Accelerated ISAV replication detection by cell culture methods combined with time-monitoring RT-qPCR.

Infectious salmon anaemia (ISA) is a viral disease that affects farmed Atlantic salmon (Salmo salar L.), often leading to mass mortalities. A quick detection of the ISA virus (ISAV) is crucial for decision-making and can prevent the occurrence of future outbreaks. Screening done by Canada's National Aquatic Animal Health Laboratory System (NAAHLS) uses quantitative reverse transcription PCR (RT-qPCR) followed by sequencing of PCR amplicons. As neither technique provides information regarding the infectivity of the virus, suspected virulent strains are subsequently tested using viral isolation. However, this stepwise process can require significant time to deliver results. To speed up this delivery, we have improved on these pre-existing techniques by combining the use of cell culture with RT-qPCR to detect replicative virus in as little as 5 days. Preliminary assays enabled the establishment of a minimal shift in Ct values over time, which is representative of viral replication in cultured cells. Subsequent blind panel analyses allowed the establishment of the optimal sampling days, as well as diagnostic sensitivity (DSe) and specificity (DSp) estimates. This method could be adopted not only by laboratories conducting diagnostic analyses for ISAV, but also for other slow-replicating viral agents that replicate through a budding mechanism.

Distribution and Pathogenicity of Two Cutthroat Trout Virus (CTV) Genotypes in Canada.

The sole member of the Piscihepevirus genus (family Hepeviridae) is cutthroat trout virus (CTV) but recent metatranscriptomic studies have identified numerous fish hepevirus sequences including CTV-2. In the current study, viruses with sequences resembling both CTV and CTV-2 were isolated from salmonids in eastern and western Canada. Phylogenetic analysis of eight full genomes delineated the Canadian CTV isolates into two genotypes (CTV-1 and CTV-2) within the Piscihepevirus genus. Hepevirus genomes typically have three open reading frames but an ORF3 counterpart was not predicted in the Canadian CTV isolates. In vitro replication of a CTV-2 isolate produced cytopathic effects in the CHSE-214 cell line with similar amplification efficiency as CTV. Likewise, the morphology of the CTV-2 isolate resembled CTV, yet viral replication caused dilation of the endoplasmic reticulum lumen which was not previously observed. Controlled laboratory studies exposing sockeye (Oncorhynchus nerka), pink (O. gorbuscha), and chinook salmon (O. tshawytscha) to CTV-2 resulted in persistent infections without disease and mortality. Infected Atlantic salmon (Salmo salar) and chinook salmon served as hosts and potential reservoirs of CTV-2. The data presented herein provides the first in vitro and in vivo characterization of CTV-2 and reveals greater diversity of piscihepeviruses extending the known host range and geographic distribution of CTV viruses.

Multiple isoforms of PAX5 are expressed in both lymphomas and normal B-cells.

The transcription factor Pax5 plays a critical role in B cell development. It has been shown that alternative splicing of its gene (PAX5) produces several distinct transcripts that modify the amino acid sequence of the putative Pax5 proteins. Subsequent studies have attempted to correlate the expression of PAX5 isoforms with certain B-cell lymphomas, the conclusions of which suggest that altered isoform expression is involved in lymphomagenesis. However, in the absence of definitive data for PAX5 isoform expression patterns in normal B cells it is difficult to confirm whether aberrant isoform expression can indeed be correlated with disease. Using a high-resolution method of analysis of reverse transcription polymerase chain reaction products, we sought to analyse the expression of the different PAX5 isoforms in normal B-cells as well as a number of B-cell lymphoma and chronic lymphocytic leukaemia cases. It was found that multiple PAX5 isoforms were expressed in both normal and malignant B cells. Immunodetection and polysomal RNA analyses also confirmed that the different PAX5 mRNAs were translated into their corresponding proteins. No consistent deregulation of PAX5 isoform expression was observed in B-cell lymphomas, but rather, complex isoform expression patterns were found in normal B cell as well as B-cell lymphoma and CLL cases.

Modified low-salt CTAB extraction of high-quality DNA from contaminant-rich tissues.

The increasing use of high-throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low-salt CTAB (MoLSC) extraction protocol to accommodate contaminant-rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in-house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open-source MoLSC protocol provides a cost-effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant-rich samples.