RESUMO
Gastroenteropancreatic (GEP) neuroendocrine neoplasm (NEN) that consists of neuroendocrine tumor and neuroendocrine carcinoma (NEC) is a lethal but under-investigated disease owing to its rarity. To fill the scarcity of clinically relevant models of GEP-NEN, we here established 25 lines of NEN organoids and performed their comprehensive molecular characterization. GEP-NEN organoids recapitulated pathohistological and functional phenotypes of the original tumors. Whole-genome sequencing revealed frequent genetic alterations in TP53 and RB1 in GEP-NECs, and characteristic chromosome-wide loss of heterozygosity in GEP-NENs. Transcriptome analysis identified molecular subtypes that are distinguished by the expression of distinct transcription factors. GEP-NEN organoids gained independence from the stem cell niche irrespective of genetic mutations. Compound knockout of TP53 and RB1, together with overexpression of key transcription factors, conferred on the normal colonic epithelium phenotypes that are compatible with GEP-NEN biology. Altogether, our study not only provides genetic understanding of GEP-NEN, but also connects its genetics and biological phenotypes.
Assuntos
Bancos de Espécimes Biológicos , Tumores Neuroendócrinos/patologia , Organoides/patologia , Animais , Cromossomos Humanos/genética , Genótipo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Intestinais/genética , Neoplasias Intestinais/patologia , Masculino , Camundongos , Modelos Genéticos , Mutação/genética , Tumores Neuroendócrinos/genética , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Fenótipo , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Transcriptoma/genética , Sequenciamento Completo do GenomaRESUMO
Recent sequencing analyses have shed light on heterogeneous patterns of genomic aberrations in human gastric cancers (GCs). To explore how individual genetic events translate into cancer phenotypes, we established a biological library consisting of genetically engineered gastric organoids carrying various GC mutations and 37 patient-derived organoid lines, including rare genomically stable GCs. Phenotype analyses of GC organoids revealed divergent genetic and epigenetic routes to gain Wnt and R-spondin niche independency. An unbiased phenotype-based genetic screening identified a significant association between CDH1/TP53 compound mutations and the R-spondin independency that was functionally validated by CRISPR-based knockout. Xenografting of GC organoids further established the feasibility of Wnt-targeting therapy for Wnt-dependent GCs. Our results collectively demonstrate that multifaceted genetic abnormalities render human GCs independent of the stem cell niche and highlight the validity of the genotype-phenotype screening strategy in gaining deeper understanding of human cancers.
Assuntos
Adenocarcinoma/patologia , Organoides/patologia , Neoplasias Gástricas/patologia , Estômago/patologia , Trombospondinas/metabolismo , Proteínas Wnt/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animais , Antígenos CD/genética , Apoptose , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caderinas/genética , Carcinogênese , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Organoides/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Trombospondinas/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Wnt/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer relapse after chemotherapy remains a main cause of cancer-related death. Although the relapse is thought to result from the propagation of resident cancer stem cells1, a lack of experimental platforms that enable the prospective analysis of cancer stem cell dynamics with sufficient spatiotemporal resolution has hindered the testing of this hypothesis. Here we develop a live genetic lineage-tracing system that allows the longitudinal tracking of individual cells in xenotransplanted human colorectal cancer organoids, and identify LGR5+ cancer stem cells that exhibit a dormant behaviour in a chemo-naive state. Dormant LGR5+ cells are marked by the expression of p27, and intravital imaging provides direct evidence of the persistence of LGR5+p27+ cells during chemotherapy, followed by clonal expansion. Transcriptome analysis reveals that COL17A1-a cell-adhesion molecule that strengthens hemidesmosomes-is upregulated in dormant LGR5+p27+ cells. Organoids in which COL17A1 is knocked out lose the dormant LGR5+p27+ subpopulation and become sensitive to chemotherapy, which suggests that the cell-matrix interface has a role in the maintenance of dormancy. Chemotherapy disrupts COL17A1 and breaks the dormancy in LGR5+p27+ cells through FAK-YAP activation. Abrogation of YAP signalling prevents chemoresistant cells from exiting dormancy and delays the regrowth of tumours, highlighting the therapeutic potential of YAP inhibition in preventing cancer relapse. These results offer a viable therapeutic approach to overcome the refractoriness of human colorectal cancer to conventional chemotherapy.
Assuntos
Neoplasias do Colo , Células-Tronco Neoplásicas , Autoantígenos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem da Célula , Proliferação de Células , Rastreamento de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Quinase 1 de Adesão Focal/metabolismo , Perfilação da Expressão Gênica , Xenoenxertos , Humanos , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/patologia , Colágenos não Fibrilares/metabolismo , Organoides/metabolismo , Organoides/patologia , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Transcrição/metabolismo , Colágeno Tipo XVIIRESUMO
During their blood-feeding process, ticks are known to transmit various viruses to vertebrates, including humans. Recent viral metagenomic analyses using next-generation sequencing (NGS) have revealed that blood-feeding arthropods like ticks harbor a large diversity of viruses. However, many of these viruses have not been isolated or cultured, and their basic characteristics remain unknown. This study aimed to present the identification of a difficult-to-culture virus in ticks using NGS and to understand its epidemic dynamics using molecular biology techniques. During routine tick-borne virus surveillance in Japan, an unknown flaviviral sequence was detected via virome analysis of host-questing ticks. Similar viral sequences have been detected in the sera of sika deer and wild boars in Japan, and this virus was tentatively named the Saruyama virus (SAYAV). Because SAYAV did not propagate in any cultured cells tested, single-round infectious virus particles (SRIP) were generated based on its structural protein gene sequence utilizing a yellow fever virus-based replicon system to understand its nationwide endemic status. Seroepidemiological studies using SRIP as antigens have demonstrated the presence of neutralizing antibodies against SAYAV in sika deer and wild boar captured at several locations in Japan, suggesting that SAYAV is endemic throughout Japan. Phylogenetic analyses have revealed that SAYAV forms a sister clade with the Orthoflavivirus genus, which includes important mosquito- and tick-borne pathogenic viruses. This shows that SAYAV evolved into a lineage independent of the known orthoflaviviruses. This study demonstrates a unique approach for understanding the epidemiology of uncultured viruses by combining viral metagenomics and pseudoinfectious viral particles.
Assuntos
Cervos , Flavivirus , Metagenômica , Carrapatos , Animais , Metagenômica/métodos , Japão/epidemiologia , Cervos/virologia , Flavivirus/genética , Flavivirus/isolamento & purificação , Flavivirus/classificação , Carrapatos/virologia , Filogenia , Viroma/genética , Vírion/genética , Sus scrofa/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Soroepidemiológicos , Genoma ViralRESUMO
With ageing, normal human tissues experience an expansion of somatic clones that carry cancer mutations1-7. However, whether such clonal expansion exists in the non-neoplastic intestine remains unknown. Here, using whole-exome sequencing data from 76 clonal human colon organoids, we identify a unique pattern of somatic mutagenesis in the inflamed epithelium of patients with ulcerative colitis. The affected epithelium accumulates somatic mutations in multiple genes that are related to IL-17 signalling-including NFKBIZ, ZC3H12A and PIGR, which are genes that are rarely affected in colon cancer. Targeted sequencing validates the pervasive spread of mutations that are related to IL-17 signalling. Unbiased CRISPR-based knockout screening in colon organoids reveals that the mutations confer resistance to the pro-apoptotic response that is induced by IL-17A. Some of these genetic mutations are known to exacerbate experimental colitis in mice8-11, and somatic mutagenesis in human colon epithelium may be causally linked to the inflammatory process. Our findings highlight a genetic landscape that adapts to a hostile microenvironment, and demonstrate its potential contribution to the pathogenesis of ulcerative colitis.
Assuntos
Colite Ulcerativa/genética , Epitélio/metabolismo , Interleucina-17/genética , Mutação , Colite Ulcerativa/metabolismo , Humanos , Interleucina-17/metabolismo , Fenótipo , Transdução de SinaisRESUMO
Precision oncology presumes an accurate prediction of drug response on the basis of the molecular profile of tumors. However, the extent to which patient-derived tumor organoids recapitulate the response of in vivo tumors to a given drug remains obscure. To gain insights into the pharmacobiology of human colorectal cancer (CRC), we here created a robust drug screening platform for patient-derived colorectal organoids. Application of suspension culture increased organoid scalability, and a refinement of the culture condition enabled incorporation of normal and precursor organoids to high-throughput drug screening. Drug screening identified bromodomain and extra-terminal (BET) bromodomain protein inhibitor as a cancer-selective growth suppressor that targets genes aberrantly activated in CRC. A multi-omics analysis identified an association between checkpoint with forkhead and ring finger domaines (CHFR) silencing and paclitaxel sensitivity, which was further validated by gene engineering of organoids and in xenografts. Our findings highlight the utility of multiparametric validation in enhancing the biological and clinical fidelity of a drug screening system.
Assuntos
Neoplasias Colorretais , Organoides , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Detecção Precoce de Câncer , Epigênese Genética , Humanos , Organoides/patologia , Medicina de PrecisãoRESUMO
BACKGROUND & AIMS: In the mouse intestinal epithelium, Lgr5+ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown. METHODS: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27+ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury. RESULTS: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5+ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5+p27+ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-ß signaling regulated the quiescent state of LGR5+ cells. Despite the plasticity of differentiated KRT20+ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5+p27+ cells prevented neighboring differentiated cells from de-differentiating. CONCLUSIONS: Our results highlight the quiescent nature of human LGR5+ colonic stem cells and their contribution to post-injury regeneration.
Assuntos
Receptores Acoplados a Proteínas G , Células-Tronco , Humanos , Camundongos , Animais , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Células-Tronco/metabolismo , Colo/metabolismo , Mucosa Intestinal/metabolismo , Fluoruracila , Fatores de Crescimento Transformadores/metabolismoRESUMO
Many Rickettsia species of the spotted fever group (SFG) cause tick-borne diseases known as "spotted fever." One of the candidate SFG Rickettsia species is "Candidatus Rickettsia kotlanii," which was first detected in Haemaphysalis concinna in Hungary in 2006. However, its precise phylogenetic position in the SFG is not clear because only single-gene sequence-based phylogenetic analyses were performed using very limited genes. Here, we present the complete genome sequences of two Japanese "Ca. R. kotlanii" isolates, which differed only by a 135 bp insertion/deletion (InDel). Using these genomes and publicly available whole genome sequences of other Rickettsia species, the precise phylogenetic position of "Ca. R. kotlanii" in Rickettsia was determined to be in a clade of the SFG. The phylogenetic relationships and average nucleotide identity of "Ca. R. kotlanii" relative to the other species indicated that "Ca. R. kotlanii" is an independent taxon in the SFG. Notably, although the genomes of the two isolates were almost identical, the isolates were obtained from different tick species in different regions and years, suggesting extremely low genomic diversity in "Ca. R. kotlanii." While the genome of "Ca. R. kotlanii" is the smallest in the transitional group and SFG Rickettsia sequenced to date, we identified genes uniquely present or absent in "Ca. R. kotlanii," but most were apparently degraded. Therefore, analyses of differences at the sequence (single nucleotide polymorphisms and small InDels) or gene expression level will be required to understand the functional or physiological features unique to "Ca. R. kotlanii."
Assuntos
Rickettsia , Rickettsiose do Grupo da Febre Maculosa , Animais , Genômica , Filogenia , Rickettsia/genética , Rickettsiose do Grupo da Febre Maculosa/genética , Rickettsiose do Grupo da Febre Maculosa/microbiologiaRESUMO
The cancer stem cell (CSC) theory highlights a self-renewing subpopulation of cancer cells that fuels tumour growth. The existence of human CSCs is mainly supported by xenotransplantation of prospectively isolated cells, but their clonal dynamics and plasticity remain unclear. Here, we show that human LGR5+ colorectal cancer cells serve as CSCs in growing cancer tissues. Lineage-tracing experiments with a tamoxifen-inducible Cre knock-in allele of LGR5 reveal the self-renewal and differentiation capacity of LGR5+ tumour cells. Selective ablation of LGR5+ CSCs in LGR5-iCaspase9 knock-in organoids leads to tumour regression, followed by tumour regrowth driven by re-emerging LGR5+ CSCs. KRT20 knock-in reporter marks differentiated cancer cells that constantly diminish in tumour tissues, while reverting to LGR5+ CSCs and contributing to tumour regrowth after LGR5+ CSC ablation. We also show that combined chemotherapy potentiates targeting of LGR5+ CSCs. These data provide insights into the plasticity of CSCs and their potential as a therapeutic target in human colorectal cancer.
Assuntos
Rastreamento de Células , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G/metabolismo , Animais , Diferenciação Celular , Linhagem da Célula , Proliferação de Células , Autorrenovação Celular , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Técnicas de Introdução de Genes , Humanos , Queratina-20/genética , Queratina-20/metabolismo , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/transplante , Organoides/metabolismo , Organoides/patologia , Organoides/transplante , Receptores Acoplados a Proteínas G/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Oz virus is a novel thogotovirus isolated from ticks that causes lethal infection in mice. We conducted serosurveillance of Oz virus infection among humans and wild mammals in Japan using virus-neutralization tests and ELISAs. Results showed that Oz virus may be naturally infecting humans and other mammalian hosts.
Assuntos
Thogotovirus , Carrapatos , Animais , Japão/epidemiologia , Mamíferos , Camundongos , ZoonosesRESUMO
Bats (order, Chiroptera) account for more than one-fifth of all mammalian species in the world and are infected by various intra-erythrocytic parasites of the family Plasmodiidae (Apicomplexa: Haemosporida), including Polychromophilus Dionisi, 1899. Recent advance in the molecular characterization of haemosporidian isolates has enabled their accurate identification, particularly in the last decade. Studies are actively conducted in tropical regions, Europe, and Australia; however, data on haemosporidian infection in bats in Asian temperate areas, including Japan, remain limited. In this study, 75 bats of 4 species (Miniopterus fuliginosus, Myotis macrodactylus, Rhinolophus nippon, and Rhinolophus cornutus) were captured at three sites in western Japan (Yamaguchi Prefecture), and haemosporidian parasites were screened microscopically and molecularly via nested polymerase chain reaction (PCR) targeting the cytochrome b (cytb), cytochrome c oxidase subunit I (cox-1), apicoplast caseinolytic protease C (clpc), and nuclear elongation factor 2 (EF2) genes. The survey detected Polychromophilus melanipherus in 15 (40.5%) miniopterid bats (M. fuliginosus) and Polychromophilus murinus in 6 (46.2%) vespertilionid bats (M. macrodactylus), whereas none of the 25 rhinolophid bats (R. nippon and R. cornutus) was infected, indicating the robust host specificity for miniopterid (P. melanipherus) and vespertilionid (P. murinus) bats regardless of orthotopic nesting. The 15 Polychromophilus cytb sequences obtained from 11 miniopterid and 4 vespertilionid bats were classified into six cytb haplotypes (three for each species), showing no region-specific variation in a phylogenetic tree of Polychromophilus isolates in the Old World. Similarly, multiple haplotypes (seven for cox-1 and nine for clpc) and genotypes (three for EF2) were characterized for the Japanese isolates of Polychromophilus, and the results were consistent with those based on a haemosporidian cytb analysis. Bat flies (Nycteribia allotopa and another undetermined Nycteribia sp.) collected from the body surface of bats harbored Polychromophilus oocysts on the external surface of the midgut. This is the first study to report the isolation and molecular characterization of Polychromophilus spp. in miniopterid and vespertilionid bats in the temperate area of Asia (western Japan). Future studies should evaluate the global prevalence of haemosporidian infections in bats.
Assuntos
Quirópteros/parasitologia , Haemosporida/genética , Haemosporida/isolamento & purificação , Infecções Protozoárias em Animais/parasitologia , Animais , Citocromos b/genética , Haemosporida/classificação , Japão/epidemiologia , Filogenia , Prevalência , Infecções Protozoárias em Animais/epidemiologiaRESUMO
The taxonomy of ruminant Trypanosoma theileri and its relatives (Kinetoplastida: Trypanosomatidae) is controversial, with recent phylogenetic studies segregating T. theileri in cattle and other ruminants worldwide into two major genetic lineages (the TthI and TthII clades) based on genetic markers. In the present study, T. theileri-like trypanosomes isolated from Honshu sika deer (Cervus nippon) in the western Japan (YMG isolate) were genetically characterized using a number of genetic markers. Sika deer trypanosomes of the YMG isolate were genetically different from the Trypanosoma sp. TSD1 isolate previously recorded from Hokkaido sika deer in northern Japan, with the former trypanosome isolate being genetically closer to European cervid trypanosomes and the bovine T. theileri TthII lineage. In contrast, the latter isolate exhibited greater relatedness to North American cervid trypanosomes and the bovine T. theileri TthI lineage, although a clear genetic distinction between these was apparent. Furthermore, trypanosomes in Honshu sika deer from the central part of Japan harboured additional genetic diversity and were closer to either TSD1 or YMG isolates, while distinct from known T. theileri-related genotypes. Importantly, cervids and wild ruminants worldwide might harbour divergent descendants of a T. theileri ancestor, which exhibit rigid host specificity to either bovines or cervid species.
Assuntos
Cervos , Trypanosoma , Animais , Bovinos , Variação Genética , Japão/epidemiologia , Filogenia , Trypanosoma/genéticaRESUMO
BACKGROUND: Borrelia bavariensis is one of the agents of Lyme Borreliosis (or Lyme disease) in Eurasia. The genome of the Borrelia burgdorferi sensu lato species complex, that includes B. bavariensis, is known to be very complex and fragmented making the assembly of whole genomes with next-generation sequencing data a challenge. RESULTS: We present a genome reconstruction for 33 B. bavariensis isolates from Eurasia based on long-read (Pacific Bioscience, for three isolates) and short-read (Illumina) data. We show that the combination of both sequencing techniques allows proper genome reconstruction of all plasmids in most cases but use of a very close reference is necessary when only short-read sequencing data is available. B. bavariensis genomes combine a high degree of genetic conservation with high plasticity: all isolates share the main chromosome and five plasmids, but the repertoire of other plasmids is highly variable. In addition to plasmid losses and gains through horizontal transfer, we also observe several fusions between plasmids. Although European isolates of B. bavariensis have little diversity in genome content, there is some geographic structure to this variation. In contrast, each Asian isolate has a unique plasmid repertoire and we observe no geographically based differences between Japanese and Russian isolates. Comparing the genomes of Asian and European populations of B. bavariensis suggests that some genes which are markedly different between the two populations may be good candidates for adaptation to the tick vector, (Ixodes ricinus in Europe and I. persulcatus in Asia). CONCLUSIONS: We present the characterization of genomes of a large sample of B. bavariensis isolates and show that their plasmid content is highly variable. This study opens the way for genomic studies seeking to understand host and vector adaptation as well as human pathogenicity in Eurasian Lyme Borreliosis agents.
Assuntos
Sequência Conservada , Genoma Bacteriano , Ixodes , Filogenia , Spirochaetales , Animais , Ásia , Grupo Borrelia Burgdorferi , Sequência Conservada/genética , Europa (Continente) , Genoma Bacteriano/genética , Genômica , Humanos , Doença de Lyme/microbiologia , Plasmídeos/genética , Federação Russa , Spirochaetales/classificação , Spirochaetales/genéticaRESUMO
Ticks are one of the arthropods that play an important role in the transmission of numerous pathogens to livestock and humans. We investigated the presence of tick-borne bacteria in 23 Amblyomma varanense that fed on a water monitor (Varanus salvator) in Indonesia. Anaplasmataceae and borreliae were detected by PCR in 17.4% and 95.7% of ticks, respectively. "Candidatus Rickettsia sepangensis", spotted fever group of Rickettsia, was detected in 21.7% of ticks. The water monitor is a common reptile that is widely encountered in city areas in Asian countries. Our results suggested that Am. varanense on water monitor in Indonesia harbored several kinds of bacteria.
Assuntos
Anaplasma/isolamento & purificação , Borrelia/isolamento & purificação , Ixodidae/microbiologia , Lagartos/microbiologia , Rickettsia/isolamento & purificação , Anaplasma/classificação , Anaplasma/genética , Animais , Borrelia/classificação , Borrelia/genética , DNA Bacteriano , DNA Ribossômico/genética , Feminino , Indonésia , Masculino , Filogenia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Rickettsia/classificação , Rickettsia/genética , Análise de Sequência de DNA/veterinária , Doenças Transmitidas por Carrapatos/microbiologiaRESUMO
Two captive cheetahs from a zoo in Japan died of a severe fever with thrombocytopenia syndrome-like illness. Severe fever with thrombocytopenia syndrome virus, an endemic tickborne phlebovirus, was detected systemically with secretion of infectious viruses into the saliva. These cases highlight the risk for exposure of captive animals to endemic arthropodborne pathogens.
Assuntos
Acinonyx , Infecções por Bunyaviridae/veterinária , Phlebovirus/isolamento & purificação , Doenças Transmitidas por Carrapatos/veterinária , Animais , Animais de Zoológico , Infecções por Bunyaviridae/diagnóstico , Diagnóstico Diferencial , Evolução Fatal , Feminino , Japão , Masculino , Phlebovirus/genética , Filogenia , Doenças Transmitidas por Carrapatos/diagnósticoRESUMO
In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014-2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.
Assuntos
Infecções por Alphavirus/epidemiologia , Infecções por Alphavirus/veterinária , Alphavirus/isolamento & purificação , Anticorpos Antivirais/sangue , Sus scrofa/virologia , Alphavirus/classificação , Alphavirus/genética , Alphavirus/imunologia , Animais , Chlorocebus aethiops , Surtos de Doenças , Ensaio de Imunoadsorção Enzimática , Cavalos/virologia , Japão/epidemiologia , Estudos Soroepidemiológicos , Células Vero , Vacinas Virais/imunologiaRESUMO
Chiropteran mammals worldwide harbour trypanosomes (Euglenozoa: Kinetoplastea: Trypanosomatida) of the subgenus 'Schizotrypanum' in the classical sense. Latterly, these trypanosomes have been referred to as members of the 'Trypanosoma cruzi clade' as their phylogenetic relationships, structure and life cycle conform to T. cruzi, parasitising various terrestrial mammals as well as humans in Latin America. Little is known, however, about the trypanosome species in Asian bats. During a survey on Borrelia spp. in the Eastern bent-winged bat (Miniopterus fuliginosus) living in a cave in Wakayama Prefecture, Japan, incidental proliferation of trypanosomes was detected in two of 94 haemocultures. Squat or slender trypanosomes that proliferated in the cultures were 7.5-20.5 µm in length between both body ends and 1.0-3.8 µm in width with/without free flagella up to 14.5 µm (n = 29). The nucleotide sequences of the small subunit ribosomal RNA gene (SSU rDNA; 2176 bp), large subunit ribosomal RNA gene (1365 bp) and glycosomal glyceraldehyde-3-phosphate dehydrogenase gene (gGAPDH; 843 bp) of the present isolates were characterized to clarify their molecular phylogenetic position in T. cruzi-like trypanosomes. The newly obtained SSU rDNA and gGAPDH nucleotide sequences showed the highest identities with Brazilian and European isolates of Trypanosoma dionisii of the T. cruzi clade, ranging between 99.4 and 99.7% or between 95.6 and 99.3% identities, respectively. Although multiple T. dionisii isolates from the North and South American continents showed the closest molecular genetic relatedness to the present Far East isolates, only short SSU rDNA segments of the former isolates were deposited. Therefore, a definitive conclusion cannot be made until full nucleotide sequencing of at least the American isolates' SSU rDNA is available. This is the first confirmation of a Far East distribution of T. dionisii, demonstrating a wide geographical distribution of the species in the Eurasian and American continents with a limited nucleotide variation.
Assuntos
Quirópteros/parasitologia , Trypanosoma cruzi/isolamento & purificação , Trypanosoma/isolamento & purificação , Animais , DNA de Protozoário , DNA Ribossômico , Feminino , Japão , Masculino , Filogenia , Análise de Sequência de DNA , Trypanosoma/classificação , Trypanosoma/genética , Trypanosoma cruzi/classificação , Trypanosoma cruzi/genéticaRESUMO
In an epidemiological study of ferret coronaviruses (FRCoVs), novel FRCoV strains (Saitama-1 and Aichi-1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of partial RNA-dependent RNA polymerase (RdRp) genes. Phylogenetic analysis indicated that these strains belonged to different clusters from other FRCoV strains. Next, the nucleotide sequence of the 3'-terminal region of Saitama-1 (8271 bases) strain was determined and compared with those of the other FRCoVs, indicating that the Saitama-1 strain differed from the previously reported MSU-1 and MSU-2 strains in the regions encoding spike (S) protein, nucleocapsid, and open reading frame 7b. Furthermore, the results of SimPlot analysis indicated that FRCoV (MSU-2 strain) emerged via a recombination event of S protein between the MSU-1 and Saitama-1 strains. This mechanism is similar to that responsible for the emergence of type II feline coronavirus. This information will be useful for understanding the pathogenesis of FRCoV in ferrets.
Assuntos
Infecções por Coronavirus/veterinária , Coronavirus Felino/genética , Furões/virologia , Recombinação Genética , Sequência de Aminoácidos , Animais , Ordem dos Genes , Fases de Leitura Aberta , Filogenia , RNA Viral , Análise de Sequência de DNARESUMO
Twenty-nine isolates of Lyme borreliosis (LB) group spirochaetes collected from ticks and rodents in China and Japan were included in a multilocus sequence analysis (MLSA). Using a different typing system, three of these strains had previously been identified as being divergent from other LB spirochaete species and the name 'Borrelia yangtze' sp. nov. was proposed. The data presented here confirm that the genetic distance, calculated using sequences of MLSA housekeeping genes, to other known LB group spirochaete species was < 95 % and to Borrelia valaisiana was 96.67 % (which represents the most closely related species within the group of LB spirochaetes). This and the fact that these strains are ecologically distinct from B. valaisiana (rodent-transmitted vs bird-transmitted) provide strong support for the validation of the proposed species status. We suggest the name Borrelia yangtzensis sp. nov. The type strain is Okinawa-CW62T ( = DSM 24625T = JCM 17189T).
RESUMO
Cattle do not generally appear to develop severe viremia when infected with Japanese encephalitis virus (JEV), and they can be infected without showing clinical signs. However, two cattle in Japan recently died from JEV infection. In this study, we investigated the presence of different species of mosquitoes and flavivirus in a cowshed in the southwest region of Japan. In this cowshed, the two most common species of mosquitoes collected were Culex tritaeniorhynchus (including Culex pseudovishnui) and Anopheles sinensis. We performed virus isolation from the collected mosquitoes and obtained two flaviviruses: JEV and a novel insect-specific flavivirus, tentatively designated Yamadai flavivirus (YDFV). Phylogenetic analysis revealed that all three JEV isolates belonged to JEV genotype I and were closely related to a JEV strain that was isolated from the brains of cattle exhibiting neurological symptoms in Japan. Genetic characterization of YDFV revealed that the full genome RNA (10,863 nucleotides) showed homology with the Culex-associated insect-specific flaviviruses Quang Binh virus (79% identity) and Yunnan Culex flavivirus (78% identity), indicating that YDFV is a novel insect-specific flavivirus.