Open Access

Discovery of variant infectious salmon anaemia virus (ISAV) of European genotype in British Columbia, Canada

  • Molly JT Kibenge1,
  • Tokinori Iwamoto1, 5,
  • Yingwei Wang2,
  • Alexandra Morton3,
  • Richard Routledge4 and
  • Frederick SB Kibenge1Email author
Virology Journal201613:3

DOI: 10.1186/s12985-015-0459-1

Received: 27 October 2015

Accepted: 28 December 2015

Published: 6 January 2016

Abstract

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Keywords

Infectious salmon anaemia virus ISAV ISAV variant European genotype

Background

Infectious salmon anaemia virus (ISAV) is an economically important pathogen of marine-farmed Atlantic salmon (Salmo salar L.). The disease infectious salmon anaemia (ISA) is arguably the most feared viral disease of the marine farmed salmon industry because it has continued to cause the Atlantic salmon farming industry severe economic losses in an increasing number of countries for the past 30 years. ISAV is the only species of the genus Isavirus, and one of the seven genera of the family Orthomyxoviridae that includes the influenza viruses [13]. A complete sequence of PB1 gene of a putative koi carp orthomyxovirus was obtained from koi carp in California with 43 % amino acid sequence identity with ISAV [4], and there is also an independent reference to an orthomyxovirus from koi carp [5], and to unknown viruses with morphology consistent with members of family Orthomyxoviridae isolated from baitfish in Wisconsin, USA [6]. The taxonomic status of these findings is not known. ISAV occurs in two basic genotypes, North American and European [7, 8]. The European genotype is more widespread [9] and shows greater genetic variation [10, 11] and greater virulence variation [1214] than the North American genotype. ISA outbreaks have only been reported in farmed Atlantic salmon, although ISAV has been detected by RT-PCR in wild fish (Table 1). It is recognized that asymptomatically ISAV-infected fish exist [15, 16]. Since 2012, only Norway, Canada and Chile have reported ISA outbreaks. The ISA outbreaks reported in Canada have occurred in the Atlantic Ocean in New Brunswick, Nova Scotia, and Newfoundland and Labrador [17].
Table 1

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 &

Test used

Country (location)

Wild fish species with ISAV (reference)

First-time outbreaks of ISA in farmed Atlantic salmon in country (reference)

1998-1999, Virus Isolation & RT-PCR

UK (Scotland)

Sea trout, Brown trout, Atlantic salmon [61]

Scotland, UK in 1998 [47]

2000, RT-PCR

Canada (New Brunswick)

Salmonids [62]

New Brunswick, Canada in 1996 [63]

2000, RT-PCR

UK (Scotland)

Atlantic salmon [49]

 

2000, RT-PCR

UK (Scotland)

Sea trout, Brown trout, Atlantic salmon [64]

 

2001, RT-PCR

West Greenland fishery

Atlantic salmon [65]

 

2001, RT-PCR

USA (Maine)

Atlantic salmon (P. Barbash, cited by [66])

Maine, USA in 2001 [67]

2000-2002, Virus Isolation & RT-PCR

USA (Maine)

Pollock*, Atlantic cod** [66]

 

1998; 2001–2003, RT-PCR

Norway (western Norway)

Salmonids (wild trout, Atlantic salmon) [31]

Norway in 1984 [68]

1995-2002, Antibody ELISA

USA (Maine & Massachusetts)

Atlantic salmon [69]

 

2010, RT-PCR

Denmark

Atlantic salmon§ [70]

 

2010, RT-PCR

Chile (an estuary in southern Chile)

free-living Salmo salar (escapees) [15]

Chile in 2007 [71]

 

Faroe Islands, Denmark

 

Faroe Islands, Denmark in 2000 [72]

*Pollock taken from inside a marine cage with ISA-disease salmon was weak RT-PCR positive;

**Atlantic cod taken from a well boat holding salmon from a marine cage with clinically diseased fish was CPE positive on SHK cell culture.

§Danish salmon produced for restocking purposes.

ISAV has a segmented genome with eight single-stranded RNA segments of negative polarity [1]. 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 [19]. When a mutation occurs in the precise region that a given primer or probe was designed to anneal, test reliability can be significantly decreased [20] producing inconsistent positive and false-negative readings between replicates [21]. 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 [24] through successful virus isolation in cell culture [25]. 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, 2629]. Gagné and Ritchie [30] 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) [31].

While virus isolation is considered the “gold standard” for virus identification [32], it can produce “false negative results” [20]. Virus isolation requires tissue heavily infected with intact virus [33], which is unlikely to be found in wild salmon which are culled by predators that target weakened fish [34]. 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 [35] 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. [36] primer-probe set targeting a 104 bp-fragment on ISAV segment 8 [37, 38]. Some laboratories and researchers have found the Plarre et al. [31] 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 [13], but are not included in the OIE Manual [38]. There is also a long standing conventional RT-PCR protocol targeting ISAV segment 8 using a primer set initially developed by Devold et al. [39], 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. [36] 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 [42]. 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

The ELF-1α controls showed a considerable variation between samples (within and between species). Fig. 1 highlights the systematic difference in the threshold cycle (C t) values for the market-sampled vs. field-sampled fish. It also highlights two outliers in each of the two groups. The log sheets for the two anomalously large values in the field-sampled group indicate potential delays in sample processing under suboptimal conditions. The anomalously low values in the market-sampled group came from a single shopping event. This in turn suggests that these fish were perhaps atypically fresh. Both these sets of outliers point to the potential for differences between sampling events to account for a substantial portion of the variability in these C t values. A formal analysis of a mixed-effects model for these values using the R package, ‘lme4’ [43], provided the following estimates: (i) that the mean C t values for the market-sampled group was 2.94 units higher than the mean for the field-sampled group, and (ii) that the standard deviation of the within-sampling-event means was 2.57, and that the residual deviation was only 1.47. Although the data set was too small to provide precise estimates of these parameters, it appears that most of the variability in the C t values can be accounted for by variation in the sample quality – much of it unavoidably associated with the necessity to rely on (i) market purchases of farmed fish and (ii) logistics of sampling remote regions of British Columbia.
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig1_HTML.gif
Fig. 1

Boxplots highlighting the systematic difference in the Ct-values for the market-sampled vs. field-sampled fish. The bold, middle line represents the median for each group; the box covers the range between the lower and upper quartiles; the whiskers extend to the last value still within 1.5 times the interquartile arrange of the relevant quartile; individual points beyond these are plotted individually as potential outliers.

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. [36] 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.

A total of 1,106 tissue samples were collected from market-sourced farmed salmon (397) and wild fish (708) obtained from (i) saltwater commercial fisheries, (ii) freshwater and saltwater sport fisheries, (iii) research sampling of juvenile salmon in seawater, and (iv) spawning adult salmon in freshwater habitat throughout British Columbia, Canada. As well, a sea louse (Lepeophtheirus salmonis) removed from a juvenile sockeye salmon was sampled. All samples were tested for ISAV using published real time RT-qPCR and/or conventional RT-PCR. The test results are detailed in Additional file 2: Table S2 and summarized in Table 2. Out of the total of 1,106 tissue samples, 20 (1.81 %) tested had a C t value (Table  2 ). Of these, only one sample (Fish # VR5, a spawning chum salmon in freshwater) was also positive in conventional RT-PCR with segment 8 and segment 6 HPR primers. One sample (Fish # TT48, an Atlantic salmon from a market) was also positive in conventional RT-PCR with segment 8, and two samples (Fish# SK20 and TT51, both Atlantic salmon from markets) were also positive in conventional RT-PCR with segment 6 HPR primers. The percentage of samples with a C t value was more than two-fold greater in farmed fish tissues (2.77 %) compared to wild fish tissues (1.3 %). There were 56/65 fish that produced positive conventional RT-PCR results with no RT-PCR C t values. All virus isolation attempts on these samples using ASK-2 and SHK-1 cell lines were negative. From a Canadian regulatory perspective, diagnostic confirmation requires virus isolation on permissive fish cell lines and virus identification [25] - hence a sample with a C t value or positive conventional RT-PCR in this study was designated as “non-negative”.
Table 2

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

Fish species

Farmed fish

Wild fish

Common name

Scientific name

n

RT-qPCR

Conventional PCR seg. 8 sequence

Conventional PCR seg. 6 sequence

n

RT-qPCR

Conventional PCR seg. 8 sequence

Conventional PCR seg. 6 sequence

Atlantic salmon

Salmo salar

334

9

18

132

    

Chinook salmon

Oncorhynchus tshawytscha

13

0

0

0

102

2

2

0

Coho salmon

Oncorhynchus kisutch

4

0

0

0

68

1

6

1

Sockeye salmon

Oncorhynchus nerka

    

256

3

7

2

Kokanee

Oncorhynchus nerka

    

1

1

1

Na

Pink salmon

Oncorhynchus gorbuscha

    

118

0

2

0

Chum salmon

Oncorhynchus keta

    

68

1

1

1

Steelhead trout

Oncorhynchus mykiss

46

2

3

0

21

0

Na

Na

Cutthroat trout

Oncorhynchus clarkii

    

18

0

83

13

Chum mackerel

Scomber japonicus

    

13

0

1

0

Pacific herring

Clupea pallasi

    

44

0

1

0

Sea louse

Lepeophtheirus salmonis

    

1

1

0

0

Total

 

397

11

21

13

709

9

29

17

1All fish tissue samples were screened by the real time RT-qPCR TaqMan® assay for ISAV. However, only some of the samples were also tested in conventional RT-PCR for segment 6 HPR or segment 8. Therefore, the numbers do not reflect a direct comparison of the 3 different RT-PCR assays. Na denotes Not applicable.

22 others with a PCR product but not sequenced.

35 others with a PCR product but not sequenced.

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.

To determine the genetic relationship between the ISAV sequences in this study and ISAV strains worldwide, we compared the segment 8 sequences using multiple alignment and phylogenetic analysis. All the 50 sequences from this study aligned well in a 221 bp-long fragment with 47 selected classical ISAV segment 8 sequences of different ISAV isolates in GenBank (GenBank Database) (Fig. 2). This alignment revealed a consistent single nucleotide mutation (5’-CAT CGT CGC TGC AGA TC-3’) in the 3’ region of the probe sequence (5’-CAT CGT CGC TGC AGT TC-3’) [36]. This mutation in the BC samples would contribute to the apparent failure of the real time RT-qPCR TaqMan® assay for ISAV in the 49 samples, positive in segment 8, but with no C t value in the real time RT-qPCR TaqMan® ISAV assay. None of the classical ISAV segment 8 sequences in the GenBank database have this mutation. While a single nucleotide variation is a minor mutation, its placement in a region that an ISAV probe sequence seeks to anneal in a standard OIE ISAV test, makes this a significant mutation that warrants recognition as a new variant. Improvements aimed at better detection of this variant are currently being developed. Recognition of, and testing for, this variant are essential prerequisites for determining how widespread it is.
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig2_HTML.gif
Fig. 2

Alignment of ISAV segment 8 nucleotide sequences in the target region of the primer-probe set of the real time RT-qPCR TaqMan® assay for ISAV [36]. The ISAV sequences belonged to a 221 bp-long PCR product amplified from 50 samples in this study and 47 selected classical ISAV segment 8 sequences of different ISAV isolates in the GenBank database. The nucleotide sequences were aligned using CLUSTAL X with the default settings [58]. Green is forward primer and reverse primer sequences and red is probe sequence. The single nucleotide mutation in the probe sequence is in blue. The reverse primer sequence includes the 9-nucleotide overlap between the reverse primer sequence of the real time RT-qPCR TaqMan® assay [36] and that of the conventional RT-PCR protocol [39]

The alignment of amino acid sequences in a 118 bp-long fragment of the same ISAV sequences in Fig. 2 without the primer sequences is shown in Fig. 3. The polypeptide aligned well. The number of mutations shown in Fig. 3 is slightly less than the number of mutations in Fig. 2. This is because of the redundancy nature of the genetic code. For example, the mutation from AAAGCCC to AAGGCCC in VT02142014-120 is not shown in Fig. 3; this is because both AAA and AAG are translated to K. The single nucleotide mutation in the probe sequence of Snow et al. [36] resulted in a single amino acid change from V to D and would therefore produce a functional full-length viral protein.
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig3_HTML.gif
Fig. 3

Alignment of ISAV segment 8 amino acid sequences in the target region of the primer-probe set of the real time RT-qPCR TaqMan® assay for ISAV [36]. The ISAV sequences are those in Fig. 2. The amino acid sequences in the region between the two primers (Forward Primer and Reverse Primer) were aligned using CLUSTAL X with the default settings [58]. The single nucleotide mutation in the probe sequence resulted in a single amino acid change from V to D

Phylogenetic analysis was used to further determine the genetic relationship between the newly discovered BC ISAV variant in the present study and the classical ISAV strains worldwide. Fig. 4 shows the phylogenetic tree generated with these sequences with satisfactory bootstrap support (bootstrapping values more than 70 % are marked). In addition to showing the relationship of the ISAV sequences from this study (all “VT” sequences), this tree also supports the well-established major division between North American genotype and European genotype ISAV. All the ISAV sequences detected in this study are of the European genotype. While these BC sequences tend to be similar to each other, their differences with other European sequences are very small. The apparent wide diversity of the BC sequences in the tree reflects the nature of the sampling, and the fact that there was no single-source-selected amplification as occurs with the classical ISAV isolates from disease outbreaks. Moreover, the branching in the tree also indicates that some (VT02142012-120, VT08092012-449, and VT08092012-402) diverted early from most of the classical European genotype sequences, while others (VT06062013-60, VT08092012-465, VT05012012-308, and VT06202012-391) have evolved separately. The data support the observation that ISAV can exist in a region for a period of years in absence of outbreaks and in a state that may not be detectable by methods designed to diagnose virulent outbreaks in moribund farmed fish.
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig4_HTML.gif
Fig. 4

Phylogenetic tree showing the genetic relationship between the 97 ISAV sequences aligned in Fig. 2. Sequences were aligned and the phylogenetic tree was generated by using CLUSTAL X with the default settings [58]. Phylogenetic analysis using Neighbor-Joining bootstrap method (1000 replicates) provided satisfactory bootstrap support (bootstrapping values are shown for branch-points with greater than 70 % bootstrap support)

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 [13], 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) [9]. ISAV-HPR0 has only been reported in apparently healthy fish and has never been associated with clinical or pathological diagnosis of ISA disease [44].

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 [47] 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 determine the genetic relationship between the segment 6 HPR sequences detected in BC and worldwide, we compared the HPR sequences using multiple alignment and phylogenetic analysis. A total of 316 sequences aligned well and the phylogenetic tree was generated depicting the overall relationship among all ISAV isolates for which segment 6 sequence is available (data not shown). Virulent ISAV isolates have a deletion in segment 6 HPR sequence [44]. Of the 316 segment 6 HPR sequences, we only identified 101 sequences that were long enough to display this deletion. We aligned these 101 sequences to show the deletion. No existing alignment software packages can align nucleotide sequences in this delicate and complex area, thus the alignment has been manually adjusted. When we prepared a figure to show these sequences and the deletion, we found it is hard to show so many sequences in a figure so that some of the sequences that behave the same in this deletion area were removed. Fig. 5 shows a portion of the alignment containing the deletion. In this figure, 71 sequences were included, representing the 101 sequences. The first five sequences of the alignment, including VT12212012-1068, are complete, i.e., they have no deletion and belong to ISAV-HPR0 [49]. The rest of the sequences have a deletion and belong to ISAV-HPRΔ [11, 44].
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig5_HTML.gif
Fig. 5

Alignment of ISAV segment 6 HPR nucleotide sequences containing the deletion. The alignment was adjusted manually as all known alignment software packages cannot align nucleotide sequences in such a delicate and complex area. Among 101 sequences analyzed, 39 of them are British Columbia isolates (ID starts with VT); the deletions of these 39 sequences are in three patterns. The first pattern is VT12212012-1068; the second pattern is VT03282012-222-C and VT02142012-90; the third pattern has 36 isolates and this pattern can be shown in VT05252012-337G. To save space in the figure, we used 6 sequences to represent the 36 sequences in this pattern

The alignment of amino acid sequences of the ISAV segment 6 HPR sequences in Fig. 5 without the primer sequences is shown in Fig. 6. Both figures show extensive deletions in the HPR. Existing software cannot produce high-quality alignments in areas with deletions. Thus this alignment was manually adjusted to reveal the deletion event. The deletions in HPR probably occur through homologous recombination (copy-choice recombination, presumably because of strand-switching by the viral RNA polymerase [50] during negative RNA strand synthesis from one nucleic acid template of one virus to another. Fig. 6 confirms the three ISAV HPR types found in the BC samples: ISAV-HPR0 (VT12212012-1068), ISAV-HPR7b (VT03282012-222-C and VT02142012-90), and ISAV-HPR5 (VT05252012-337G and 5 other samples) and also reveals the relationship between these HPR types and ISAV HPR types in other countries.
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig6_HTML.gif
Fig. 6

Alignment of ISAV segment 6 HPR amino acid sequences containing the deletion. The alignment was adjusted manually as all known alignment software packages cannot align amino acid sequences in such a delicate and complex area in Fig. 5. The alignment confirms the three ISAV HPR patterns in Fig. 5: The first pattern VT12212012-1068 belongs to ISAV-HPR0; the second pattern VT03282012-222-C and VT02142012-90 belongs to ISAV-HPR7b; the third pattern with 36 isolates and shown in VT05252012-337G belongs to ISAV-HPR5. Similarly to Fig. 5, only 6 of the 36 sequences in the third pattern are included in Fig. 6

Fig. 7 shows the phylogenetic tree generated with the sequences in Fig. 4 with satisfactory bootstrap support (bootstrapping values more than 70 % are marked); where NBISA01/98 is used as an outgroup. This tree contains only the isolates from Fig. 5. It was not created to reflect the whole evolutionary history of ISAV segment 6, but to help with the analysis of the HPR deletion. Comparing Figs. 5, 6 and 7, we can find some consistency, i.e. isolates that show the same deletion pattern tend to be closer inside the tree. Although such consistency only exists for a few groups, the combination of these two approaches may reveal more insights on ISAV’s evolutionary history.
https://static-content.springer.com/image/art%3A10.1186%2Fs12985-015-0459-1/MediaObjects/12985_2015_459_Fig7_HTML.gif
Fig. 7

Phylogenetic tree showing the genetic relationship between the 71 representative segment 6 HPR sequences aligned in Fig. 4. Sequences were aligned and the phylogenetic tree was generated by using CLUSTAL X with the default settings [58]. Phylogenetic analysis using Neighbor-Joining bootstrap method (1000 replicates) provided satisfactory bootstrap support (bootstrapping values are shown for branch-points with greater than 70 % bootstrap support)

Conclusions

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 [41], and in British Columbia [17] 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.

Methods

Sampling

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 [51]. 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.

Real-time RT-qPCR

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 [52]. 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 [51] was carried out using the primer-probe set developed by Snow et al. [36] targeting segment 8 and described in the OIE Aquatic Manual [38]. 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

Samples testing positive by real-time RT-qPCR were further tested using conventional one step RT-PCR targeting segments 6 and 8 to obtain PCR products for DNA sequencing. The ISAV conventional one step RT-PCR used ISAV-specific primers FA-3/RA-3 and the conditions described by Devold et al. [39] for RNA segment 8, and the following primers segment 6 HPR primers, Fwd 5'-GCC CAG ACA TTG ACT GGA GTA G-3', and Rev 5'-AGA CAG GTT CGA TGG TGG AA-3' described by Kibenge et al. [9] for RNA segment 6, and was run in a Bio-Rad thermal cycler (Bio-Rad). Briefly, amplification was performed using 50 μl reaction mixture utilizing One-step RT-PCR kit (QIAGEN) as follows: the reaction mixture contained 2 μl of total RNA, 10 μl of 5X QIAGEN OneStep RT-PCR buffer, 2 μl of dNTPs, 10 units of RNAse inhibitor (Life Technologies), 0.6 μM (final concentration) of each primer pair, and 2 μl of QIAGEN OneStep RT-PCR enzyme mix in a final volume of 50 μl. The RT-PCR amplification conditions were 1 cycle at 50 °C for 30 min, one cycle at 95 °C for 15 min, 40 cycles at 94 °C for 30 s, 60 °C for 60 s and 72 °C for 90 s and 1 cycle at 72 °C for 10 min before soaking at 4 °C. Amplified products were analyzed by electrophoresis on 1 % agarose gel and purified using High Pure PCR Product Purification Kit (Roche). The PCR products were cloned into the pCRII vector using a TOPO TA cloning kit (Invitrogen) in preparation for nucleotide sequencing, although in some cases the RT-PCR products were sequenced directly without cloning. Plasmid DNA for sequencing was prepared as per Kibenge et al. [53], and DNA sequencing as per Kibenge et al. [10] by ACGT Corporation (Toronto, Ontario, Canada). Sequence analysis used the BLAST programs [54] against the latest release at GenBank [55], the Sequence Manipulation suite version 2 [56], and the FASTA program package for microcomputers [57]. Sequences are available through GenBank and their accession numbers are listed in Table 3.
Table 3

GenBank Accession numbers used in the multiple alignments and phylogenetic analyses and of new sequences from this study

Isolate or sample ID

Segment 6

Segment 8

Reference

VT02142012-120

JQ857081

JQ857078

This study

VT05252012-334H

KR998473

KR998431

This study

VT05252012-336G

KR998474

KR998432

This study

VT08092012-449

KR998475

KR998433

This study

ISAV2/89

DQ785246

  

96/09/734

DQ785250

DQ785278

 

96/09/768

DQ785249

DQ785277

 

96/09/1729

DQ785251

DQ785279

 

96/09/1712

DQ785245

DQ785273

 

93/09/2264

DQ785255

DQ785283

 

93/09/2163

AF427049

DQ785281

 

HPR4_Gullesfjord/94_Norway

AF302801

AF262384

 

HPR14_Vedoy/99_Norway

AF302803

AF262383

 

Glesvaer/90

AF220607

AF262382

 

90/09/400

DQ785248

DQ785276

 

H2143/89

DQ785247

DQ785275

 

SK779/06

EU118820

EU118822

 

NBISA01/98

AF283996

AF315063

 

VT03282012-222-C

KR998476

 

This study

VT03282012-222-E

KR998477

 

This study

VT05252012-337G

KR998478

 

This study

VT12212012-1068

KR998479

 

This study

ADL-PM3205

HQ011267

  

MR104/05

DQ108607

  

MR102/05

DQ108605

  

MR60/01

AY127876

  

ST25/97

AF364885

  

SF47/99

AF364888

  

97/09/615/97

DQ785252

  

MR46/99

AF364896

  

HI/87

AF364893

  

N32/98

AF364883

  

H36/98

AF302799

  

T22/96

AF364889

  

SF48/99

AF364878

  

H07/92

AF364898

  

SK-05/90/05

FM203287

  

MR105/05

DQ108608

  

MR52/00

AF364892

  

N09/93

AF364895

  

N49/99

AF364876

  

CH01/08

EU851043

  

H56/00

AF364880

  

ST21/96

AF364886

  

SK-05/144/05

FM203274

  

MR103/05

DQ108606

  

N75/03

AY971661

  

SF18/96

AF364869

  

ST27/97

AF364897

  

ST26/97

AF364879

  

H97/04

DQ108604

  

NT92B/04

AY973188

  

200304B

FM203244

  

1490/98

AF391126

  

6/91

AF364894

  

SF70/02

AY127880

  

F72d/02

AY971657

  

F72b/02

AY971656

  

SF63/01

AY127879

  

SF57/00

AF364890

  

SF54/00

AF364884

  

MR62/01

AY127878

  

MR61/01

AY127877

  

MR71/02

AY127881

  

MR14/95

AF364873

  

ISAV10/95

DQ785254

  

N5/89

AY127882

  

NS2003/03

AY973182

  

HPR7a_CGA/220-5/14

KJ944288

  

96/09/768

DQ785249

  

H17/96

AF364891

  

VT03202012-194

 

KR998434

This study

VT03202012-195

 

KR998435

This study

VT03202012-196

 

KR998436

This study

VT03202012-199

 

KR998437

This study

VT03202012-200

 

KR998438

This study

VT03202012-202

 

KR998439

This study

VT03202012-204A

 

KR998440

This study

VT03202012-215

 

KR998441

This study

VT03202012-216

 

KR998442

This study

VT03202012-217

 

KR998443

This study

VT03202012-218

 

KR998444

This study

VT03202012-219

 

KR998445

This study

VT05012012-308

 

KR998424

This study

VT05252012-336H

 

KR998425

This study

VT05252012-338G

 

KR998446

This study

VT06042012-341G

 

KR998447

This study

VT06042012-341H

 

KR998448

This study

VT06042012-343H

 

KR998449

This study

VT06042012-345H

 

KR998450

This study

VT08092012-402

 

KR998451

This study

VT08092012-406

 

KR998452

This study

VT08092012-419

 

KR998453

This study

VT08092012-420

 

KR998454

This study

VT08092012-421

 

KR998455

This study

VT08092012-423

 

KR998456

This study

VT08092012-425

 

KR998457

This study

VT08092012-431

 

KR998426

This study

VT08092012-433

 

KR998427

This study

VT08092012-434

 

KR998458

This study

VT08092012-446

 

KR998428

This study

VT08092012-447

 

KR998459

This study

VT08092012-450

 

KR998460

This study

VT08092012-451

 

KR998461

This study

VT08092012-465

 

KR998462

This study

VT08092012-466

 

KR998463

This study

VT08092012-468

 

KR998464

This study

VT08092012-476

 

KR998465

This study

VT08092012-478

 

KR998466

This study

VT08092012-481

 

KR998467

This study

VT06202012-379

 

KR998468

This study

VT06202012-380

 

KR998469

This study

VT06202012-387

 

KR998470

This study

VT06202012-388

 

KR998429

This study

VT06202012-391

 

KR998471

This study

VT01222013-03

 

KR998430

This study

VT06062013-60

 

KR998472

This study

Vir22_96/09/724

 

DQ785286

 

97/09/393

 

DQ785284

 

97/09/615

 

DQ785280

 

94/09/579

 

DQ785285

 

SF83/04

 

AY744395

 

Svolvaer/96

 

AF262381

 

Bremnes/98

 

AF262385

 

Brekke/98

 

AF262380

 

Eikelandsosen/89

 

AF262386

 

810/9/99

 

DQ022085

 

95

 

DQ785282

 

901

 

GU830910

 

752

 

GU830902

 

390/98

 

DQ003602

 

485/9/97

 

DQ003605

 

U5575-1

 

DQ003603

 

04-085-1

 

DQ058660

 

01-0973-3

 

DQ003607

 

02-1179-4

 

DQ003601

 

00-0593-1

 

DQ003606

 

02-0775-14

 

DQ003604

 

98-049-1

 

DQ003600

 

RPC/NB-280-2

 

AF312317

 

AF109304

 

AF109304

 

RPC/NB-877

 

AF312316

 

7833-1

 

AF312315

 

Fundy/97

 

AF262389

 

AF404340

 

AF404340

 

NB458

 

AY151798

 

Phylogenetic analyses

Sequences were aligned and phylogenetic trees were generated using CLUSTAL X with the default settings [58]. 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.

Virus isolation

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 [59] and ASK-2 cells [39] were grown as previously described [12]. Homogenized tissues were inoculated on monolayers of SHK-1 and/or ASK-2 cell lines following standard protocols in the OIE Aquatic Manual [38]. 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 [60]. 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.

Ethics

The in vitro work was approved by the UPEI Biosafety Committee.

Declarations

Acknowledgements

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.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Pathology and Microbiology, Atlantic Veterinary College, University of Prince Edward Island
(2)
Department of Computer Science, University of Prince Edward Island
(3)
Raincoast Research Society
(4)
Department of Statistics and Actuarial Science, Simon Fraser University
(5)
Current address: Diagnostic Services Unit, Atlantic Veterinary College, University of Prince Edward Island

References

  1. McCauley J, Hongo S, Kaverin N, Kochs G, Lamb R, Matrosovich M, et al. Family Orthomyxoviridae. In: King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ, editors. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego, CA: Academic; 2011. p. 749–61.
  2. Adams S, Xing Z, Li J, Kristelle M, Perez D, Reed K, et al. The effect of avian influenza virus NS1 allele on virus replication and innate gene expression in avian cells. Mol Immunol. 2013;56:358–68.View ArticlePubMed
  3. Hause B, Collin E, Liu R, Huang B, Sheng Z, Lu W, et al. Characterization of a novel influenza virus strain in cattle and swine: proposal for a new genus in the Orthomyxoviridae family. MBio. 2014;5:1–14.View Article
  4. Anonymous. Fish Diseases; Emerging viruses. 2013 http://wfrc.usgs.gov/fieldstations/hq/emerging.html (Consulted on Oct 1, 2015).
  5. Granzow H, Fichtner D, Schütze H, Lenk M, Dresenkamp B, Nieper H, et al. Isolation and partial characterization of a novel virus from different carp species suffering gill necrosis – ultrastructure and morphogenesis. J Fish Dis. 2014;37:559–69.View ArticlePubMed
  6. McCann R Viral survey of fathead minnows, golden shiners, and white suckers from baitfish dealers in Wisconsin. Master’s Degree Thesis, 2012 College of Science and Health, University of Wisconsin, La Crosse, USA.
  7. Blake S, Bouchard D, Keleher W, Opitz M, Nicholson B. Genomic relationships of the North American isolate of infectious salmon anemia virus (ISAV) to the Norwegian strain of ISAV. Dis Aquat Organ. 1999;35:139–44.View ArticlePubMed
  8. Kibenge FSB, Kibenge MJ, McKenna PK, Stothard P, Marshall R, Cusack RR, et al. Antigenic variation among isolates of infectious salmon anaemia virus (ISAV) correlates with genetic variation of the viral haemagglutinin gene. J Gen Virol. 2001;82:2869–79.View ArticlePubMed
  9. Kibenge FSB, Godoy MG, Wang Y, Kibenge MJT, Gherardelli V, Mansilla S, et al. Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences. Virol J. 2009;6:88.View ArticlePubMedPubMed Central
  10. Kibenge FSB, Kibenge MJT, Wang Y, Qian B, Hariharan S, McGeachy S. Mapping of putative virulence motifs on infectious salmon anaemia virus surface glycoprotein genes. J Gen Virol. 2007;88:3100–11.View ArticlePubMed
  11. Nylund A, Plarre H, Karlsen M, Fridell F, Ottem KF, Bratland A, et al. Transmission of infectious salmon anaemia virus (ISAV) in farmed populations of Atlantic salmon (Salmo salar). Arch Virol. 2007;152:151–79.View ArticlePubMed
  12. Kibenge FSB, Kibenge MJT, Groman D, McGeachy S. In vivo correlates of infectious salmon anaemia virus pathogenesis in fish. J Gen Virol. 2006;87:2645–52.View ArticlePubMed
  13. Christiansen D, Østergaard PS, Snow MI, Dales OB, Falk K. A low-pathogenic variant of infectious salmon anemia virus (ISAV1 - HPR0) is highly prevalent and causes a non-clinical transient infection in farmed Atlantic salmon (Salmo salar L.) in the Faroe Islands. J Gen Virol. 2011;92:909–18.View ArticlePubMed
  14. Plarre H, Nylund A, Karlsen M, Brevik Ø, Sæther PA, Vike S. Evolution of infectious salmon anaemia virus (ISA virus). Arch Virol. 2012;157:2309–26.View ArticlePubMed
  15. González RR, Ruiz P, Llanos-Rivera A, Cruzat F, Silva J, Astuya A, et al. ISA virus outside the cage: Ichthyofauna and other possible reservoirs to be considered for marine biosafety management in the far-southern ecosystems of Chile. Aquaculture. 2011;318:37–42.View Article
  16. Marshall SH, Ramírez R, Labra A, Carmona M, Muñoz C. Bona fide evidence for natural vertical transmission of infectious salmon anemia virus in freshwater brood stocks of farmed Atlantic salmon (Salmo salar) in Southern Chile. J Virol. 2014;88:6012–8.View ArticlePubMedPubMed Central
  17. CFIA: ARCHIVED - 2012 – 2013 Status Update on the Surveillance of Wild and Enhanced Anadromous Salmonids in British Columbia. 2014 http://www.inspection.gc.ca/animals/aquatic-animals/diseases/reportable/isa/wild-anadromous-salmonids/eng/1410057185143/1410057186002.
  18. Sepúlveda D, Cárdenas C, Carmona M, Marshall SH. Novel strategy to evaluate infectious salmon anemia virus variants by high resolution melting. PLoS One. 2012;7:e37265.View ArticlePubMedPubMed Central
  19. Abdelwhab EM, Erfan AM, Grund C, Ziller M, Arafa A-S, Beer M, et al. Simultaneous detection and differentiation by multiplex real time RT-PCR of highly pathogenic avian influenza subtype H5N1 classic (clade 2.2.1 proper) and escape mutant (clade 2.2.1 variant) lineages in Egypt. Virol J. 2010;7:1–8.View Article
  20. Klungthong C, Chinnawirotpisan P, Hussem K, Phonpakobsin T, Manasatienkij W, Ajariyakhajorn C, et al. The impact of primer and probe-template mismatches on the sensitivity of pandemic influenza A/H1N1/2009 virus detection by real-time RT-PCR. J Clin Virol. 2010;48:91–5.View ArticlePubMed
  21. Kumar S, Henrickson KJ. Update on influenza diagnostics: lessons from the novel H1N1 influenza A pandemic. Clin Microbiol Rev. 2012;25:344–61.View ArticlePubMedPubMed Central
  22. Caraguel C, Stryhn H, Gagné N, Dohoo I, Hammell L. A modeling approach to predict the variation of repeatability and reproducibility of a RT-PCR assay for infectious salmon anaemia virus across infection prevalences and infection stages. Prev Vet Med. 2012;103:63–73.View ArticlePubMed
  23. Nérette P, Dohoo I, Hammell L, Gagné N, Barbash P, Maclean S. Estimation of the repeatability and reproducibility of three diagnostic tests for infectious salmon anaemia virus. J Fish Dis. 2005;28:101–10.View ArticlePubMed
  24. CFIA: Canada completes infectious salmon anaemia testing: No confirmed cases in BC salmon – December 02, 2011. http://www.inspection.gc.ca/english/corpaffr/newcom/2011/20111202e.shtml.
  25. DFO: Accepted Testing Methods for ISAV – November 2011. http://www.dfo-mpo.gc.ca/media/back-fiche/2011/20111108-eng.htm.
  26. Kibenge FSB, Munir K, Kibenge MJT, Joseph T, Moneke E. Infectious salmon anaemia virus: causative agent, pathogenesis and immunity. Anim Health Res Rev. 2004;5:65–78.View ArticlePubMed
  27. Rimstad E, Mjaaland S. Infectious salmon anaemia virus – an orthomyxovirus causing an emerging infection in Atlantic salmon. Apmis. 2002;110:273–82.View ArticlePubMed
  28. Mjaaland S, Hungnes O, Teig A, Dannevig BH, Thorud K, Rimstad E. Polymorphism in the Infectious Salmon Anemia Virus Hemagglutinin Gene: Importance and Possible Implications for Evolution and Ecology of Infectious Salmon Anemia Disease. Virology. 2002;304:379–91.View ArticlePubMed
  29. Lyngstad TM, Kristoffersen AB, Hjortaas MJ, Devold M, Aspehaug V, Larssen RB, et al. Low virulent infectious salmon anaemia virus (ISAV-HPR0) is prevalent and geographically structured in Norwegian salmon farming. Dis Aquat Organ. 2012;101:197–206.View ArticlePubMed
  30. Gagné N, Ritchie RJ. Development of a strand-specific RT-PCR assay for ISAV. Can Tech Rep Fish Aquat Sci. 2006;2675:1–32.
  31. Plarre H, Devold M, Snow M, Nylund A. Prevalence of infectious salmon anaemia virus (ISAV) in wild salmonids in Western Norway. Dis Aquat Organ. 2005;66:71–9.View ArticlePubMed
  32. Leland DS, Ginocchio CC. Role of cell culture for virus detection in the age of technology. Clin Microbiol Rev. 2007;20:49–78.View ArticlePubMedPubMed Central
  33. Knüsel R, Bergmann SM, Casey J, Segner H, Wahli T. Virus isolation vs RT-PCR: which method is more successful in detecting VHSV and IHNV in fish tissue sampled under field conditions? J Fish Dis. 2007;30:559–68.View ArticlePubMed
  34. Bergh O. The dual myths of the healthy wild fish and the unhealthy farmed fish. Dis Aquat Org. 2007;75:159–64.View ArticlePubMed
  35. Garibyan L, Avashia N. Polymerase Chain Reaction. J Invest Dermatol. 2013;133:e6.View ArticlePubMedPubMed Central
  36. Snow M, McKay P, McBeath AJ, Black J, Doig F, Kerr R, et al. Development, application and validation of a Taqman® real-time RT-PCR assay for the detection of infectious salmon anaemia virus (ISAV) in Atlantic salmon (Salmo salar). Dev Biol. 2006;126:133–45.
  37. Kibenge FSB, Kibenge MJT, Masaoud E. Infectious Salmon Anaemia Virus (ISAV) Ringtest: Validation of the ISAV Diagnostic Process using Virus-spiked Fish Tissues and ISAV TaqMan® Real-time RT-PCR. J Aquac Res and Devel. 2011;2:1–8.
  38. OIE: Manual of Diagnostic Tests for Aquatic Animals 2015, http://www.oie.int/international-standard-setting/aquatic-manual/access-online/
  39. Devold M, Krossøy B, Aspehaug V, Nylund A. Use of RT-PCR for diagnosis of infectious salmon anaemia virus (ISAV) I carrier sea trout Salmo trutta after experimental infection. Dis Aquat Org. 2000;40:9–18.View ArticlePubMed
  40. Casselman A: More salmon shenanigans in British Columbia. Nature News Blog, December 02, 2011 http://blogs.nature.com/news/2011/12/more_salmon_shenanigans_in_bri.html.
  41. Amos KH, Gustafson L, Warg J, Whaley J, Purcell M, Rolland J, et al. U.S. Response to a Report of Infectious Salmon Anemia Virus in Western North America. Fisheries. 2014;39:501–6.View Article
  42. Cohen Commission: Exhibits1549, 2040, 2042, 2043, 2044, 2045, 2051, 2052, 2053, 2060 https://web.archive.org/web/20131114212437/http://www.cohencommission.ca/en/Exhibits.php
  43. Bates D, Mächler M, Bolker BM, Walker, SC: Fitting Linear Mixed-Effects Models using lme4. https://cran.r-project.org/web/packages/lme4/vignettes/lmer.pdf.
  44. EFSA: European Food Safety Authority (EFSA) Panel on Animal Health and Welfare (AHAW): Scientific opinion on infectious salmon anaemia. EFSA Journal 2012, 10:2971. http://www.efsa.europa.eu/en/efsajournal/pub/2971.htm.
  45. Nylund A, Devold M, Plarre H, Isdal E, Aarseth M. Emergence and maintenance of infectious salmon anaemia virus (ISAV) in Europe: a new hypothesis. Dis Aquat Org. 2003;56:11–24.View ArticlePubMed
  46. Lyngstad TM, Jansen PA, Sindre H, Jonassen CM, Hjortaas MJ, Johnsen S, et al. Epidemiological investigation of infectious salmon anaemia (ISA) outbreaks in Norway 2003–2005. Prev Vet Med. 2008;84:213–27.View ArticlePubMed
  47. Rodger HD, Turnbull T, Muir F, Millar S, Richards RH. Infectious salmon anaemia (ISA) in the United Kingdom. B Eur Assoc Fish Pathol. 1998;18:115–6.
  48. Cottet L, Cortez-San Martin M, Tello M, Olivares E, Rivas-Aravena A, Vallejos E, et al. Bioinformatic analysis of the genome of infectious salmon anaemia virus associated with outbreaks with high mortality in Chile. J Virol. 2010;84:11916–28.View ArticlePubMedPubMed Central
  49. Cunningham CO, Gregory A, Black J, Simpson I, Raynard RS. A novel variant of infectious salmon anaemia virus (ISAV) haemagglutinin gene suggests mechanisms for virus diversity. Bull Eur Ass Fish Pathol. 2002;22:366–74.
  50. Castro-Nallar E, Martín MC-S, Mascayano C, Molina C, Crandall KA. Molecular Phylodynamics and protein modeling of infectious salmon anaemia virus (ISAV). BMC Evol Biol. 2011;11:349.View ArticlePubMedPubMed Central
  51. Workenhe ST, Kibenge MJT, Iwamoto T, Kibenge FSB. Absolute quantitation of infectious salmon anaemia virus using different real-time reverse transcription PCR chemistries. J Virol Methods. 2008;154:128–34.View ArticlePubMed
  52. Kibenge MJT, Iwamoto T, Wang Y, Morton A, Godoy MG, Kibenge FSB. Whole-genome analysis of piscine reovirus (PRV) shows PRV represents a new genus in family Reoviridae and its genome segment S1 sequences group it into two separate sub-genotypes. Virol J. 2013;10:230.View ArticlePubMedPubMed Central
  53. Kibenge FSB, Dybing JK, McKenna KP. Rapid procedure for large-scale isolation of plasmid DNA. Biotechniques. 1991;11:65–7.PubMed
  54. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403–10.View ArticlePubMed
  55. GenBank Database: http://www.ncbi.nlm.nih.gov/Genbank
  56. Stothard P. The Sequence Manipulation Suite: JavaScript Programs for Analyzing and Formatting Protein and DNA Sequences. Biotechniques. 2000;28:1102–4. http://www.ualberta.ca/~stothard/javascript/index.html.PubMed
  57. Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1998;85:2444–8.View Article
  58. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl Acids Res. 1997;25:4876–82.View ArticlePubMedPubMed Central
  59. Dannevig BH, Falk K, Namork E. Isolation of the causal virus of infectious salmon anemia (ISA) in a long-term cell line from Atlantic head kidney. J Gen Virol. 1995;76:1353–9.View ArticlePubMed
  60. Munir K, Kibenge FSB. Detection of infectious salmon anaemia virus by real-time RT-PCR. J Virol Methods. 2004;117:37–47.View ArticlePubMed
  61. Raynard RS, Murray AG, Gregory A. Infectious salmon anemia virus in wild fish from Scotland. Dis Aquat Organ. 2001;46:93–100.View ArticlePubMed
  62. Olivier G. Disease interactions between wild and cultured fish - Perspectives from the American Northeast (Atlantic Provinces). Bull Eur Assoc Fish Pathol. 2002;22:103–9.
  63. Mullins JE, Groman D, Wadowska D. Infectious salmon anaemia in salt water Atlantic salmon (Salmo salar L.) in New Brunswick, Canada. Bull Eur Assoc Fish Pathol. 1998;18:110–4.
  64. Raynard RS, Murray AG, Gregory A. Infectious salmon anaemia virus in wild fish from Scotland. Dis Aquat Org. 2001;46:93–100.View ArticlePubMed
  65. MacLean SA, Brown RW: ISA outbreaks and disease testing of Atlantic salmon in the United States and the West Greenland fishery. Work. Pap. 2002/20. Copenhagen: International Council for the Exploration of the Sea North Atlantic Salmon Working Group. 3 p.
  66. MacLean SA, Bouchard DA, Ellis SK. Survey of non-salmonid marine fishes for detection of infectious salmon anaemia virus and other salmonid pathogens. In: Miller O, Cipriano RC, editors. International response to infectious salmon anemia: prevention, control, and eradication: Symposium Proceedings. 3–4 September 2002, New Orleans, LA. Tech Bull. Washington, DC: US Dept Agriculture; 2003. p. 135–44.
  67. Bouchard DA, Brockway K, Giray C, Keleher W, Merrill PL. First report of infectious salmon anemia (ISA) in the United States. Bull Eur Assoc Fish Pathol. 2001;21:86–8.
  68. Thorud KE, Djupvik HO. Infectious salmon anaemia in Atlantic salmon (Salmo salar L). Bull Eur Assoc Fish Pathol. 1988;8:109–11.
  69. Cipriano RC. Antibody against infectious salmon anaemia virus among feral Atlantic salmon (Salmo salar). ICES J Mar Sci. 2009;66:865–70.View Article
  70. Skall HF: Detection of HPR0 in Denmark and criteria for diagnosis of ISA. In Report on the 15th Annual Meeting of the National Reference Laboratories for Fish Diseases. Copenhagen, Denmark. 2011:48–49.
  71. Godoy MG, Aedo A, Kibenge MJT, Groman DB, Yason CV, Grothusen H, et al. First detection, isolation and molecular characterization of infectious salmon anaemia virus associated with clinical disease in farmed Atlantic salmon (Salmo salar) in Chile. BMC Vet Res. 2008;4:1–13.View Article
  72. Anonymous: ISA hits the Faroes. Fish Farming Int 2000, 47.

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