Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada
© Kibenge et al. 2016
Received: 27 October 2015
Accepted: 28 December 2015
Published: 6 January 2016
Infectious salmon anaemia (ISA) virus (ISAV) belongs to the genus Isavirus, family Orthomyxoviridae. ISAV occurs in two basic genotypes, North American and European. The European genotype is more widespread and shows greater genetic variation and greater virulence variation than the North American genotype. To date, all of the ISAV isolates from the clinical disease, ISA, have had deletions in the highly polymorphic region (HPR) on ISAV segment 6 (ISAV-HPRΔ) relative to ISAV-HPR0, named numerically from ISAV-HPR1 to over ISAV-HPR30. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish. It is recognized that asymptomatically ISAV-infected fish exist. There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the primer-probe set targeting a 104 bp-fragment on ISAV segment 8. Some laboratories and researchers have found a primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive.
In this study, we tested 1,106 fish tissue samples collected from (i) market-bought farmed salmonids and (ii) wild salmon from throughout British Columbia (BC), Canada, for ISAV using real time RT-qPCR targeting segment 8 and/or conventional RT-PCR with segment 8 primers and segment 6 HPR primers, and by virus isolation attempts using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. The sequences from the conventional PCR products were compared by multiple alignment and phylogenetic analyses.
Seventy-nine samples were “non-negative” with at least one of these tests in one or more replicates. The ISAV segment 6 HPR sequences from the PCR products matched ISAV variants, HPR5 on 29 samples, one sample had both HPR5 and HPR7b and one matched HPR0. All sequences were of European genotype. In addition, alignment of sequences of the conventional PCR product segment 8 showed they had a single nucleotide mutation in the region of the probe sequence and a 9-nucleotide overlap with the reverse primer sequence of the real time RT-qPCR assay. None of the classical ISAV segment 8 sequences in the GenBank have this mutation in the probe-binding site of the assay, suggesting the presence of a novel ISAV variant in BC. A phylogenetic tree of these sequences showed that some ISAV sequences diverted early from the classical European genotype sequences, while others have evolved separately. All virus isolation attempts on the samples were negative, and thus the samples were considered “negative” in terms of the threshold trigger set for Canadian federal regulatory action; i.e., successful virus isolation in cell culture.
This is the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. These sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines.
Timeline (chronological history) of the detection of ISAV in wild fish related to first-time outbreaks of ISA in farmed Atlantic salmon
Year of sample &
Wild fish species with ISAV (reference)
First-time outbreaks of ISA in farmed Atlantic salmon in country (reference)
1998-1999, Virus Isolation & RT-PCR
Sea trout, Brown trout, Atlantic salmon 
Scotland, UK in 1998 
Canada (New Brunswick)
New Brunswick, Canada in 1996 
Atlantic salmon 
Sea trout, Brown trout, Atlantic salmon 
West Greenland fishery
Atlantic salmon 
Atlantic salmon (P. Barbash, cited by )
Maine, USA in 2001 
2000-2002, Virus Isolation & RT-PCR
Pollock*, Atlantic cod** 
1998; 2001–2003, RT-PCR
Norway (western Norway)
Salmonids (wild trout, Atlantic salmon) 
Norway in 1984 
1995-2002, Antibody ELISA
USA (Maine & Massachusetts)
Atlantic salmon 
Atlantic salmon§ 
Chile (an estuary in southern Chile)
free-living Salmo salar (escapees) 
Chile in 2007 
Faroe Islands, Denmark
Faroe Islands, Denmark in 2000 
ISAV has a segmented genome with eight single-stranded RNA segments of negative polarity . The Orthomyxoviridae family is known to exhibit high mutation rates, and ISAV occurs in at least 30 recognized HPR variants [9, 18]. When viruses mutate, ‘drift variants’ arise that can escape detection by real-time RT-qPCR tests due to mismatches in the primer-probe binding sites . When a mutation occurs in the precise region that a given primer or probe was designed to anneal, test reliability can be significantly decreased  producing inconsistent positive and false-negative readings between replicates . There is no scientific standard for interpretation of high, or inconsistent threshold cycle (C t) values, and so these kinds of results are interchangeably reported as “negative,” “suspicious” or “positive” [22, 23]. For the purposes of this work, we simply designated our results as negative or non-negative.
In Canada, a federally reportable fish disease such as ISA must be confirmed at the Fisheries and Oceans (DFO) Canada National Reference Laboratory  through successful virus isolation in cell culture . However, ISAV-HPRΔ strains of low virulence and the non-pathogenic ISAV-HPR0 strains grow poorly or not at all in currently available fish cell lines [15, 26–29]. Gagné and Ritchie  report an increasing number of ISAV positive results by RT-PCR in Canada that cannot be confirmed by other diagnostic tests. It is also recognized in Norway that ISAV may be present even when attempts at virus isolation are negative as ISAV has never been isolated from a wild salmon despite positive RT-PCR results (Table 1) .
While virus isolation is considered the “gold standard” for virus identification , it can produce “false negative results” . Virus isolation requires tissue heavily infected with intact virus , which is unlikely to be found in wild salmon which are culled by predators that target weakened fish . As well, intact, infective ISAV may not reliably occur in healthy salmon that have been harvested for several days, such as fish found in markets. Molecular tests, however, have the capacity to detect low levels of virus fragments  making them ideally suited for the types of samples available to this study.
There is no universally accepted ISAV RT-qPCR TaqMan® assay. Most diagnostic laboratories use the Snow et al.  primer-probe set targeting a 104 bp-fragment on ISAV segment 8 [37, 38]. Some laboratories and researchers have found the Plarre et al.  primer-probe set targeting ISAV segment 7 to be more sensitive. Other researchers have published different ISAV segment 8 primer-probe sets that are highly sensitive , but are not included in the OIE Manual . There is also a long standing conventional RT-PCR protocol targeting ISAV segment 8 using a primer set initially developed by Devold et al. , which is less sensitive than real time RT-qPCR. This yields a PCR product of 221 bp, which includes the first 94 bp of the 104 bp-PCR amplicon of the Snow et al.  RT-qPCR TaqMan® assay, with the reverse primer sequences of both assays overlapping in 9 nucleotides. While preliminary results from this study were interpreted as controversial [40, 41], they are consistent with the nature of both the tests and the samples, i.e., wild fish and fish from markets. The findings in the present study are supported by the many unpublished ISAV RT-PCR positive results in farmed and wild salmon in British Columbia, which exist as unpublished federal laboratory exhibits released by the Cohen Commission into the Decline of the Sockeye Salmon of the Fraser River . Here we present more complete test results demonstrating that the ISAV sequences detected in British Columbia (BC) fish, both ISAV-HPRΔ and ISAV-HPR0, are of European genotype, with a mismatch in segment 8 that contributes to the inconsistent results of the RT-qPCR TaqMan® assay, and represents a new ISAV variant that appears to occur in BC in absence of high losses to the salmon farming industry. It would add to the knowledge of ISAV to test fresh moribund farmed salmon using the methods we describe here.
Results and Discussion
Sample RNA quality was based on real-time RT-PCR for ELF-1α as internal control for all samples
Fewer than 2.0 % of British Columbia fish tested were “non-negative” in the real time RT-qPCR TaqMan® assay for ISAV
In the present study, we used the Snow et al.  primer-probe set targeting segment 8 with a cut-off C t value established as the mean C t value in the highest virus dilution for which all 30 replicates were positive (Additional file 1: Table S1). Thus for the purposes of this study, samples were considered “non-negative” when the fluorescence signal increased above the C t, and if the C t value was ≤ 34.20. Samples with C t > 34.2 to ≤ 39.9 were considered weak “non-negative” and > 40, suspicious as C t of the last five cycles has higher uncertainity. Where the C t value was zero, the result was deemed to be negative.
Number of samples for each species that (i) tested non-negative for infectious salmon anaemia virus (ISAV) by RT-qPCR1 and that produced sequences by conventional PCR for (ii) segment 6 and (iii) segment 8
Conventional PCR seg. 8 sequence
Conventional PCR seg. 6 sequence
Conventional PCR seg. 8 sequence
Conventional PCR seg. 6 sequence
ISAV sequences detected in British Columbia fish have a mismatch in segment 8 compared to classical ISAV and represent a new ISAV variant of European genotype
Whereas all fish tissue samples were screened by the real time RT-qPCR TaqMan® assay for ISAV, only a portion of these samples was additionally tested by conventional RT-PCR for segment 8 or segment 6 HPR. Table 2 lists all non-negative test results by species and by farmed vs. wild status. This study did not attempt a direct comparison of the 3 different RT-PCR assays. Such an effort would require standardizing sample quality, which would require direct access to salmon in the farms.
ISAV sequences detected in British Columbia fish include both ISAV-HPRΔ and ISAV-HPR0 and are of European genotype
The detection of ISAV-HPR0 in British Columbia fish (Fish# SK20) was designated a suspect result by the Canadian Food Inspection Agency (CFIA), because of the inability for follow up by the federal authorities. With the widespread occurrence of ISAV-HPR0 variants in many parts of the world and its potential as a precursor to the virulent strains of ISAV , it is essential that RT-PCR positive results based on segment 8 primers be followed up with conventional RT-PCR using segment 6 primers targeting the HPR. Sequencing of the PCR product is also essential in order to determine the ISAV HPR type present (ISAV-HPRΔ or ISAV-HPR0 or both) . ISAV-HPR0 has only been reported in apparently healthy fish and has never been associated with clinical or pathological diagnosis of ISA disease .
Of the fish tested in conventional RT-PCR for segment 6 HPR, sequences of the PCR product were obtained from 13 farmed fish samples (13 Atlantic salmon) and 17 from wild fish samples (1 coho, 2 Sockeye, 1 Chum and 13 Cutthroat) (Table 2). In contrast to conventional RT-PCR for segment 8, where 49 samples positive in segment 8 had no C t value in the real time RT-qPCR TaqMan® assay for ISAV, only 3 samples (Fish# SS132, MQ06, and P113, Additional file 2: Table S2) were positive in conventional RT-PCR for segment 6, with no C t value. These samples were also negative by conventional RT-PCR for segment 8.
The sequences of ISAV segment 6 obtained from the PCR products matched ISAV-HPR5 on 29 samples, one had both ISAV-HPR5 and ISAV-HPR7b and one sample matched ISAV-HPR0 (Additional file 2: Table S2). All were of European genotype. ISAV-HPRΔ strains of HPR5 and HPR7b types have been associated with ISA outbreaks in Norway [45, 46], Scotland  and Chile [9, 48]. Thus our data indicate that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation, which is in agreement with other reports [15, 16].
To our knowledge the present work constitutes the first published report of the detection of ISAV sequences in fish from British Columbia, Canada. The sequences detected, both of ISAV-HPRΔ and ISAV-HPR0 are of European genotype. The virus in these samples has a mismatch in segment 8 that can account for failure of the real time RT-qPCR TaqMan® assay for ISAV recommended in the OIE Aquatic Manual. Furthermore, these sequences are different from the classical ISAV segment 8 sequences, and this difference suggests the presence of a new ISAV variant of European genotype in BC. Our results further suggest that ISAV-HPRΔ strains can be present without clinical disease in farmed fish and without being detected by virus isolation using fish cell lines. Recent reports on ISAV surveillance in Washington, USA , and in British Columbia  report no ISAV detection. However, neither of these studies report on samples from the known target host of ISAV, farmed Atlantic salmon, and it is unreported whether weak RT-PCR positives similar to ours were found, and interpreted as “negative”. More research on the source of this variant ISAV sequence is critically important for assessing the risks to both farmed and wild salmon in the region, its origin and to better understand ISAV evolution.
Wild fish were collected from freshwater spawning grounds, fresh and saltwater sport fisheries, saltwater commercial fisheries, and saltwater scientific fisheries. Wild fish samples included all species of Pacific salmon (Oncorhynchus sp.), Atlantic salmon (Salmo salar), steelhead (Oncorhynchus mykiss), cutthroat trout (Oncorhynchus clarkii), kokanee (Oncorhynchus nerka), Pacific chub mackerel (Scomber japonicus) and Pacific herring (Clupea pallasi) (Table 2). All fish and the sampled organs were photographed in situ. Gill and heart were sampled from the whole wild fish. Gill and remnant head kidney were sampled from the gutted, head-on farmed salmon and also the farmed salmon heads purchased from markets. The hearts were not available from these samples. All samples were placed immediately in sterile Whirl-Pak® bags (Nasco Inc., Fort Atkinson, WI) on ice with replicate samples preserved in RNALater® (Ambion Inc., Foster City, CA) and shipped overnight by courier to the testing laboratory. At the laboratory, samples were immediately stored at −80 °C until they were analyzed. The testing laboratory ran tests exclusively on the samples and did not participate in the collection of the samples or in the custody of the samples prior to receipt of the samples.
Total RNA preparation
Total RNA was isolated using a modified total RNA extraction protocol with the RNeasy® mini Kit (QIAGEN). Briefly, each tissue (or pool of tissues) was weighed and macerated to a 10 % suspension w/v in phosphate buffered saline (PBS) with 10x antibiotics. The specimen supernatant was used for RNA extraction. Samples preserved in RNAlater® were first washed three times with PBS and then homogenized as described above prior to total RNA extraction. Total RNA was isolated from samples using 1.25 ml of TRIZOL Reagent (Invitrogen) and 375 μl of sample volume as previously described . The Viral RNA mini Kit (QIAGEN) was also utilized on selected samples following the manufacturer’s recommended protocol. In all cases, the extracted RNA was eluted in 20–50 μl of nuclease-free water, and RNA yields were quantified and purity analysed using the OD260/280 ratio and a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific). The eluted RNA was tested immediately following quantitation, or was stored frozen at −80 °C prior to use in RT-PCR.
RT-qPCR was run on the LightCycler 480 (Roche Applied Science), version 4.0. The crossing point (Cp) or threshold cycle (C t) was determined by use of the maximum-second-derivative function on the LightCycler software release 1.5.0. The Roche LightCycler® 480 RNA master Hydrolysis Probe kit (Roche Diagnostics) was employed for all RT-qPCR reactions according to the manufacturer’s specifications. Sample RNA quality was based on RT-qPCR for elongation factor 1 alpha (ELF-1α) as internal control targeting either Atlantic salmon ELF-1α (GenBank accession number BT072490) or Chinook salmon ELF-1α (GenBank accession number FJ890356) using primers, probes, and RT-qPCR thermal cycling parameters as previously reported . RNA quality varied, with the higher C t values generally occurring in farmed salmon from markets where the interval between harvest and sampling was on the order of days, not minutes as was the case for most wild fish samples. Nonetheless, some wild fish, such as the cutthroat trout and LaP1, were caught by fishermen with unavoidable delays in processing the sampled fish. Such delays may have contributed to the higher C t values in some of these samples. Results from tests with C t values above 40 or at 0 were designated as negative. In addition, these samples would be considered unfit for further testing if after re-extraction and repeated RT-qPCR the same results were obtained.
Detection of ISAV with the one-step real-time RT-qPCR  was carried out using the primer-probe set developed by Snow et al.  targeting segment 8 and described in the OIE Aquatic Manual . However, there is no defined C t value cut off to aid interpretation of results. In this study, the cut-off C t value for this probe was set at ≤34.20 ± 1.05 based on 10-fold dilutions of cell culture ISAV (ADL 2007) each tested in 5 replicates and repeated 6 times for a total of 30 replicates, and denotes the mean C t value in the highest virus dilution for which all 30 replicates were positive (Additional file 1: Table S1). The same preparations were also tested in the conventional RT-PCR methods below, allowing for a correlation of the cut-off C t value with the conventional RT-PCR tests on cell culture virus.
Conventional RT-PCR and nucleic acid sequencing
GenBank Accession numbers used in the multiple alignments and phylogenetic analyses and of new sequences from this study
Isolate or sample ID
Sequences were aligned and phylogenetic trees were generated using CLUSTAL X with the default settings . Alignment regions containing gaps were excluded from the analysis. The results were analyzed by using the bootstrap method (1000 replicates) to provide confidence levels for the tree topology. We then used different outgroup sequences to determine and verify the root of each tree.
Primary virus isolation was attempted on some of the RT-PCR “non-negative” samples using Salmon head kidney (SHK-1 and ASK-2) cell line monolayers. SHK-1  and ASK-2 cells  were grown as previously described . Homogenized tissues were inoculated on monolayers of SHK-1 and/or ASK-2 cell lines following standard protocols in the OIE Aquatic Manual . Briefly, each tissue was weighed and macerated to a 10 % homogenate w/v in PBS with 10x antibiotics. The homogenates were centrifuged at 205.3 g for 15 min at 4 °C. The supernatants were individually filtered using 0.45 μM syringe filters to remove any bacteria prior to use in virus isolation attempts. 24 hr-old cell monolayers in tissue culture flasks free of medium were inoculated with the sample supernatant diluted 1:10 in serum-free medium, and incubated for 2 hr at room temperature to allow for virus adsorption. Maintenance medium was then added and the inoculated cells were then incubated at 16 °C and infection was allowed to proceed with daily monitoring using an inverted light microscope until the CPE was evident or 21 days and the flasks were frozen at −80 °C. Virus isolation was monitored by RT-PCR on the cell lysates since virus replication may occur without development of apparent CPE . CPE negative and RT-PCR negative cultures were passaged on fresh cell monolayers. A sample was considered negative if no CPE or positive RT-PCR was observed after three blind passages.
The in vitro work was approved by the UPEI Biosafety Committee.
This work was supported by the Virology Research Laboratory at the Atlantic Veterinary College, University of Prince Edward Island, Charlottetown, PE, Canada, and a Natural Sciences and Engineering Research Council (NSERC) of Canada Discovery Grant to F.S.B.K. Van City, Rudy North, James Corbett, Patagonia, Tony and Kim Allard, Dick and Val Bradshaw, Vancouver Foundation, the Tula Foundation, Elizabeth Borak, Wheeler Family Foundation, Yvon Chouinard, Eden Conservation Trust, Sarah Haney, Tides Foundation, Jaymac Foundation, an NSERC grant to R.R. and many smaller donors. Thank you to the First Nations who assisted and allowed us to sample their fish - Boston Bar, Gitanyow Fisheries, Gitxsan Fisheries Authorities, Heiltsuk, Kwikwasut’inuxw Haxwa’mis, Lake Babine Nation Fisheries, Lake Cowichan, Mowachaht/Muchalaht, N'Quatqua, Nicola, Seton Lake, Shuswap, Skeena First Nations, Snuneymuxw, Splatsin, St’at’imc, Stellaquo, Stó:lō , Takla Lake, Xeni Gwet'in, Office of the Wet'suwet'en Fisheries and Wuikinuxv First Nations. We also thank Rob, Nola, Krystal and Amber-Bachen, Sandy Bodrug, Farlyn and Tavish Campbell, Tamara and Roy Campbell, Brad Crowther, Roger Dunlop, Jody Eriksson, Randy Ericksen, Dave and Nicole Gerbrandt, Nicole, Donna and Bill Mackay, Jennifer Parkhouse, Stan Proboszcz, Anissa Reed, Dave Rolston, Louise Routledge, Steve Sharron, Tsylos Park Lodge, Ted and Duane Walkus, Monica Woods, and Sabra Woodworth.
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