Computational protocol: First description of clinical presentation of piscine orthoreovirus (PRV) infections in salmonid aquaculture in Chile and identification of a second genotype (Genotype II) of PRV

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Protocol publication

[…] For DNA sequencing, we included samples that were positive to PRV with Ct value ≤ 30 and ELF1α Ct value ≤ 20. The full length segment S1 of PRV was amplified using the primers and conditions described by Kibenge et al. []. Conventional RT-PCR was carried out using the EXPRESS qPCR SuperMix® with Premixed ROX® (Invitrogen), in a SwiftTM MaxPro Thermal Cycler® (Esco Healthcare Pte. Ltd.). The PCR products were then purified by agarose gel electrophoresis. Sequencing was performed using the ABI 310 instrument in ETECMA, CIBA Laboratory, using the BigDye Terminator V. 3.1 kit according to the laboratory procedure. Duplicate samples were sequenced commercially by Macrogen Inc. (South Korea). Duplicate total RNA samples were precipitated in 100 % ethanol and submitted to the Virology Research Laboratory (University Prince Edward Island, Canada) for confirmation of the sequencing results.The sequences were analyzed with Geneious v6.0.4 software [] and subjected to a BLAST search using programs available via the National Center for Biotechnology Information [] against the latest release at GenBank []. Analysis to identify putative ORFs and their predicted amino acid sequences and other protein characteristics was conducted using the Sequence Manipulation suite, version 2 []. Pairwise sequence comparision was done with EMBOSS Water on-line program [], which uses the Smith-Waterman algorithm (modified for speed enhancements) to calculate the local alignment of two sequences. The evolutionary distance of PRV-S1 gene sequenced in this study, together with some other PRV-S1 sequences accessible in GenBank [] was estimated using Maximum Likelihood method (software package: PhyML 3.0) [] and Neighbor-Joining phylogenetic analysis []. A bootstrap analysis to investigate the stability of the trees was performed on 1000 replicates (only the bootstrapping values that are greater than 60 % are marked). […]

Pipeline specifications

Software tools Geneious, EMBOSS, PhyML
Application Phylogenetics
Organisms Oncorhynchus kisutch, Oncorhynchus mykiss, Salmo salar, Papaya ringspot virus
Diseases Infection, Muscular Diseases, Myocarditis, Myositis, HIV Infections, Chemical and Drug Induced Liver Injury
Chemicals Amino Acids, Nucleotides