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      De novo assembly of Sockeye salmon kidney transcriptomes reveal a limited early response to piscine reovirus with or without infectious hematopoietic necrosis virus superinfection

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          Abstract

          Background

          Piscine reovirus (PRV) has been associated with the serious disease known as Heart and Skeletal Muscle Inflammation (HSMI) in cultured Atlantic salmon Salmo salar in Norway. PRV is also prevalent in wild and farmed salmon without overt disease manifestations, suggesting multifactorial triggers or PRV variant-specific factors are required to initiate disease. In this study, we explore the head kidney transcriptome of Sockeye salmon Oncorhynchus nerka during early PRV infection to identify host responses in the absence of disease in hopes of elucidating mechanisms by which PRV may directly alter host functions and contribute to the development of a disease state. We further investigate the role of PRV as a coinfecting agent following superinfection with infectious hematopoietic necrosis virus (IHNV) – a highly pathogenic rhabdovirus endemic to the west coast of North America.

          Results

          Challenge of Sockeye salmon with PRV resulted in high quantities of viral transcripts to become present in the blood and kidney of infected fish without manifestations of disease. De novo transcriptome assembly of over 2.3 billion paired RNA-seq reads from the head kidneys of 36 fish identified more than 320,000 putative unigenes, of which less than 20 were suggested to be differentially expressed in response to PRV at either 2 or 3 weeks post challenge by DESeq2 and edgeR analysis. Of these, only one, Ependymin, was confirmed to be differentially expressed by qPCR in an expanded sample set. In contrast, IHNV induced substantial transcriptional changes (differential expression of > 20,000 unigenes) which included transcripts involved in antiviral and inflammatory response pathways. Prior infection with PRV had no significant effect on host responses to superinfecting IHNV, nor did host responses initiated by IHNV exposure influence increasing PRV loads.

          Conclusions

          PRV does not substantially alter the head kidney transcriptome of Sockeye salmon during early (2 to 3 week) infection and dissemination in a period of significant increasing viral load, nor does the presence of PRV change the host transcriptional response to an IHNV superinfection. Further, concurrent infections of PRV and IHNV do not appear to significantly influence the infectivity or severity of IHNV associated disease, or conversely, PRV load.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s12864-016-3196-y) contains supplementary material, which is available to authorized users.

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          Most cited references54

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          The Air noncoding RNA epigenetically silences transcription by targeting G9a to chromatin.

          A number of large noncoding RNAs (ncRNAs) epigenetically silence genes through unknown mechanisms. The Air ncRNA is imprinted--monoallelically expressed from the paternal allele. Air is required for allele-specific silencing of the cis-linked Slc22a3, Slc22a2, and Igf2r genes in mouse placenta. We show that Air interacts with the Slc22a3 promoter chromatin and the H3K9 histone methyltransferase G9a in placenta. Air accumulates at the Slc22a3 promoter in correlation with localized H3K9 methylation and transcriptional repression. Genetic ablation of G9a results in nonimprinted, biallelic transcription of Slc22a3. Truncated Air fails to accumulate at the Slc22a3 promoter, which results in reduced G9a recruitment and biallelic transcription. Our results suggest that Air, and potentially other large ncRNAs, target repressive histone-modifying activities through molecular interaction with specific chromatin domains to epigenetically silence transcription.
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            YY1 tethers Xist RNA to the inactive X nucleation center.

            The long noncoding Xist RNA inactivates one X chromosome in the female mammal. Current models posit that Xist induces silencing as it spreads along X and recruits Polycomb complexes. However, the mechanisms for Xist loading and spreading are currently unknown. Here, we define the nucleation center for Xist RNA and show that YY1 docks Xist particles onto the X chromosome. YY1 is a "bivalent" protein, capable of binding both RNA and DNA through different sequence motifs. Xist's exclusive attachment to the inactive X is determined by an epigenetically regulated trio of YY1 sites as well as allelic origin. Specific YY1-to-RNA and YY1-to-DNA contacts are required to load Xist particles onto X. YY1 interacts with Xist RNA through Repeat C. We propose that YY1 acts as adaptor between regulatory RNA and chromatin targets. Copyright © 2011 Elsevier Inc. All rights reserved.
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              Count-based differential expression analysis of RNA sequencing data using R and Bioconductor

              , , (2013)
              RNA sequencing (RNA-seq) has been rapidly adopted for the profiling of transcriptomes in many areas of biology, including studies into gene regulation, development and disease. Of particular interest is the discovery of differentially expressed genes across different conditions (e.g., tissues, perturbations), while optionally adjusting for other systematic factors that affect the data collection process. There are a number of subtle yet critical aspects of these analyses, such as read counting, appropriate treatment of biological variability, quality control checks and appropriate setup of statistical modeling. Several variations have been presented in the literature, and there is a need for guidance on current best practices. This protocol presents a "state-of-the-art" computational and statistical RNA-seq differential expression analysis workflow largely based on the free open-source R language and Bioconductor software and in particular, two widely-used tools DESeq and edgeR. Hands-on time for typical small experiments (e.g., 4-10 samples) can be <1 hour, with computation time <1 day using a standard desktop PC.
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                Author and article information

                Contributors
                Mark.Polinski@dfo-mpo.gc.ca
                Julia.Bradshaw@dfo-mpo.gc.ca
                r92smi@mun.ca
                Jon.Richard@dfo-mpo.gc.ca
                camilla.fritsvold@vetinst.no
                trygve.poppe@nmbu.no
                mrise@mun.ca
                Kyle.Garver@dfo-mpo.gc.ca
                Stewart.Johnson@dfo-mpo.gc.ca
                Journal
                BMC Genomics
                BMC Genomics
                BMC Genomics
                BioMed Central (London )
                1471-2164
                2 November 2016
                2 November 2016
                2016
                : 17
                : 848
                Affiliations
                [1 ]Fisheries and Oceans Canada, Pacific Biological Station, 3190 Hammond Bay Rd, Nanaimo, BC V9T6N7 Canada
                [2 ]Department of Ocean Sciences, Memorial University, St. John’s, NF A1C5S7 Canada
                [3 ]Department of Pathology, Norwegian Veterinary Institute, Oslo, NO-0106 Norway
                [4 ]Department of Basic Sciences and Aquatic Medicine (Basam), Norwegian University of Life Sciences, P.O. Box 8146, Dep, N-0033 Oslo, Norway
                Article
                3196
                10.1186/s12864-016-3196-y
                5094019
                27806699
                f727cb1a-e53c-42ef-8642-132b7a12fe8c
                © The Author(s). 2016

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 4 June 2016
                : 22 October 2016
                Funding
                Funded by: Program for Aquaculture Regulatory Research
                Award ID: PARR-2014-P-13
                Funded by: FundRef http://dx.doi.org/10.13039/501100002790, Canadian Network for Research and Innovation in Machining Technology, Natural Sciences and Engineering Research Council of Canada;
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2016

                Genetics
                sockeye salmon,piscine reovirus, infectious hematopoietic necrosis virus,rna-seq,transcriptome,de novo assembly

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