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      Heart and Skeletal Muscle Inflammation of Farmed Salmon Is Associated with Infection with a Novel Reovirus

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          Abstract

          Atlantic salmon ( Salmo salar L.) mariculture has been associated with epidemics of infectious diseases that threaten not only local production, but also wild fish coming into close proximity to marine pens and fish escaping from them. Heart and skeletal muscle inflammation (HSMI) is a frequently fatal disease of farmed Atlantic salmon. First recognized in one farm in Norway in 1999 [1], HSMI was subsequently implicated in outbreaks in other farms in Norway and the United Kingdom [2]. Although pathology and disease transmission studies indicated an infectious basis, efforts to identify an agent were unsuccessful. Here we provide evidence that HSMI is associated with infection with piscine reovirus (PRV). PRV is a novel reovirus identified by unbiased high throughput DNA sequencing and a bioinformatics program focused on nucleotide frequency as well as sequence alignment and motif analyses. Formal implication of PRV in HSMI will require isolation in cell culture and fulfillment of Koch's postulates, or prevention or modification of disease through use of specific drugs or vaccines. Nonetheless, as our data indicate that a causal relationship is plausible, measures must be taken to control PRV not only because it threatens domestic salmon production but also due to the potential for transmission to wild salmon populations.

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          Comparison of DNA sequences with protein sequences.

          The FASTA package of sequence comparison programs has been expanded to include FASTX and FASTY, which compare a DNA sequence to a protein sequence database, translating the DNA sequence in three frames and aligning the translated DNA sequence to each sequence in the protein database, allowing gaps and frameshifts. Also new are TFASTX and TFASTY, which compare a protein sequence to a DNA sequence database, translating each sequence in the DNA database in six frames and scoring alignments with gaps and frameshifts. FASTX and TFASTX allow only frameshifts between codons, while FASTY and TFASTY allow substitutions or frameshifts within a codon. We examined the performance of FASTX and FASTY using different gap-opening, gap-extension, frameshift, and nucleotide substitution penalties. In general, FASTX and FASTY perform equivalently when query sequences contain 0-10% errors. We also evaluated the statistical estimates reported by FASTX and FASTY. These estimates are quite accurate, except when an out-of-frame translation produces a low-complexity protein sequence. We used FASTX to scan the Mycoplasma genitalium, Haemophilus influenzae, and Methanococcus jannaschii genomes for unidentified or misidentified protein-coding genes. We found at least 9 new protein-coding genes in the three genomes and at least 35 genes with potentially incorrect boundaries.
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            Panmicrobial Oligonucleotide Array for Diagnosis of Infectious Diseases

            To facilitate rapid, unbiased, differential diagnosis of infectious diseases, we designed GreeneChipPm, a panmicrobial microarray comprising 29,455 sixty-mer oligonucleotide probes for vertebrate viruses, bacteria, fungi, and parasites. Methods for nucleic acid preparation, random primed PCR amplification, and labeling were optimized to allow the sensitivity required for application with nucleic acid extracted from clinical materials and cultured isolates. Analysis of nasopharyngeal aspirates, blood, urine, and tissue from persons with various infectious diseases confirmed the presence of viruses and bacteria identified by other methods, and implicated Plasmodium falciparum in an unexplained fatal case of hemorrhagic feverlike disease during the Marburg hemorrhagic fever outbreak in Angola in 2004–2005.
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              Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon

              Background Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. Results The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription levels of genes encoding 18S rRNA, S20 ribosomal protein, β-actin, glyceraldehyde-3P-dehydrogenase (GAPDH), and two paralog genes encoding elongation factor 1A (EF1AA and EF1AB) were quantified in gills, liver, head kidney, spleen, thymus, brain, muscle, and posterior intestine in six untreated adult fish, in addition to a group of individuals that went through smoltification. Based on calculations performed with the geNorm VBA applet, which determines the most stable genes from a set of tested genes in a given cDNA sample, the ranking of the examined genes in adult Atlantic salmon was EF1AB>EF1AA>β-actin>18S rRNA>S20>GAPDH. When the same calculations were done on a total of 24 individuals from four stages in the smoltification process (presmolt, smolt, smoltified seawater and desmoltified freshwater), the gene ranking was EF1AB>EF1AA>S20>β-actin>18S rRNA>GAPDH. Conclusion Overall, this work suggests that the EF1AA and EF1AB genes can be useful as reference genes in qRT-PCR examination of gene expression in the Atlantic salmon.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2010
                9 July 2010
                : 5
                : 7
                : e11487
                Affiliations
                [1 ]Center for Infection and Immunity, Columbia University, New York, New York, United States of America
                [2 ]National Veterinary Institute, Oslo, Norway
                [3 ]454 Life Sciences, Branford, Connecticut, United States of America
                [4 ]Ministry of Fisheries and Coastal Affairs, Oslo, Norway
                [5 ]Department of Biomedical Informatics and Center for Computational Biology and Bioinformatics, Columbia University, New York, New York, United States of America
                [6 ]Norwegian School of Veterinary Science, Oslo, Norway
                Yale University, United States of America
                Author notes

                Conceived and designed the experiments: GP ML TT MH ME ER WIL. Performed the experiments: GP ML MH SH JH RTK NS AVB CC DLH. Analyzed the data: GP TT MH AS ABK CS VT RR. Contributed reagents/materials/analysis tools: RTK CS VT RR. Wrote the paper: GP ML TT MH ER WIL.

                Article
                10-PONE-RA-18083R1
                10.1371/journal.pone.0011487
                2901333
                20634888
                2a9fe282-d0b4-4bcf-aabd-ea609fbdc8db
                Palacios et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 19 April 2010
                : 4 June 2010
                Page count
                Pages: 7
                Categories
                Research Article
                Infectious Diseases
                Molecular Biology/Bioinformatics
                Virology/Diagnosis
                Virology/Emerging Viral Diseases
                Marine and Aquatic Sciences/Fisheries

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                Uncategorized

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