Comparison of complete polyprotein sequences of two isolates of salmon alphavirus (SAV) type I and their behaviour in a salmonid cell line

Matějusová, Iveta; Lester, Katherine; Li, Ziduo; Bravo, J. S.; Bland, Fiona; Collet, Bertrand

doi:10.1007/s00705-013-1689-4

Cited by 9 publications

(6 citation statements)

References 16 publications

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“…Mortality due these conditions varies, but all of them can also cause severe growth rate reduction and downgrading at harvest, with consequential impact on commercial value. These infections exhibit differences in cardiac immunopathological responses [25,26] and some important insights into host pathogen interactions have been gained through in vitro studies on non-cardiac cell lines [12,27,28]. However, understanding the basis of cardiac diseases in fish, as with other hosts, has been hampered by the lack of appropriate models that mimic the complexity of the organ [29].…”

Section: Introductionmentioning

confidence: 99%

Atlantic salmon cardiac primary cultures: An in vitro model to study viral host pathogen interactions and pathogenesis

et al. 2017

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Development of Salmon Cardiac Primary Cultures (SCPCs) from Atlantic salmon pre-hatch embryos and their application as in vitro model for cardiotropic viral infection research are described. Producing SCPCs requires plating of trypsin dissociated embryos with subsequent targeted harvest from 24h up to 3 weeks, of relevant tissues after visual identification. SCPCs are then transferred individually to chambered wells for culture in isolation, with incubation at 15–22°. SCPCs production efficiency was not influenced by embryo’s origin (0.75/ farmed or wild embryo), but mildly influenced by embryonic developmental stage (0.3 decline between 380 and 445 accumulated thermal units), and strongly influenced by time of harvest post-plating (0.6 decline if harvested after 72 hours). Beating rate was not significantly influenced by temperature (15–22°) or age (2–4 weeks), but was significantly lower on SCPCs originated from farmed embryos with a disease resistant genotype (F = 5.3, p<0.05). Two distinct morphologies suggestive of an ex vivo embryonic heart and a de novo formation were observed sub-grossly, histologically, ultra-structurally and with confocal microscopy. Both types contained cells consistent with cardiomyocytes, endothelium, and fibroblasts. Ageing of SCPCs in culture was observed with increased auto fluorescence in live imaging, and as myelin figures and cellular degeneration ultra-structurally. The SCPCs model was challenged with cardiotropic viruses and both the viral load and the mx gene expression were measurable along time by qPCR. In summary, SCPCs represent a step forward in salmon cardiac disease research as an in vitro model that partially incorporates the functional complexity of the fish heart.

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Section: Introductionmentioning

confidence: 99%

Atlantic salmon cardiac primary cultures: An in vitro model to study viral host pathogen interactions and pathogenesis

et al. 2017

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“…The sequencing of sample SCO/4640/08 was stopped after 3-hours producing over 400 Mb of ‘pass’ reads (Q-score ≥ 7), resulting in almost 40,000x coverage throughout the genome (Table 3 ). By mapping against the Sanger sequenced SAV1 reference sequence for SCO/4640/08 32 …”

Section: Resultsmentioning

confidence: 99%

“…Assuming an SAV genome was tiled across 7 PCR amplicons (e.g. 32 ) and sequenced directly using Sanger with no cloning step (which would add further costs), we estimate a cost of approx. £100 per SAV consensus genome, including all reagents and bi-directional sequencing.…”

Section: Resultsmentioning

confidence: 99%

“…Seven overlapping PCRs were performed in triplicates for five SAV isolates (Supplementary Table 2 ) according to the methods published by Matejusova et al . 32 . The complete SAV genomes were generated by Sanger sequencing, assembled using Sequencher v5.4.6 and used in the phylogenetic analysis presented in Fig.…”

Section: Methodsmentioning

confidence: 99%

“…Base-called FASTQ files were loaded into Geneious v.10 59 for mapping and analysis. SAV1 (SCO/4640/08) sample reads were mapped to the SAV1 Sanger-reference sequence 32 . SAV6 (F1045-96) sample reads were individually mapped to the partial gene E2 and NsP3 sequences of SAV6 28 and reference genomes for SAV1 32 , SAV2 29 , SAV3 33 , SAV4 (generated in this study; isolate SAV 04-44) and SAV5 (generated in this study; isolate SCO10-684).…”

Section: Methodsmentioning

confidence: 99%

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Nanopore sequencing for rapid diagnostics of salmonid RNA viruses

Gallagher

Matějusová

Nguyen

et al. 2018

Sci Rep

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Analysis of pathogen genome variation is essential for informing disease management and control measures in farmed animals. For farmed fish, the standard approach is to use PCR and Sanger sequencing to study partial regions of pathogen genomes, with second and third-generation sequencing tools yet to be widely applied. Here we demonstrate rapid and accurate sequencing of two disease-causing viruses affecting global salmonid aquaculture, salmonid alphavirus (SAV) and infectious salmon anaemia virus (ISAV), using third-generation nanopore sequencing on the MinION platform (Oxford Nanopore Technologies). Our approach complements PCR from infected material with MinION sequencing to recover genomic information that matches near perfectly to Sanger-verified references. We use this method to present the first SAV subtype-6 genome, which branches as the sister to all other SAV lineages in a genome-wide phylogenetic reconstruction. MinION sequencing offers an effective strategy for fast, genome-wide analysis of fish viruses, with major potential applications for diagnostics and robust investigations into the origins and spread of disease outbreaks.

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Status assessment and opportunities for improving fish welfare in animal experimental research according to the 3R-Guidelines

Grunow

Strauch

2023

Rev Fish Biol Fisheries

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In mammalian research, the implementation of the 3Rs is ubiquitous. However, the adaptation of procedures for experimental work on fish seems less extensive in comparison, even though fish model organisms are common in a broad range of research fields already. To strengthen animal welfare in experimental research, we provide an overview of current research results, including studies on the nociception of fishes. Furthermore, we describe the potential of implementing the 3Rs in fish experimental research. In the context of "Reduction", we show alternative research methods to lethal sampling. Considering "Refinement", we point out possibilities to improve fish handling and indicate that adaptations to the individual species ecology are necessary. Under the aspect of "Replacement", we describe the high potential of cell cultures that can be obtained from fish tissue and give an overview of the already extensive use in ecotoxicology and virology. In addition, we illustrate that cell cultures could also be increasingly used for basic research. Graphical abstract

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Comparison of complete polyprotein sequences of two isolates of salmon alphavirus (SAV) type I and their behaviour in a salmonid cell line

Cited by 9 publications

References 16 publications

Atlantic salmon cardiac primary cultures: An in vitro model to study viral host pathogen interactions and pathogenesis

Atlantic salmon cardiac primary cultures: An in vitro model to study viral host pathogen interactions and pathogenesis

Nanopore sequencing for rapid diagnostics of salmonid RNA viruses

Status assessment and opportunities for improving fish welfare in animal experimental research according to the 3R-Guidelines

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