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Frequently asked questions about Infectious Salmon Anemia virus (ISAv)

Frequently asked questions about Infectious Salmon Anemia Virus (ISAv)

Note: this list of questions will be updated as necessary to clarify new developments in ISAv research in the Pacific Northwest.  Newer questions will be added at the top.
[Answers provided by Todd Sandell, Ph.D., Disease Ecologist at the Wild Fish Conservancy]

Click the question below to go directly to the answer.

What is ISAv, how does it affect fish, and what are the risks?


Detecting ISAv


What is ISAv, how does it affect fish, and what are the risks?

Question: What is ISAv?
ISAv is an orthomyxovirus (related to influenza viruses) that is thought to have evolved in Atlantic salmon; the first incidence of disease was reported among salmon farms in Norway in 1984.  The virus is extremely infectious and can result in high mortalities (in excess of 70%).  The main route of infection is “horizontal” (fish-to-fish), though it is also suspected that ISAv can be passed “vertically” (through the eggs) because it was introduced into Chile, where no adult salmon were imported or previously existed.  ISAv may also infect herring, cod, Pollock, sablefish, and other marine species.  These species, which do not typically show signs of the infection, serve as a “reservoir” that makes eliminating the virus, once present, impossible because they can shed virus that will re-infect salmon.

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How is ISAv introduced, and how does it spread?
ISAv can be introduced by the shipment of infected salmon tissue and eggs; water containing the virus could also be infectious.  Since it emerged in virulent form from a native strain found in Norway, ISAv has been introduced by the salmon aquaculture industry into Ireland, Scotland, the Faroe Islands and Chile.  The detection of the European strain in eastern Canada is likely also due to the movement of infected fish or eggs, although it is possible that is was brought to eastern Canada by migrating Atlantic salmon.

The primary method of ISAv transmission is horizontal (fish-to-fish) because infected fish shed the virus in mucous, urine, feces and ovarian fluid.  Sea lice have been implicated as a vector for ISAv, and the wounds they make on a salmon’s skin may allow the virus to infect new hosts.  It also appears to spread vertically (from an infected female to her eggs), although it is unclear if it is simply found on the outside of eggs or whether it manages to infect the young, unhatched offspring.

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Was ISAv introduced to the Pacific Northwest by the salmon aquaculture industry?
At present we do not have enough information to determine if the recent detections of ISAv are from European strains, which would suggest introduction by the salmon aquaculture industry.   However, once the recently detected virus is sequenced, we will be able to identify its origin.  It may be that a strain of ISAv has been present in the Pacific Northwest for some time, or even be native.

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If ISAv is a native virus, why should we be worried about it?

Even if the strain of ISAv found in the Pacific Northwest is native there is still cause for concern.  In locations where intensive Atlantic salmon aquaculture is found, highly virulent strains of ISAv have developed (see Can ISAv evolve and what are the implications?).   In the Pacific Northwest, ISAv could develop into a virulent form, likely first in Atlantic salmon in aquaculture facilities, and then could spread to native Pacific salmon stocks - potentially from California to the Bering Sea, jeopardizing populations that are already endangered or in decline.  The salmon in the net pens would serve as a continuous source of viral particles, creating a long-term exposure to ISAv for native salmonids (salmon and trout) and other marine fishes.  Crowded conditions such as those found in aquaculture facilities would allow the virus to mutate and become infectious for new species.

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Is there more than one type of ISAv?
There are several “strains” (variants) of ISAv.  These have previously fallen into two groups, based on their genetic similarity: one in the North Atlantic (Europe) and the other on the Atlantic coast of North America (although the European strain has also been found in eastern Canada).  The virus was also introduced into Chile, where there are no native salmon, on several different occasions by the salmon aquaculture industry.  The introduction of a Norwegian strain in 2007 led to disastrous losses in Chilean aquaculture, and killed both net pen Atlantic and coho salmon.  Some of those fish had recently been infected by another pathogen (Piscirickettsia, a bacterium), and both may have played a role in the high mortalities observed.

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How do the strains of ISAv differ?
The original strain of ISAv is thought to have a full-length “highly polymorphic region” (HPR), which is a gene segment associated with virulence; this strain (HPR0) is not commonly associated with disease.  This strain was naturally occurring in the North Atlantic and only resulted in disease after Atlantic salmon net pens were established on the Norwegian coast.  Other strains with higher virulence (the ability to spread and cause disease, “pathogenic”) have mutations in this region that result in shorter HPR gene segments- these are usually referred to as HPR1, HPR2b, etc.  Like many RNA viruses, ISAv mutates very quickly and mutations in other regions may also be important.

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Can ISAv evolve and what are the implications?
One of the key features of ISAv is its ability to mutate rapidly and increase its virulence; there is evidence that this has happened independently on several occasions in association with fish farming around the world.  We now know that mutation events that lead to a shortening of the highly polymorphic region (HPR) can result in increased pathogenicity.   We also have evidence that ISAv can evolve virulence for Pacific salmon species (in the 2007 Chilean outbreak, farmed coho salmon were also infected and killed by ISAv; however, other infections may have contributed to the high mortality).  Since all salmonids (salmon and trout) are relatively closely related genetically, it is possible that a chronic exposure to ISAv in intensive culture conditions would eventually lead to the emergence of a more virulent form of the virus that could infect native Pacific salmon species.

The following quote summarizes the situation (from Christiansen et al., 2011, Journal of General Virology, volume 92): “Arguably the most important question for fish-health managers regards the risk that the presence of low pathogenic ISAv-HPR0 presents for the development of highly pathogenic ISAv and ISA disease. ISAv-HPR0 has been proposed to be the ancestor of highly pathogenic ISAv that is capable of causing disease in Atlantic salmon farms, and probably undergoes an adaptation event in association with intensive aquaculture…The virus is thus well equipped to adapt to the highly selective environment associated with aquaculture, which includes evolutionary pressures such as high host abundance and continuous availability, high rearing densities and exposure of naive hosts to new pathogens. Such factors are known to drive the faster evolution of other fish RNA viruses within aquaculture, such as viral hemorrhagic septicemia virus (VHSV).  In this case, adaptation of the virus to an alternate ‘pathogenic’ lifestyle has occurred several times within aquaculture” (see original for citations).

The only way to remove this threat is to require salmon aquaculture facilities to be moved onto land, where escapees can be prevented and the effluent from the land-based farms can be sterilized and monitored to prevent the release of pathogens.

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I thought ISAv wasn’t dangerous for Pacific salmon?
A widely cited paper entitled “Relative resistance of Pacific salmon to ISAv”(Rolland and Winton, 2003, Journal of Fish Diseases, volume 26, pages 511-520) indicated that Chinook, coho, chum and steelhead experimentally infected with two virulent strains of ISAv did not develop disease; these two strains killed Atlantic salmon in the same experiment.  It is important to note that sockeye salmon were not included in the study, so we have no information about their susceptibility to infection or disease caused by ISAv.  While this is good news for Pacific salmon, it does not mean they are not at risk.  After roughly 2 weeks, ISAv was still recovered from the Pacific salmon species, indicating that the virus was able to replicate in these fish, although it did not cause disease.  The ability to infect a new species is the first hurdle that a pathogen needs to overcome to become virulent in that species, and that step was readily accomplished by ISAv.  The authors went on to conclude: “The results indicate that while Oncorhynchus spp.[Pacific salmon] are quite resistant to ISAv relative to Atlantic salmon, the potential for ISAv to adapt to Oncorhynchus spp. should not be ignored.”

It is also worth noting that laboratory studies on the effect of infections, while highly valuable, do not tell the whole story.  The fish used in such “controlled” studies are usually pathogen-free at the outset so that the effects of one particular infection can be examined.  However, in natural systems it is expected that any given salmon will have multiple concurrent infections from naturally occurring, native parasites and other pathogens (whether viral or bacterial).  The presence of multiple infections may have additive or synergistic effects on the health of the fish- in effect, the fish’s immune system is fighting off several invaders at once, depleting its energy stores and potentially leading to disease.  In addition, salmon arising from areas with human impacts on the environment (which includes most of Washington, Oregon and California) - pollutants, chemicals that affect hormone balance, or other issues with water quality – are also impaired by these exposures.  The net effect is that a fish may be resistant to a low-pathogenic strain of ISAv in the lab, but in the wild, the presence of other pathogens and/or water quality issues may allow that strain to lead to disease and kill the host.

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Can ISAv have an impact on the food chain?  Does it affect human health?
Because the virus evolved in cold water fishes, it is inactivated by warmer body temperatures like those found in birds and mammals.  It is not thought to bioaccumulate in the food chain, and it does not infect humans.

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Detecting ISAv

How is ISAv detected?

There are 3 common tests (“assays”) for ISAv:

  1. The classic test is cell culture; salmon cells are grown in the laboratory and tissues from infected fish are ground up and added to the cell culture.  If pathogenic (disease causing) viruses are present, they will infect and kill the cells, resulting in “plaques” or cytopathic effects (CPE)- areas where the cells have died.  This indicates that a virus is present.  Once CPE are detected (under a microscope), other assays are used to figure out what type of virus is infecting the cells.  Cell culture can yield results slowly and, if the virus present is not pathogenic or the cell type used is not “permissive” (does not support viral infection by that particular strain of virus), no CPE will be observed (a “false negative” result).
  2. A second test uses antibodies to detect viral particles (for ISAv, the test is called “indirect fluorescent antibody test,” IFAT).  IFAT tests have problems with low sensitivity and/or reacting to other things besides ISAv (a “false positive”) - sometimes the antibodies are not specific for ISAv alone, creating problems.  For these reasons IFAT is not widely used to detect ISAv.
  3. The third and most recent assay type developed relies on molecular biology: the polymerase chain reaction (PCR) test.  PCR assays detect DNA (because the ISAv genome is encoded as RNA, the first step is to convert this to DNA; this additional step, called “reverse transcription”, means the assay has the acronym RT-PCR).  PCR tests are extremely sensitive and, in theory, do not generate false positive results.  Standard PCR tests yield a positive or negative result; either the virus is present or it is not.  Another type of PCR, “quantitative” PCR (qPCR, or qRT-PCR), yields an estimate of the number of viral genomes present in the sample.  If the virus is present in large quantities, this assay detects it in fewer steps – so a low number indicates a highly infected sample.  A problem with all PCR assays is contamination because the tests are very sensitive.  Because of this drawback, samples positive by PCR are typically tested multiple times, and the product should also be sequenced to make sure that the “target” (viral) DNA was present.  Often, cell culture is also used to confirm a PCR positive sample, but studies published in 2001, 2004 and 2009 show that there are virulent strains of ISAv that do not grow in culture, indicating that this method doesn’t always work for ISAv.

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What is the best assay to detect ISAv?
The best type of assay, in terms of speed and sensitivity, is the RT-PCR assay.  However, there is a great deal of confusion about which specific PCR assay is best – ISAv has 8 genome segments, and various researchers have developed different RT-PCR assays, some even for the same segment of the genome.  Until very recently it was commonly accepted that RT-PCRs aimed at segment 8 would detect all strains of ISAv, but some of the recently detected Fraser River fish samples were positive only by segment 7 RT-PCR.  The selection of the correct RT-PCR assay is critical; if the wrong test is run, ISAv will not be detected even if it is present in great abundance - a false negative result.  PCR results should be confirmed by another assay and/or by sequencing the PCR product to make sure it matches the ISAv sequence.

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When was ISAv first detected in the Pacific Northwest?
The recent detections, in juvenile sockeye from the West Coast of Vancouver Island, B.C., were reported on October 15, 2011.  Within 10 days, the virus had also been detected in adult Chinook, coho and chum salmon from the Fraser River.  Both of these results were reported by the laboratory of Dr. Fred Kibenge, the OIE (World Organization for Animal Health) reference lab for ISAv in the western hemisphere.  Shortly thereafter, these results were confirmed by Dr. Are Nylund, the head of the OIE reference lab for ISAv in the eastern hemisphere (Norway).  The result was a weak positive that was not reproducible in subsequent tests, and only the segment 7 RT-PCR was positive.  The Canadian government then took the samples from the Kibenge lab and reran them in an attempt to confirm these results, and could not do so.  However, they specifically cited that the samples were in poor condition, which may have affected their results, and they have not yet explained which tests they ran; if they chose the wrong assay, they would not have detected the virus even if it were present.

On November 28, 2011, a manuscript dating from 2004 on ISAv detection in the Pacific Northwest was leaked to the press.  The work was performed by Molly Kibenge (at the time a postdoctoral student at the Pacific Biological Station in Nanaimo) but was never submitted for publication because of the objections of her supervisor, who works for the Department of Fisheries and Oceans, Canada (DFO).  The paper describes the detection of ISAv in Pacific salmon species (as well as some farmed Atlantic salmon) by RT-PCR from the Bering Sea south to the Fraser River and the west coast of Vancouver Island.  The infected fish were caught at sea and did not have overt signs of infection or disease (“asymptomatic”), suggesting a less virulent strain of virus was present.  Kibenge used RT-PCR assays aimed at both segment 7 and 8 of the ISAv genome and also sequenced the gene segments that were amplified to confirm ISAv.  Importantly, she also sequenced her positive controls (which are run to make sure that the assay is working correctly), which had different sequences than the virus that was detected in the salmon sampled.  This makes the work compelling, even though she could not get the virus to infect tissue culture cells.  Although this manuscript was not entirely complete, it should have provoked enough interest to be shared with the broader community and pursued further.

On December 15, 2011, during the reconvened Cohen Commission hearings, Dr. Kristi Miller, head of molecular genetics at the federal Pacific Biological Station in Nanaimo, provided testimony that suggested ISAv has been present in BC salmon as far back as 1986

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Canadian Food Inspection Agency and other agencies have done sampling/testing to try to find ISAv. Why haven't they been able to find a case? Are they doing different tests than the laboratory of Dr. Fred Kibenge at the Atlantic Veterinary College? Why are there conflicting laboratory results?
At present the B.C. government/Canadian Food Inspection Agency (CFIA)/Department of Fisheries and Oceans (DFO) have not released the methods that they are employing to look for ISAv in the samples that were reported positive.  Without this information we cannot interpret their results.  Insistence on finding the virus in culture is not scientifically valid; we have known since 2001 that there are strains of ISAv that do not grow well or at all in tissue culture, including some virulent strains (in fact, this finding was first reported in the scientific literature in 1999, and has since been confirmed several times).  If the Canadian agencies are using RT-PCR to test for ISAv (see “How is ISAv detected?”), we need to know which specific assay is being used and what gene segment it targets.  If they are not using a segment 7 assay for detection, or the primers used by M. Kibenge in the unpublished 2004 manuscript, they may be testing samples that contain ISAv and they would not detect it (false negatives). [see also “What is the best assay to detect ISAv?”]

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What are the OIE regulations for reporting ISAv?
[from http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/2.3.05_ISA%20.pdf]
7.2. Definition of confirmed case
The following criteria in i) should be met for confirmation of ISA. The criteria given in ii) and iii) should be met for the confirmation of ISAv infection.
i) Mortality, clinical signs and pathological changes consistent with ISA (Section 4.2), and detection of ISAv in tissue preparations by means of specific antibodies against ISAv (IFAT on tissue imprints [Section 4.3.1.1.2] or fixed sections as described in Section 4.3.1.1.3) in addition to either:
a) isolation and identification of ISAv in cell culture from at least one sample from any fish on the farm, as described in Section 4.3.1.2.1
or
b) detection of ISAv by RT-PCR by the methods described in Section 4.3.1.2.3;
ii) Isolation and identification of ISAv in cell culture from at least two independent samples (targeted or routine) from any fish on the farm tested on separate occasions as described in Section 4.3.1.2.1;
iii) Isolation and identification of ISAv in cell culture from at least one sample from any fish on the farm with corroborating evidence of ISAv in tissue preparations using either RT-PCR (Section 4.3.1.2.3) or IFAT (Sections 4.3.1.1.2 and 4.3.1.1.3).

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