Validation of a TaqMan one-step real-time RT-PCR assay targeting ISAV segment 7 spliced mRNA

Infectious salmon anaemia virus (ISAV) can cause severe systemic infection in Atlantic salmon (Salmo salar L.), and a timely diagnosis is critical. Conventional real-time reverse transcription PCR (RT-qPCR) assays target unspliced RNA from either ISAV segment 7 or 8 and provide data on viral load. Here, we evaluate a TaqMan one-step RT-qPCR assay that detects explicitly a spliced messenger RNA (mRNA) of ISAV segment 7, thus providing evidence of active viral transcription. Assay performance was comparable with existing unspliced segment 7 and segment 8 assays. PCR efficiency as evaluated from dilutions of a synthetic DNA fragment was 98 % (R² = 1.00). The assay also performed well on clinical heart samples with PCR efficiency of 108 % (R² = 1.00). Finally, evaluation on kidney samples from experimental infection revealed higher levels of active transcription for high-virulent compared to low-virulent ISAV. At early, peak, and late infection, mean ratios of spliced to unspliced segment 7 RNA were 3.0 % (± 0.7), 1.7 % (± 0.3), and 1.5 % (± 0.1) for the low virulent and 9.4 % (± 2.2), 4.7 % (± 0.8), and 6.2 % (± 0.1) for the high virulent isolate, respectively. By detection and quantification of active ISAV transcription, this assay may provide a more detailed understanding of ISAV infection dynamics.