Project description:To evaluate targeted MinION next generation sequencing as a diagnostic method for detection of pathogens in human blood and plasma, human blood or plasma samples were spiked with measured amounts of viruses, bacteria, protozoan parasites or tested pathogen-free as negative controls. Nucleic acid was extracted from samples and PCR amplification performed in multiplex primer pools with a procedure described in ArrayExpress experiment submission ID 18379. The PCR products were used for library preparation. The libraries sequenced on an Oxford Nanopore MinION. The passed reads aligned with a custom reference file to determine the identity of the pathogen in the sample.
Project description:S. meliloti strains with a bi- and monopartite genome configuration were constructed by consecutive Cre/lox-mediated site-specific fusions of the secondary replicons. Beside the correct genomic arrangements, these strains and precursors were tested for variations in the nucleotide sequence. Futher, a marker fequency analysis was performed to test if replication is initiated at all origins and to determine the replication termination regions of the triple replicon fusion molecule. To gain the sequence data for these analyses, respective strains were applied to whole genome sequencing using an Illumina MiSeq-System and Oxford Nanopore (MinION) sequencing technology.
Project description:The genome of two isogenic lines from Aedes aegypti from Ile Royale, French Guiana, with a marked difference in resistance to deltamethrin was investigated in order to understand the genetic basis of this phenotypic difference. Genomic sequencing was performed both with Illumina short, paired reads and with Minion long reads.
Project description:Aedes aegypti mosquitoes are important vectors of viral diseases. Mosquito host factors play important roles in virus control and it has been suggested that Dengue virus replication is regulated by Dnmt2-mediated DNA methylation. However, recent studies have shown that Dnmt2 is a tRNA methyltransferase and that Dnmt2-dependent methylomes lack defined DNA methylation patterns, thus necessitating a systematic re-evaluation of the mosquito genome methylation status. We have now searched the A. aegypti genome for candidate DNA modification enzymes. This failed to reveal any known (cytosine-5) DNA methyltransferases, but identified homologues for the Dnmt2 tRNA methyltransferase, the Mettl4 (adenine-6) DNA methyltransferase, and the Tet DNA demethylase. All genes were expressed at variable levels throughout mosquito development. Mass spectrometry demonstrated that DNA methylation levels were several orders of magnitude below the levels that are usually detected in organisms with DNA methylation-dependent epigenetic regulation. Furthermore, whole-genome bisulfite sequencing failed to reveal any evidence of defined DNA methylation patterns. These results suggest that the A. aegypti genome is unmethylated. Interestingly, additional RNA bisulfite sequencing provided evidence for Dnmt2-mediated tRNA methylation in mosquitoes. These findings have important implications for understanding the mechanism of Dnmt2-dependent virus control.
Project description:Microarray analysis on days 1, 2 and 7 post-infection with dengue, yellow fever and West Nile virus in Aedes aegypti Rockefeller strain mosquitoes RNA was purified and hybridized with Nimblegen X4 microarray chips using 81-mer probes designed from 18,000 open reading frames (ORF) found in the Ae. aegypti genome, with 2 different probes per ORF
Project description:Zika virus (ZIKV) of the Flaviviridae family is a recently emerged mosquito-borne virus that has been implicated in the surge of the number of microcephaly instances in south America. The virus is transmitted mainly by the mosquito Aedes aegypti that also vectors dengue virus. Considering rather recent rapid spread of the virus and its declaration as a global health emergency by the World Health Organization, little is known about the interactions of the virus with the mosquito vector. In this study, we investigated the transcriptome profiles of whole Ae. aegypti mosquitoes in response to ZIKV infection at 2, 7 and 14 days post-infection using deep sequencing. Results showed a large number of transcripts were altered at each time point following infection, but 18 transcripts were commonly changed at the three time points. The outcomes provide a basic understanding of Ae. aegypti responses to ZIKV and help determining host factors involved in replication or anti-viral response against the virus.
Project description:Efficient virus replication in its vector, Aedes mosquitoes, is essential for the transmission of arboviral diseases like dengue virus (DENV) in populations. In order to identify RNA-independent host factors involved in DENV replication in mosquitoes, we established a system expressing all non-structural proteins within the context of the macro protein complex as observed during viral infections. We GFP-tagged Loqs to purify it's interactors by label-free mass spectrometry.
Project description:The midgut of hematophagous insects is the initial site of infection by arthropod-borne viruses (arboviruses) and plays a crucial role in vector competence. To further understand processes that occur in the midgut in response to infection by an arbovirus, DNA microarrays were used to analyze gene expression changes following infection by the alphavirus, Sindbis (MRE16 Malaysian strain). Midgut transcription profiles from mosquitoes fed blood containing 108 pfu/ml of virus were compared with those from mosquitoes ingesting blood meals having no virus. Transcription profiles from both experimental groups were analyzed at 1, 4, and 8 days post feeding. Among the many transcription changes observed by microarray analysis, the most dramatic involved three genes that had twenty-five to forty-fold increases in transcript levels in virus infected mosquitoes at 4 days post infection . These genes were synaptic vesicle protein-2 (SV2), potassium-dependent sodium/calcium exchanger (NCKX), and a homologue of C. elegans Unc-93, a putative component of a two-pore potassium channel. We speculate that these changes represent changes in vesicle transport processes. In addition to these observations, transcript changes were observed in infected mosquitoes that suggested involvement of Toll and JNK immune cascades as a response to viral infection
Project description:MicroRNAs (miRNA) have alternative forms known as isomiRs, which differ from each other by a few nucleotides. Next generation sequencing platforms facilitate identification of these isomiRs and recent discoveries regarding their functional importance have increased our understandings of the regulatory complexities of the microRNAome. Observed changes in the miRNA profiles in mosquitoes infected with flaviviruses have implicated small RNAs in the interactions between viruses and their vectors. Here we analysed the isomiR profiles of both uninfected and infected blood fed Aedes aegypti mosquitoes with a major human pathogen, Dengue virus at two time points post-infection. We found noticeable changes to the isomiR expression profile in response to infection and aging. Data analysis revealed a distinct bias towards isomiR production in the mature miRNA as opposed to the star strand. Furthermore, we noticed that only in 40% of Ae. aegypti miRNAs, the most abundant reads for each particular miRNA match the exact sequence reported in the miRbase. The isomiR expression variations between an Ae. aegypti embryonic cell line (Aag2) and whole mosquitoes demonstrated a tissue-specific pattern of isomiR production. Our results illustrated a bias for certain types of isomiRs for each miRNA. The findings presented in this study also provide evidence that isomiR production is not a random phenomenon and may be important in DENV colonisation of its vector.