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This formal comment is in response to Siah et al. [1], Piscine Reovirus: Genomic and Molecular Phylogenetic Analysis from Farmed and Wild Salmonids Collected on the Canada/US Pacific Coast, with a subsequent correction (Siah et al. 2016 [2]). Although a correction for this paper was published on Oct. 12, 2016, (Siah et al. 2016 [2]), there continues to be inadequate supporting evidence for the primary conclusion that PRV genetic sequences are temporally and spatially homogeneous in salmonid species across the northeastern Pacific region.  The evidence in this paper warrants thorough consideration. Piscine orthoreovirus (PRV) causes acute infection of the red blood cells in salmon (Finstad et al. 2014 [3]; Haatveit et al. 2017 [4]). It is the causative agent of the emerging farm salmon disease Heart and Skeletal Muscle Inflammation (HSMI) (Wessel et al. 2017 [5]) with clinical symptoms which can include lethargy, anemia, anorexia and mortality (Kongtorp et al. 2004 [6]). Palacios et al. [7] expressed concerns about the transfer of PRV from farmed to wild fish due to its contagious nature. PRV is now considered ubiquitous in farmed Atlantic salmon (Haatveit et al. 2017 [4]) and has an estimated 80% prevalence rate among BC farmed salmon (Kibenge et al. 2013 [8]). HSMI has recently been diagnosed in British Columbia (BC), Canada (Di Cicco et al. 2017 [9]). Hence, release of PRV from salmon farms into Pacific salmon habitat is a significant management concern in the eastern Pacific Ocean.  In the correction, Siah et al. [2] acknowledge that the conclusion that PRV has not been recently introduced to BC was overstated. However their supporting evidence that "… salmonids from western North America Pacific waters carried PRV RNA sequences for at least 13 years with little genetic differentiation among sequence types in selected samples spanning 2001 to 2014" remains insufficient.  Their conclusion appears to be highly dependent on six unique sequences of PRV segment S1, detected by Siah et al. [1]:   {"type":"entrez-nucleotide","attrs":{"text":"KR478642","term_id":"931323276","term_text":"KR478642"}}KR478642: collected in May 2001   {"type":"entrez-nucleotide","attrs":{"text":"KR478643","term_id":"931323280","term_text":"KR478643"}}KR478643: collected in Aug. 2001   {"type":"entrez-nucleotide","attrs":{"text":"KR478644","term_id":"931323283","term_text":"KR478644"}}KR478644: collected in Aug. 2001   {"type":"entrez-nucleotide","attrs":{"text":"KR347078","term_id":"931316047","term_text":"KR347078"}}KR347078: collected in Aug. 2001   {"type":"entrez-nucleotide","attrs":{"text":"KR347079","term_id":"931316050","term_text":"KR347079"}}KR347079: collected in Aug. 2001   {"type":"entrez-nucleotide","attrs":{"text":"KR347080","term_id":"931316053","term_text":"KR347080"}}KR347080: collected in Mar. 2005    These six Siah et al. [1] sequences collected in 2001 and 2005 (submitted to GenBank April—May 2015) predate those collected by Kibenge et al. [10,11] by seven years and are nearly identical to the isolates Siah et al. [1] collected in 2013 and 2014. Thus, these six PRV isolates appear to be highly resistant to mutation over a 13-year interval 2001–2014, which is atypical for RNA viruses, generally known to exhibit a high mutation rate (Chao et al. [12]). Drake and Holland [13] estimate the genomic mutation rate (U) to be between 1 and 0.1 for most RNA viruses, where U is G x u, G is the genome size in nucleotides, and u is the per-nucleotide mutation rate.  Weight of evidence for longer-term PRV presence in BC Siah et al. [1] cite detection of PRV in a wild Steelhead trout (O. mykiss) collected in 1977 in support of longer-term PRV presence in BC. This result is cited from Marty et al. [14], who provided no S1 segment sequence information to verify the PRV strain identity as per the sequence groupings reported by both Kibenge et al. [8] and Garseth et al. [15]. The recent discovery of the widespread occurrence of PRV-2 across the North Pacific (Takano et al. 2016 [16]) raises the question: Was the 1977 steelhead infected with PRV-2 or PRV? In absence of S1 sequencing this uncertainty cannot be resolved.  Furthermore, this result could not be replicated by a second laboratory (Purcell and Thompson 2014 [17]) and therefore warrants qualification as a non-repeatable result and a suspect positive lacking sufficient robustness to provide evidence critical to the temporal presence of PRV in BC.

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Formal comment on: Piscine reovirus: Genomic and molecular phylogenetic analysis from farmed and wild salmonids collected on the Canada/US Pacific Coast