Project description:This experiment investigates the gene expression differences upon Orsay virus infection in the Caenorhabdits elegans strains N2 and CB4856. Assays measuring viral load found that the N2 strain displays higher viral loads upon infection than the CB4856 strain. The goal of the experiment was to identify gene-expression differences that could explain the differences in viral load. We (mock-)infected 26h-old C. elegans populations with Orsay virus and took samples after 30h of infection. For each treatment-strain combination 8 samples were collected. Thereafter RNA was isolated, labelled, and hybridized on microarray.
Project description:This experiment investigates the temporal dynamics in gene expression upon Orsay virus infection in the nematode Ceanorhabditis elegans. Three different strains were infected: JU1580, N2, and CB4856. Of these, JU1580 is highly susceptible, N2 moderately susceptible, and CB4856 the most resistant strain. The goal of the experiment was to identify genes that show different expression dynamics over the time-course of the infection. We (mock-)infected 40h-old C. elegans populations with Orsay virus and took samples over a 32-hour spanning time-course. For each treatment-strain combination 12 samples were collected. Thereafter RNA was isolated, labelled, and hybridized on microarray. The gene-expression dynamics of previously identified genes (e.g. from literature and from a highly replicated N2 versus CB4856 experiment) were analyzed.
Project description:This experiment investigates changes in gene expression upon Orsay virus infection and between males and hermaphrodites in the nematode Ceanorhabditis elegans. The laboratory reference strain N2 was used. For this strain, males are less susceptible to Orsay virus infection than hermaphrodites. The goal of the experiment was to identify genes that show different expression patterns for both sexes upon Orsay virus infection. We mock-treated or infected 48h-old C. elegans populations with Orsay virus and took samples 30-hours after infection. For each treatment-sex combination 8 samples were collected. Thereafter RNA was isolated, labelled, and hybridized on microarray. The gene-expression dynamics of previously identified genes (e.g. from literature and from a highly replicated N2 versus CB4856 experiment) were analyzed.
Project description:C. elegans has served as a laboratory model organism due to its ease of manipulation and the availability of both forward and reverse genetics. In recent years, efforts to study host-pathogen interactions in C. elegans have increased. For example, analysis of infections by bacteria such as Pseudomonas, Salmonella or Serratia has revealed the existence of innate immune pathways in C. elegans that are also conserved in vertebrates. To date, there has been no natural virus infection reported in C. elegans or C. briggsae. Here we describe evidence of natural virus infection in wild isolates of both C. elegans and C. briggsae. Two highly divergent but related RNA viruses in the family Nodaviridae, tentatively named Orsay nodavirus and Santeuil nodavirus, were detected and their genomes partially sequenced. Infected worm lysates passed through 0.2 um filters could be used to infect uninfected worms, which could be further passaged for many generations. Furthermore, the viruses were subject to processing by the RNAi machinery as evidenced by the detection of virally derived small RNAs. Infection of mutant worms defective in small RNA pathways yielded more robust levels of viral RNA as compared to infection of isogenic N2 reference worms. These data demonstrate that nodaviruses are natural parasites of nematodes in the wild. Further study of the interactions between these viruses and nematodes is likely to provide insight into the natural ecology of nematodes and may reveal novel innate immune mechanisms that respond to viral infection.
Project description:C. elegans has served as a laboratory model organism due to its ease of manipulation and the availability of both forward and reverse genetics. In recent years, efforts to study host-pathogen interactions in C. elegans have increased. For example, analysis of infections by bacteria such as Pseudomonas, Salmonella or Serratia has revealed the existence of innate immune pathways in C. elegans that are also conserved in vertebrates. To date, there has been no natural virus infection reported in C. elegans or C. briggsae. Here we describe evidence of natural virus infection in wild isolates of both C. elegans and C. briggsae. Two highly divergent but related RNA viruses in the family Nodaviridae, tentatively named Orsay nodavirus and Santeuil nodavirus, were detected and their genomes partially sequenced. Infected worm lysates passed through 0.2 um filters could be used to infect uninfected worms, which could be further passaged for many generations. Furthermore, the viruses were subject to processing by the RNAi machinery as evidenced by the detection of virally derived small RNAs. Infection of mutant worms defective in small RNA pathways yielded more robust levels of viral RNA as compared to infection of isogenic N2 reference worms. These data demonstrate that nodaviruses are natural parasites of nematodes in the wild. Further study of the interactions between these viruses and nematodes is likely to provide insight into the natural ecology of nematodes and may reveal novel innate immune mechanisms that respond to viral infection. Two small RNA libraries (18-30 nt) from nodavirus-infected and cured C. elegans wild isolate JU1580 were sequenced on the Illumina Genome Analyzer II platform. Samples were treated with tobacco acid pyrophosphatase to allow cloning of small RNA molecules with 5'-triphosphates. Each sample was labelled with a unique four base pair barcode and libraries were multiplexed together with a third library (not included in this submission). The multiplexed libraries were sequenced in triplicate.
Project description:Background information: According to our observations, cde-1 (tm1021) null C. elegans animals show increased susceptibility to the Orsay virus (which has a bipartite positive RNA genome), compared to WT (N2 strain). Viral infection seems limited to intestinal cells. CDE-1 is a terminal uridylyltransferase (TUT). TUTs have been shown to promote RNA decay by 3’ end uridylation in various contexts and organisms. We hypothesize that CDE-1 serves as an antiviral factor by uridylating the viral RNA genome. Aims: We question small RNA populations in cde-1 mutants versus WT after two days of infection by the Orsay virus. Methods: Approximately 200 C. elegans animals were infected for 48 hours from the L2 stage to the adult stage with 20 µl filtrate of the Orsay virus on 50mm plate seeded with HB101 bacteria, in biological duplicates, for the following genetic backgrounds: N2 (+), drh-1 (ok3495), rde-1 (ne219), cde-1 (mj414), cde-1 (tm1021), cde-1 (tm1021);drh-1 (ok3495), cde-1 (tm1021);rde-1 (ne219). Animals were then washed in M9 and resuspended in 1 ml TRIsure (Bioline). Samples were freeze-thaw three times using liquid nitrogen and RNA purification was performed according to Bioline’s guidelines. Purified RNA was either directly used for library preparation (5’ dependent libraries) or was first submitted to 5’ polyphosphatase treatment (5’ independent libraries) as in Ashe, et al. 2013. sRNA libraries were prepared using the TruSeq Small RNA Library Preparation Kit (Illumina) according to manufacturer’s instructions (with size selection between 20 and 35 nt, adapters excluded) and deep sequencing was produced with an Illumina HiSeq machine (single read 36).