refereed][Not invited]
  • Youn-Kyoung Goo, Honglin Jia, Mohamad Alaa Terkawi, Gabriel Oluga Aboge, Junya Yamagishi, Yoshifumi Nishikawa, Suk Kim, Hyung-Kwan Jang, Kozo Fujisaki, Xuenan Xuan
    EXPERIMENTAL PARASITOLOGY 123 (3) 273 - 276 0014-4894 2009/11 [Refereed][Not invited]
     
    Babesia gibsoni causes canine babesiosis. Here, we describe the identification and characterization of a novel gene, BgP22, containing an open reading frame of 621 bp and encoding a 22-kDa protein from B. gibsoni, as a serodiagnostic candidate. The recombinant BgP22 (rBgP22) was expressed and used as an antigen to produce anti-rBgP22 sera in mice. Using these anti-rBgP22 sera, a native 22-kDa protein was recognized by Western blot analysis and observed in the membrane of the parasites by immunofluorescent antibody tests (IFAT). The enzyme-linked immunosorbent assay (ELISA) using the rBgP22 detected specific antibodies to this protein in the sera of dogs experimentally and naturally infected with B. gibsoni in chronic stage. Furthermore, it did not show a cross reaction with the closely related apicomplexan parasites, indicating that the rBgP22 could be used as a diagnostic antigen for a detection of the chronic carrier stages of B. gibsoni infection. (C) 2009 Elsevier Inc. All rights reserved.
  • Terkawi MA, Amornthep A, Ooka H, Aboge G, Jia H, Goo YK, Nelson B, Yamagishi J, Nishikawa Y, Igarashi I, Kawazu SI, Fujisaki K, Xuan X
    Parasitology 10 136 (10) 1147 - 1160 0031-1820 2009/09 [Refereed][Not invited]
  • Jia H, Terkawi MA, Aboge GO, Goo YK, Luo Y, Li Y, Yamagishi J, Nishikawa Y, Igarashi I, Sugimoto C, Fujisaki K, Xuan X
    Parasitology 9 136 (9) 945 - 952 0031-1820 2009/08 [Refereed][Not invited]
  • Terkawi MA, Aboge G, Jia H, Goo YK, Ooka H, Yamagishi J, Nishikawa Y, Yokoyama N, Igarashi I, Kawazu SI, Fujisaki K, Xuan X
    Parasite immunology 6 31 328 - 340 0141-9838 2009/06 [Refereed][Not invited]
  • Houshuang Zhang, Oriel M. M. Thekisoe, Gabriel O. Aboge, Hisako Kyan, Junya Yamagishi, Noboru Inoue, Yoshifumi Nishikawa, Satoshi Zakimi, Xuenan Xuan
    EXPERIMENTAL PARASITOLOGY 122 (1) 47 - 50 0014-4894 2009/05 [Refereed][Not invited]
     
    Loop-mediated isothermal amplification (LAMP) method amplifies DNA with high specificity, sensitivity and rapidity. In this study, we used a conserved sequence in the 200- to 300-fold repetitive 529 bp gene of Toxoplasma gondii to design primers for LAMP test. Detection limit of T. gondii LAMP assay with the primers is 1 pg/mu L of T. gondii DNA, which was evaluated using 10-fold serially diluted DNA of cultured parasites. Furthermore, LAMP and conventional PCR methods were applied for amplification of the T. gondii DNA extracted from the lymph nodes taken from pigs which were suspected to be Toxoplasma infection. As a result, 76.9% (70/91) and 85.7% (78/91) of the samples were positive on PCR and LAMP analyzes, respectively. Therefore, the LAMP has a potential to be applied as an alternative molecular diagnostic tool for detection of T gondii infection from veterinary samples. This is the first study, which applies the LAMP method to diagnose Toxoplasma from veterinary samples. (C) 2009 Elsevier Inc. All rights reserved.
  • Youn-Kyoung Goo, Honglin Jia, G. Oluga Aboge, M. Alaa Terkawi, Eung-Goo Lee, Junya Yamagishi, Yoshifumi Nishikawa, Hyung-Kwan Jang, Fujiko Sunaga, Kazuhiko Namikawa, Kozo Fujisaki, Xuenan Xuan
    PARASITOLOGY INTERNATIONAL 58 (1) 55 - 60 1383-5769 2009/03 [Refereed][Not invited]
     
    A novel gene, BgP12, encoding a 12-kDa protein was identified from Babesia gibsoni. The full-length cDNA of BgP12 contains an open reading frame of 378 bp, corresponding to 126 amino acid (aa) residues consisting of a putative 26 aa signal peptide and a 100 aa mature protein. The recombinant BgP12 (rBgP12) lacking the N-terminal signal peptide was expressed in Escherichia coli as a soluble glutathione S-transferase (GST) fusion protein (rBgP12) that produced all anti-rBgP12 serum in mice after immunization. Using this anti-rBgP12 serum, a native 12-kDa protein in B. gibsoni was recognized by Western blot analysis. Immunofluorescent antibody tests (IFAT) revealed that BgP12 was mainly seen during the ring stage of B. gibsoni trophozoite. An indirect enzyme-linked immunosorbent assay (ELISA) using the rBgP12 detected specific antibodies in the sequential sera of a dog experimentally infected with B. gibsoni beginning 10 days post-infection to 442 clays post-infection, even when the dog became chronically infected and showed a low level of parasitemia, Moreover, the antigen did not show cross-reaction with antibodies to the closely related apicomplexan parasites, indicating that the rBgP12 might be all immunodominant antigen for B. gibsoni infection that Could be used as a diagnostic antigen for B. gribsoni infection with high specificity and sensitivity. (C) 2008 Elsevier Ireland Ltd. All rights reserved.
  • Junya Yamagishi, Erik D. Burnett, Steven H. Harwood, Gary W. Blissard
    VIROLOGY 365 (1) 34 - 47 0042-6822 2007/08 [Refereed][Not invited]
     
    The pp31 gene of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) encodes a phosphorylated DNA binding protein that associates with virogenic stroma in the nuclei of infected cells. Prior studies of pp31 by transient late expression assays suggested that pp3l may play an important role in transcription of AcMNPV late genes [Todd, J. W., Passarelli, A. L., and Miller, L. K. (1995). Eighteen baculovirus genes, including lef-11, p35, 39K, and p47, support late gene expression. J. Virol. 69, 968-974] although genetic studies of the closely related BmNPV pp3l gene suggested that pp31 may be dispensable [Gomi, S., Zhou, C. E., Yih, W., Majima, K., and Maeda, S. (1997). Deletion analysis of four of eighteen late gene expression factor gene homologues of the baculovirus, BmNPV. Virology 230 (1), 35-47]. In the current study, we examined the role of the pp3l gene in the context of the AcMNPV genome during infection. We used a BACmid-based system to generate a pp3l knockout in the AcMNPV genome. The pp3l knockout was subsequently rescued by reinserting the pp3l gene into the polyhedrin locus of the same virus genome. We found that pp3l was not essential for viral replication although the absence of pp3l resulted in a lower viral titer. Analysis of viral DNA replication in the absence of pp3l showed that the kinetics of viral DNA replication were unaffected. An AcMNPV oligonucleotide microarray was used to compare gene expression from all AcMNPV genes in the presence or absence of pp31. In the absence of pp31, a modest reduction in transcripts was detected for many viral genes (99 genes) while no substantial increase or decrease was observed for 43 genes. Transcripts from 6 genes (p6.9, ORF 97, ORF 60, ORF 98, ORF 102 and chitinase) were reduced by 66% or more compared to the levels detected from the control virus. Microarray results were further examined by qPCR analysis of selected genes. In combination, these data show that deletion of the pp3l gene was not lethal and did not appear to affect viral DNA replication but resulted in an apparent modest down-regulation of a subset of AcMNPV genes that included both early and late genes. (c) 2007 Elsevier Inc. All rights reserved.
  • R Fujita, T Matsuyama, J Yamagishi, K Sahara, S Asano, H Bando
    JOURNAL OF VIROLOGY 80 (5) 2390 - 2395 0022-538X 2006/03 [Refereed][Not invited]
     
    The gene expression of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was examined in two types of mammalian cells, human HeLa14 and hamster BHK cells. DNA microarray analysis followed by reverse transcription-PCR identified at least 12 viral genes transcribed in both HeLa14 cells and BHK cells inoculated with AcMNPV. 5' rapid amplification of cDNA ends was carried out to examine the transcriptional fidelity of these genes in HeLa14 cells. The transcription of ie-1, ie-0 and gp64 was initiated at a baculovirus early gene motif, CAGT, accompanied by a TATA motif. In addition, the same splicing observed for ie-0 mRNA in Sf9 cells occurred in HeLa14 cells. While the transcription initiation sites for pe38 and p6.9 were not located in the CAGT motif, most of them were in a typical eukaryotic RNA polymerase II promoter structure (a conventional TATA motif and/or an initiator). Interestingly, the expression of beta-actin was upregulated in the mammalian cells inoculated with AcMNPV. Subsequent experiments using UV-inactivated virus confirmed the upregulation, suggesting that de novo synthesis of viral products is not required for the event. These results indicated that the AcMNPV genome acts as a template for transcription in mammalian cells through the usual infection pathway, though there is no evidence for the functional expression of viral genes at present.
  • SP Assenga, M You, CH Shy, J Yamagishi, T Sakaguchi, JL Zhou, MK Kibe, XN Xuan, K Fujisaki
    PARASITOLOGY RESEARCH 98 (2) 111 - 118 0932-0113 2006/01 [Refereed][Not invited]
     
    Baculoviruses are specific insect pathogens used as selective biological insecticides on lepidopteran insects. We have tested a recombinant baculovirus expressing a chitinase gene for its efficacy as a tick bioacaricide. The recombinant Autographa californica multiple nuclear polyhedrosis virus expressing a chitinase enzyme (AcMNPV-CHT1) from the hard tick, Haemaphysalis longicornis, was constructed and found to have a novel bioacaricidal effect against ticks. The recombinant baculovirus was used to express the chitinase enzyme in Spodoptera frugiperda (Sf9) insect cells. Topical application of the supernatant harvested from the insect cell culture was found to cause mortality in nymphal ticks of H. longicornis. High temperature (> 30 degrees C) and infrared radiation affected the chitinase enzyme activity and recombinant baculovirus infectivity by reducing the speed of tick killing by 60%. A mixture of recombinant virus and chitinase was found to kill ticks faster (p < 0.01) than pure chitinase and recombinant virus alone. Thus, the recombinant virus showed a synergistic effect with the foreign chitinase gene. In order to reduce the excessive use and cost of acaricides, it was found that a mixture of recombinant virus and flumethrin could halve the dose of the chemical acaricide used. These findings are important for the safe use of the recombinant virus expressing chitinase as a bioacaricide against ticks.
  • Tani H, Abe T, Limn CK, Mochizuki R, Yamagishi J, Kitagawa Y, Watanabe R, Moriishi K, Matsuura Y
    Uirusu 53 (2) 185 - 193 0042-6857 2003/12 [Refereed][Not invited]
  • J Yamagishi, R Isobe, T Takebuchi, H Bando
    ARCHIVES OF VIROLOGY 148 (3) 587 - 597 0304-8608 2003/03 [Refereed][Not invited]
     
    We describe, for the first time, the generation of a viral DNA chip for simultaneous expression measurements of nearly all known open reading frames (ORFs) in the best-studied members of the family Baculoviridae, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV). In this study, a viral DNA chip (Ac-BmNPV chip) was fabricated and used to characterize the viral gene expression profile for AcMNPV in different cell types. The viral chip is composed of microarrays of viral DNA prepared by robotic deposition of PCR-amplified viral DNA fragments on glass for ORFs in the NPV genome. Viral gene expression was monitored by hybridization to the DNA fragment microarrays with fluorescently labeled cDNAs prepared from infected Spodoptera frugiperda, Sf9 cells and Trichoplusia ni, TnHigh-Five cells, the latter a major producer of baculovirus and recombinant proteins. A comparison of expression profiles of known ORFs in AcMNPV elucidated six genes (ORF150, p10, pk2, and three late gene expression factor genes lef-3, p35 and lef-6) the expression of each of which was regulated differently in the two cell lines. Most of these genes are known to be closely involved in the viral life cycle such as in DNA replication, late gene expression and the release of polyhedra from infected cells. These results imply that the differential expression of these viral genes accounts for the differences in viral replication between these two cell lines. Thus, these fabricated microarrays of NPV DNA which allow a rapid analysis of gene expression at the viral genome level should greatly speed the functional analysis of large genomes of NPV.
  • J Yamagishi, Y Hu, J Zheng, H Bando
    ARCHIVES OF VIROLOGY 144 (11) 2111 - 2124 0304-8608 1999 [Refereed][Not invited]
     
    We determined the complete nucleotide sequence of an infectious clone of the cockroach small spherical virus (CSSV) genome. Analysis of the genome organization and the predicted viral protein sequences showed clearly that this virus should be classified as a new member of the subfamily Densovirinae, genus Densovirus, and should be designated as PfDNV. However, our data revealed some differences between the gene expression strategies used by PfDNV and other DNVs. An internal promoter, in addition to the promoter (p3) at the genome terminus, was observed at map unit 18 (p18), implying transcriptional regulation of generation of the nonstructural proteins of PfDNV. Furthermore, the structural analysis of cDNAs complementary to mRNAs from the region coding for structural proteins suggested alternative splicing and polyadenylation as means for generation of the structural proteins of PfDNV.

MISC

  • Junya Yamagishi, Anna Natori, Mohammed E M Tolba, Arthur E Mongan, Chihiro Sugimoto, Toshiaki Katayama, Shuichi Kawashima, Wojciech Makalowski, Ryuichiro Maeda, Yuki Eshita, Josef Tuda, Yutaka Suzuki  Genome research  28-  (8)  1253  -1253  2018/08  [Not refereed][Not invited]
  • 江下優樹, 江下優樹, 江下優樹, 江下優樹, 和田雄治, 林田京子, 山岸潤也, 杉本千尋, 佐々木道仁, 大場靖子, 澤洋文, 飛弾野真也, 神山長慶, 小林隆志, 高崎智彦, 加藤文博, 田島茂, 倉根一郎, LIM Chang Kweng  衛生動物  69-  (Supplement)  51  2018/03/30  [Not refereed][Not invited]
  • Chao Yang, Shivani Pasricha, Sze Ying Ong, Andrew Stephen Brown, Junya Yamagishi, Chihiro Sugimoto, Sammy Bedoui, Ian R. van Driel, Elizabeth L. Hartland  CYTOKINE  100-  149  -149  2017/12  [Not refereed][Not invited]
  • 林田京子, 林田京子, 白藤梨可, SIVAKUMAR Thillaiampalam, 山岸潤也, 鈴木穣, 杉本千尋, 横山直明  日本寄生虫学会大会プログラム・抄録集  86th-  89  2017  [Not refereed][Not invited]
  • 山岸潤也, 麻田正仁, HASSAN Hakimi, 田中健, 田中健, 杉本千尋, 河津信一郎  日本獣医学会学術集会講演要旨集  159th-  334  -334  2016/08/30  [Not refereed][Not invited]
  • 田中健, 山岸潤也, HASSAN Hakimi, 麻田正仁, 河津信一郎  日本獣医学会学術集会講演要旨集  158th-  304  -304  2015/08/30  [Not refereed][Not invited]
  • Babesia ovataの短時間かつ安定的な遺伝子導入系の確立(Establishment of transient and stable transfection system for Babesia ovata)
    Hakimi Hassan, 山岸 潤也, 外川 裕人, 田中 健, 金子 修, 河津 信一郎, 麻田 正仁  日本獣医学会学術集会講演要旨集  158回-  304  -304  2015/08  [Not refereed][Not invited]
  • 薄井美帆, 増田(菅沼)裕乃, 元岡大祐, 中村昇太, 山岸潤也, 福本晋也, 井上昇, 堀井俊宏, 河津信一郎  日本寄生虫学会大会プログラム・抄録集  84th-  79  2015/02  [Not refereed][Not invited]
  • 江下 優樹, Runtuwene Lucky R, 牧野 芳大, 野口 香緒里, 川上 絵理, 福田 昌子, 小林 隆志, 小西 英二, 山中 敦史, Srisawat Raweewan, Komalamisra Narumon, 成田 弘成, 牛島 廣治, Mon-gan Arthur E, 今田 美穂子, 山岸 潤也, 鈴木 穣, 中井 謙太, 前田 龍一郎, 杉本 千尋, 倉根 一郎, 高崎 智彦  都市有害生物管理  4-  (2)  120  -121  2014/12/20  [Not refereed][Not invited]
  • SHINOZAKI NATSUKO, YAMAGISHI JUN'YA, SATO YUKUTO, YAMASHITA RIU, YAMADA TAKUJI, NAGASAKI MASAO, TSUBOI AKITO  日本ゲノム微生物学会年会要旨集  8th-  66  2014  [Not refereed][Not invited]
  • RUNTUWENE LUCKY R, KAWASHIMA SHUICHI, YAMAGISHI JUNYA, SUZUKI YUTAKA, SUGANO SUMIO, NAKAI KENTA, MAEDA RYUICHIRO, SUGIMOTO CHIHIRO, TAKASAKI TOMOHIKO, KURANE ICHIRO, KOBAYASHI TAKASHI, ESHITA YUKI  衛生動物  64-  (2)  138  2013/06/15  [Not refereed][Not invited]
  • 山崎詩乃, 菅沼啓輔, 麻田正仁, NGASAMAN Ruttayaporn, 周末, 山岸潤也, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  155th-  216  -216  2013/03/04  [Not refereed][Not invited]
  • 菅沼啓輔, 山崎詩乃, 山岸潤也, 麻田正仁, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  155th-  118  -118  2013/03/04  [Not refereed][Not invited]
  • congolense epimastigote specific protein(CESP)のEMF特異的発現は3'非翻訳領域によって調節されている
    菅沼 啓輔, 山崎 詩乃, 山岸 潤也, 麻田 正仁, 河津 信一郎, 井上 昇  獣医寄生虫学会誌  11-  (1)  49  -49  2012/10  [Not refereed][Not invited]
  • Trypanosoma congolenseハプトグロビン-ヘモグロビン複合体レセプター様タンパク質(TcHpHbR)に関する研究
    山崎 詩乃, 菅沼 啓輔, 山岸 潤也, 麻田 正仁, 河津 信一郎, 井上 昇  獣医寄生虫学会誌  11-  (1)  50  -50  2012/10  [Not refereed][Not invited]
  • 山岸潤也, 鈴木穣, 五十嵐郁男, 河津信一郎, 玄学南  日本熱帯医学会大会プログラム抄録集  53rd-  56  -57  2012/09  [Not refereed][Not invited]
  • 菅沼啓輔, 山崎詩乃, 山岸潤也, 麻田正仁, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  153rd-  224  -224  2012/03/01  [Not refereed][Not invited]
  • 山崎詩乃, 菅沼啓輔, 山岸潤也, 麻田正仁, 河津信一郎, 井上昇  日本獣医学会学術集会講演要旨集  153rd-  225  -225  2012/03/01  [Not refereed][Not invited]
  • 山岸潤也, 若栗浩幸, 河津信一郎, 鈴木穣, XUAN Xuenan  日本寄生虫学会大会プログラム・抄録集  81st-  87  2012/02  [Not refereed][Not invited]
  • 山岸潤也, 山下理宇, 鈴木穣, 五十嵐郁男, 河津信一郎, XUAN Xuenan  日本分子生物学会年会プログラム・要旨集(Web)  35th-  1P-0029 (WEB ONLY)  2012  [Not refereed][Not invited]
  • 麻田正仁, 山岸潤也, 後藤康之, 横山直明, 井上昇, 河津信一郎  日本寄生虫学会東日本支部大会プログラム・講演要旨  72nd-  19  2012  [Not refereed][Not invited]
  • 名取杏奈, TUDA Josef, MONGAN Arthur, TOLBA Mohammed, TOLBA Mohammed, 今田美穂子, 山岸潤也, XUAN Xuenan, 若栗浩幸, 菅野純夫, 杉本千尋, 鈴木穣  日本分子生物学会年会プログラム・要旨集(Web)  35th-  WEB ONLY 4P-0211  2012  [Not refereed][Not invited]
  • 【ウイルスベクター 最新の話題】 バキュロウイルスベクター 哺乳動物細胞への遺伝子導入
    谷 英樹, 阿部 隆之, 林 昌宏, 望月 理加, 山岸 潤也, 北川 善紀, 渡辺 理恵, 宮本 大伸, 森石 恆司, 松浦 善治  ウイルス  53-  (2)  185  -193  2003/12  [Not refereed][Not invited]
  • Junya Yamagishi, Shinichiro Asano, Ken Sahara, Toshihiko Iizuka, Hisanori Bando  Journal of Sericultural Science of Japan  69-  (4)  271  -276  2000  [Not refereed][Not invited]
     
    Alternative splicing observed in the mRNAs for the structural proteins of PfDNV seems to be necessary to generate five structural proteins from two separated ORFs. Involvement of several splicing donor sites and acceptor sites in the alternative splicing complicates understanding of the splicing mechanism of the PfDNV. First we developed a method for detection of spliced RNA molecules using the combination of the different two techniques, RT-PCR and primer extension. This splicing-detection method demonstrated that the sites of alternative splicing occurred within the PfDNV RNA was also recognized in S2 cells (a Drosophila cell line). Interestingly, a drastic suppression of the splicing and the accumulation of the unspliced RNA molecules were observed in S2 cells transfected with the PfDNV non-structural proteins (γ and β)-expression plasmids. These results suggested that the cellular factors play an important role on the selection of the specific splicing sites and that the viral nonstruc ural proteins may regulate it in a suppressive manner. © 2000, The Japanese Society of Sericultural Science. All rights reserved.
  • Junya Yamagishi, Shinichiro Asano, Ken Sahara, Toshihiko Iizuka, Hisanori Bando  Journal of Sericultural Science of Japan  69-  (4)  271  -276  2000  [Not refereed][Not invited]
     
    Alternative splicing observed in the mRNAs for the structural proteins of PfDNV seems to be necessary to generate five structural proteins from two separated ORFs. Involvement of several splicing donor sites and acceptor sites in the alternative splicing complicates understanding of the splicing mechanism of the PfDNV. First we developed a method for detection of spliced RNA molecules using the combination of the different two techniques, RT-PCR and primer extension. This splicing-detection method demonstrated that the sites of alternative splicing occurred within the PfDNV RNA was also recognized in S2 cells (a Drosophila cell line). Interestingly, a drastic suppression of the splicing and the accumulation of the unspliced RNA molecules were observed in S2 cells transfected with the PfDNV non-structural proteins (γ and β)-expression plasmids. These results suggested that the cellular factors play an important role on the selection of the specific splicing sites and that the viral nonstruc ural proteins may regulate it in a suppressive manner. © 2000, The Japanese Society of Sericultural Science. All rights reserved.

Educational Activities

Teaching Experience

  • 感染症学特別研究Ⅰ
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Advanced Seminar on Analytical Machines(Intergraded Grade)
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 感染症学特別演習
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Advanced Seminar on Analytical Machines(Intergraded Grade)
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 感染症学特別研究ⅡA
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Advanced Lecture on Microbiology
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
    キーワード : 細菌、ウイルス、グラム陽性菌、グラム陰性菌、抗酸性菌、感染症、ワクチン、プリオン、寄生虫、原虫、マイコプラズマ、クラミジア、スピロヘータ、真菌
  • 感染症学特別研究ⅡB
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Advanced Lecture on Microbiology
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
    キーワード : 細菌、ウイルス、グラム陽性菌、グラム陰性菌、抗酸性菌、感染症、ワクチン、プリオン、寄生虫、原虫、マイコプラズマ、クラミジア、スピロヘータ、真菌
  • 研究機器演習
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • Advanced Lecture on Zoonosis Control
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学特別研究
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • Advanced Lecture on Pathogen Genomics
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 国際感染症学院
  • 獣医科学特論演習
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 研究機器演習
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目B 獣医微生物学特論
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 獣医科学基礎科目A 人獣共通感染症制御学特論
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学研究科
  • 研究機器演習
    開講年度 : 2020
    課程区分 : 博士後期課程
    開講学部 : 獣医学院
  • 人獣共通感染症学
    開講年度 : 2020
    課程区分 : 学士課程
    開講学部 : 獣医学部


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