Project description:Piscine reovirus (PRV) is a causative agent of heart and skeletal muscle inflammation in Atlantic salmon, which is propagated in red blood cells (RBC). Here, transcriptome analyses of PRV infected erythrocytes showed strong and complex innate antiviral responses.
Project description:To study in vitro the epithelial cells and PrV interactions during infection, we followed PrV and PK15 cells transcriptome modifications during time-course infection (I) and mock-infection (MI).Six time points were studied: just after I and MI, 1h, 2h, 4h, 8h and 12h post-I and MI. For this study, a pig DNA/cDNA microarray containing genes of the SLA region, additional genes encoding other important immunological molecules and all the PrV genes was constructed. Keywords: infection time course
Project description:Circular RNAs (circRNAs) and microRNAs (miRNAs) participate in regulating many biological processes. However, their roles in PrV-II pathogenicity are largely unknown. Here, we analyzed the expression profile of circRNAs and miRNAs in the PrV-DX, a wild-type (WT) strain of PRV-II, and its attenuated gE-TK- PRV-DX infected cells by high-throughput sequencing.
Project description:To study in vitro the epithelial cells and PrV interactions during infection, we followed PrV and PK15 cells transcriptome modifications during time-course infection (I) and mock-infection (MI). Four time points were studied: 1h, 2h, 4h and 8h post-I and MI. Four replicates of I and MI were analysed. Keywords: Pig, PrV, Pk15 cells, kinetics
Project description:To study in vitro the epithelial cells and PrV interactions during infection, we followed PrV and PK15 cells transcriptome modifications during time-course infection (I) and mock-infection (MI). Four time points were studied: 1h, 2h, 4h and 8h post-I and MI. Four replicates of I and MI were analysed. 32 samples - The hybridization scheme which can be define as dye-switch was chosen. A balanced loop design with two independent loops, each loop containing 2 replicates of PrV infection and MI, was used. In total, 32 slides were used in this experiment.
Project description:Purpose: Evaluation of the m6A modification of PRV and PK15 transcripts during PRV infection Methods: Porcine kidney cell line PK15 was uninfected or infected with PRV for 24 hours. Total RNA from each sample were extracted. Intact mRNA was isolated from total RNA samples and then chemically fragmented to 300-nucleoside-long fragments. Fragmented mRNAs were immunoprecipitated with anti-N6-methyadenosine (m6A) antibody (a part of the fragmented mRNAs was kept as input). Both m6A enriched mRNAs and input mRNAs were concentrated for RNA-seq libraries construction. The libraries were forwarded to sequencing run on Illumina NovaSeq 6000. Results: PRV transcripts were m6A modified during PRV infection and PRV infection changed m6A modification profiles of PK15 transcripts.
Project description:Piscine orthoreovirus (PRV) belongs to the Reoviridae family and is the only known fish virus related to the Orthoreovirus genus. The virus is the causative agent of heart and skeletal muscle inflammation (HSMI), an emerging disease in farmed Atlantic salmon (Salmo salar L.). PRV is ubiquitous in farmed Atlantic salmon and high loads of PRV in the heart are consistent findings in HSMI. The mechanism by which PRV infection causes disease remains largely unknown. In this study we investigated the presence of PRV in blood and erythrocytes using an experimental cohabitation challenge model. We found that in the early phases of infection, the PRV loads in blood were significantly higher than in any other organ. Most virus was found in the erythrocyte fraction, and in individual fish more than 50% of erythrocytes were PRV-positive, as determined by flow cytometry. PRV was condensed into large cytoplasmic inclusions resembling viral factories, as demonstrated by immunofluorescence and confocal microscopy. By electron microscopy we showed that these inclusions contained reovirus-like particles. The PRV particles and inclusions also had a striking resemblance to previously reported viral inclusions described as Erythrocytic inclusion body syndrome (EIBS). We conclude that the erythrocyte is a major target cell for PRV infection. These findings provide new information about HSMI pathogenesis, and show that PRV is an important factor of viral erythrocytic inclusions.
Project description:Piscine orthoreovirus (PRV) infects farmed and wild salmon and trout species in North America, South America, Europe, and East Asia. PRV groups into three distinct genotypes (PRV-1, PRV-2, and PRV-3) that can vary in distribution, host specificity, and/or disease potential. Detection of the virus is currently restricted to genotype specific assays such that surveillance programs require the use of three assays to ensure universal detection of PRV. Consequently, herein, we developed, optimized, and validated a real-time reverse transcription quantitative PCR assay (RT-qPCR) that can detect all known PRV genotypes with high sensitivity and specificity. Targeting a conserved region at the 5' terminus of the M2 segment, the pan-PRV assay reliably detected all PRV genotypes with as few as five copies of RNA. The assay exclusively amplifies PRV and does not cross-react with other salmonid viruses or salmonid host genomes and can be performed as either a one- or two-step RT-qPCR. The assay is highly reproducible and robust, showing 100% agreement in test results from an inter-laboratory comparison between two laboratories in two countries. Overall, as the assay provides a single test to achieve highly sensitive pan-specific PRV detection, it is suitable for research, diagnostic, and surveillance purposes.
Project description:The myxozoan Tetracapsuloides bryosalmonae is the causative agent of proliferative kidney disease (PKD) – a disease of salmonid fishes, notably of the commercially farmed rainbow trout Oncorhynchus mykiss. Both wild and farmed salmonids are threatened by this virulent/deadly disease, which is often expressed by swollen kidneys. In order to understand the causes and consequences of the disease, we studied the immune response towards the parasite. To profile the influence of the disease on these cells, we produced a transcriptome of teleost RBCs in health and disease. Compared to erythrocytes originating from healthy fish, PKD fundamentally altered RBCs in their metabolism, adhesion, and response to inflammation.