Project description:Primary objectives: The primary objective is to investigate circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Primary endpoints: circulating tumor DNA (ctDNA) via deep sequencing for mutation detection and by whole genome sequencing for copy number analyses before start (baseline) with regorafenib and at defined time points during administration of regorafenib for treatment efficacy in colorectal cancer patients in terms of overall survival (OS).
Project description:BackgroundThe genus Orbivirus includes a number of important pathogenic viruses, including Bluetongue virus (BTV), African horse sickness virus (AHSV), and Epizootic hemorrhagic disease virus (EHDV). In this study we describe the isolation and characterization of an Orbivirus strain isolated from Anopheles maculatus mosquitoes collected in Tibet, China.Methods and resultsInitial viral screening identified a viral strain (XZ0906) that caused significant cytopathic effect (CPE) in BHK-21 cells, including rounding, cell rupture, and floating. Although CPE was not observed in insect cells (C6/36), these cells supported viral replication. Polyacrylamide gel analysis revealed a genome consisting of 10 segments of double-stranded RNA (dsRNA), with a distribution pattern of 3-3-3-1. 454 high throughput sequencing of culture supernatant was used for viral identification. Complete genome sequencing was performed by Sanger sequencing in combination with 5'-RACE and 3'-RACE. Sequence analysis demonstrated that all 5'- and 3'- untranslated regions (UTRs) for each of the 10 genome segments contained a series of six highly conserved nucleotides. In addition, homology analysis and phylogenetic analysis based on amino acid sequence was completed, and all results show that virus XZ0906 was not a member of any known species or serotype of Orbivirus, indicating it to be a new species within the genus Orbivirus.ConclusionsThe isolated Orbivirus strain was designated Tibet Orbivirus, TIBOV to denote the location from which it was isolated. TIBOV is a novel orbivirus species which is isolated from Anopheles maculatus mosquitoes collected in Tibet, China.
Project description:Tibet orbivirus (TIBOV) was first isolated from Anopheles maculatus mosquitoes in Xizang, China, in 2009. In recent years, more TIBOV strains have been isolated in several provinces across China, Japan, East Asia, and Nepal, South Asia. Furthermore, TIBOVs have also been isolated from Culex mosquitoes, and several midge species. Additionally, TIBOV neutralizing antibodies have been detected in serum specimens from several mammals, including cattle, sheep, and pigs. All of the evidence suggests that the geographical distribution of TIBOVs has significantly expanded in recent years, with an increased number of vector species involved in its transmission. Moreover, the virus demonstrated infectivity towards a variety of animals. Although TIBOV is considered an emerging orbivirus, detailed reports on its genome and molecular evolution are currently lacking. Thus, this study performed the whole-genome nucleotide sequencing of three TIBOV isolates from mosquitoes and midges collected in China in 2009, 2011, and 2019. Furthermore, the genome and molecular genetic evolution of TIBOVs isolated from different countries, periods, and hosts (mosquitoes, midges, and cattle) was systematically analyzed. The results revealed no molecular specificity among TIBOVs isolated from different countries, periods, and vectors. Meanwhile, the time-scaled phylogenetic analysis demonstrated that the most recent common ancestor (TMRCA) of TIBOV appeared approximately 797 years ago (95% HPD: 16-2347) and subsequently differentiated at least three times, resulting in three distinct genotypes. The evolutionary rate of TIBOVs was about 2.12 × 10-3 nucleotide substitutions per site per year (s/s/y) (95% HPD: 3.07 × 10-5, 9.63 × 10-3), which is similar to that of the bluetongue virus (BTV), also in the Orbivirus genus. Structural analyses of the viral proteins revealed that the three-dimensional structures of the outer capsid proteins of TIBOV and BTV were similar. These results suggest that TIBOV is a newly discovered and rapidly evolving virus transmitted by various blood-sucking insects. Given the potential public health burden of this virus and its high infectious rate in a wide range of animals, it is significant to strengthen research on the genetic variation of TIBOVs in blood-feeding insects and mammals in the natural environment and the infection status in animals.
Project description:While serological and virological evidence documents the exposure of bats to medically-important arboviruses, their role as reservoirs or amplifying hosts is less well-characterized. We describe a novel orbivirus (Reoviridae:Orbivirus) isolated from an Egyptian fruit bat (Rousettus aegyptiacus leachii) trapped in 2013 in Uganda and named Bukakata orbivirus. This is the fifth orbivirus isolated from a bat, however genetic information had previously only been available for one bat-associated orbivirus. We performed whole-genome sequencing on Bukakata orbivirus and three other bat-associated orbiviruses (Fomede, Ife, and Japanaut) to assess their phylogenetic relationship within the genus Orbivirus and develop hypotheses regarding potential arthropod vectors. Replication kinetics were assessed for Bukakata orbivirus in three different vertebrate cell lines. Lastly, qRT-PCR and nested PCR were used to determine the prevalence of Bukakata orbivirus RNA in archived samples from three populations of Egyptian fruit bats and one population of cave-associated soft ticks in Uganda. Complete coding sequences were obtained for all ten segments of Fomede, Ife, and Japanaut orbiviruses and for nine of the ten segments for Bukakata orbivirus. Phylogenetic analysis placed Bukakata and Fomede in the tick-borne orbivirus clade and Ife and Japanaut within the Culicoides/phlebotomine sandfly orbivirus clade. Further, Bukakata and Fomede appear to be serotypes of the Chobar Gorge virus species. Bukakata orbivirus replicated to high titers (10⁶⁻10⁷ PFU/mL) in Vero, BHK-21 [C-13], and R06E (Egyptian fruit bat) cells. Preliminary screening of archived bat and tick samples do not support Bukakata orbivirus presence in these collections, however additional testing is warranted given the phylogenetic associations observed. This study provided complete coding sequence for several bat-associated orbiviruses and in vitro characterization of a bat-associated orbivirus. Our results indicate that bats may play an important role in the epidemiology of viruses in the genus Orbivirus and further investigation is warranted into vector-host associations and ongoing surveillance efforts.
Project description:An extensive comparative analysis of orbivirus genomes revealed four cases of unclear numeration and protein designation, due to confused reference to protein size or segment size by which they are encoded. A concise nomenclature based on type species, sequence homology and functional characteristics independent of segment or protein size is suggested.
Project description:modENCODE_submission_5014 This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The White Lab is aiming to map the association of all the Transcription Factors (TF) on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Stat92E-GFP; Developmental Stage: Embryo 12-24h; Genotype: PBac{y[+]-attP-3B}VK00037; Transgene: Stat92E genomic coding region; EXPERIMENTAL FACTORS: Developmental Stage Embryo 12-24h; Target gene Stat92E; Strain Stat92E-GFP; Antibody GFP ab290 (target is Green Fluorescent Protein)
Project description:modENCODE_submission_5023 This submission comes from a modENCODE project of Kevin White. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: The White Lab is aiming to map the association of all the Transcription Factors (TF) on the genome of Drosophila melanogaster. One technique that we use for this purpose is chromatin immunoprecipitation coupled with deep sequencing (ChIP-seq) utilizing an Illumina next generation sequencing platform. The data generated by ChIP-seq experiments consist basically of a plot of signal intensity across the genome. The highest signals correspond to positions in the genome occupied by the tested TF. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf EXPERIMENT TYPE: CHIP-seq. BIOLOGICAL SOURCE: Strain: Hr46-GFP; Developmental Stage: Embryo 0-8; Genotype: PBac{y[+]-attP-3B}VK00033; Sex: Unknown; Transgene: Hr46 genomic coding region; EXPERIMENTAL FACTORS: Developmental Stage Embryo 0-8; Strain Hr46-GFP; Antibody tgo mouse Crews (target is tango)