Infectious Salmon Anemia Virus

A small project about ISAVirus brought to you by a group of Year 2 MBIO students from Ngee Ann Polytechnic.

Click on the pastel orange box on the left for the names of the students involved in this project.

Thank you.

Introduction

Thursday, 26 January 2012 @ 12:51

Infectious Salmon Anemia Virus (ISAV) is a piscine orthomyxovirus. This virus causes high mortalities in aqua life worldwide. Its structure is an enveloped virus with a genome consisting of eight negative sense single-stranded RNA segments encoding at least 10 proteins. The ISAV has small projections on its surface that is similar to a mushroom-shape.

ISAV contains 4 major polypeptides of putative viral origins: estimated molecular sizes of 71, 53, 43, 24 kDa. The diameter of virion can be in a range of 130nm to 140nm(slightly larger than influenza viruses). ISAV shows higher functional similarity to bovine or porcine torovirus and bovine coronavirus HE proteins and influenza C virus HE fusion protein. ISAV can reach maximum replication at 15˚C(slightly lower than influenza viruses).

Trypsin, which are commonly found in cell culture medium enhances viral replication. Viral replication occurs in the nucleus of host cell(salmon). The replication cycle of ISAV is similar to the replication cycle of the Influenza A virus. Structural, morphological, physiochemical properties have determined where it belongs in the viral classification: Orthomyxoviridae.

Life Cycle of ISAV

The virus enters the host cell(salmon) by binding its hemagglutinin to the sialic acid found on glycolipids or glycoproteins on the surface of the host cell.
The host cell then endocytoses the virus.
Due to the low pH environment in the endosome, the virus mutates and fuses its small projections(envelope) with the endosomal membrane.
A signal is received by the endosome to release the viral nucleoclaspid into the cytoplasm of the host cell.
The viral nucleoclaspid will then proceed to the nucleus of the host cell where the replication occurs.
In the nucleus of the host cell, the viral nucleoclaspid will go through transcription to prepare itself with necessary proteins needed for replication.
The primary transcription involves “cap-snatching”.
The viral endonuclease(PB2) “steals” the 5’methyguanosine cap and around 10 to 13 nucleotides from the host cell mRNA.
This is used as the primer for the transcription of the PB1 protein(viral transcriptase).
The different types of proteins produced that are necessary for its life cycle can be found in the Key Protein segment(e.g Hemagglutinin, neuraminidase)
Once the initial proteins for the virus are produced, the RNA strands will then be produced. Since ISAV is more similar to the Influenza C virus, it produces 7 complementary positive sense RNA strands from 8 complementary negative sense RNA segments.
These positive sense RNA or cRNA strands lack of 5’capped primer and the 3’poly A tail found in the mRNA(The strands were incomplete). Once the strands are completed, it becomes negative sense RNA strands.
A variety of proteins will then help the negative sense RNA strands to exit the nucleus and finally into the cytoplasm.
Hemagglutinin and neuraminidase(key proteins) have already gone through glycosylation, polymerization, acylation. These proteins, as well as matrix protein 2(M2) will travel together heading towards the plasma membrane.
Together, these proteins, the eight negative mRNA strands plus a matrix protein 1(M1) will start the budding process at the plasma membrane of the host cell.
Once the virus buds, the neuraminidase destroys the sialic acid receptors on the plasma membrane and the virus leaves the host cell, which will eventually die.

Key Proteins

A 38-43 kDa Hemagglutinin erastase(HE)(a surface glycoprotein)- Responsible for viral attachment and release(receptor binding and receptor destroying enzyme activities(RDE activity)(performed by neuraminidase))
Major surface proteins: a 50kDa(a protein encoded by gene segment 5). Functions: responsible for fusion of viral and cellular membranes
Tyrosine protein kina FRK: stimulation and suppression of cell proliferation
D-type cyclins: coupling of extracellular cues with the regulation of cell cycle, govern entrance and progression through G1 phase via phosphorylation of antiproliferation retinoblastoma protein(protein is inactivated to allow for cell proliferation)
Histone deacetylase-1 and high mobility group protein 2(hmgb2): Biosynthesis of nucleotides and regulation of transcription at different levels.
Enzyme inhibition studies indicate that the acetylesterase of the ISAV HE contains a serine in its active site.
The acetylesterase responsible for the RDE activity allows the virus to detach (elute) from these erythrocytes.
The ISAV protein with highest sequence variation is the HE containing a highly polymorphic region (HPR) in the stalk of the protein, near the transmembrane domain.
HPR0(no deletions)-> Found in healthy wild atlantic salmon fishes
HPR(has differential deletions)->Found in infected atlantic salmon fishes
Therefore it is concluded that variations in the length of the HPR may affect the structure of the molecule(whether it is infectious or not).

Detection of ISAV

Infectious Salmon Anemia Virus (ISAV) can be quantitatively detected by using a one-tube real-time reverse transcription–polymerase chain reaction (RT–PCR) that utilizes SYBR Green. Primers targeted at ISAV RNA segment 8 is used and ISAV isolate U5575-1 is utilized as a template.

The process includes one cycle of reverse transcription, 50 cycles of cDNA amplification as well as data analysis took only 80 minutes. The melting curve and gel electrophoresis analysis of real-time RT–PCR showed harmony with each other as a virus-specific single melting peak and a product of the expected size of 211 bp were obtained.

Materials & Methods:

· RNA extraction

-Fish tissues are obtained to extract total RNA by macerating and homogenizing the sample.
- 1:1 dilution in sterile PBS is then carried out.
- centrifugation at 3000rpm for 15 minutes and supernatant is used for RNA extraction.
-TRIZOL reagent is used to purify virus

· Virus purification

-Infectious Salmon Anemia Virus undergoes centrifugation at 4430 rpm for 30 minutes at 4˚C.
-Ammonium sulphate added to concentrate the viral supernatant
-RNA from purified virus extracted using TRIZOL reagent

· Preparation of ISAV RNA standards

-viral RNA diluted 10 times in RNase-free PCR grade water
-Sample quantitated using spectrophotometry

Table 1. Optimized reagent concentrations in 20 μl reaction volume for ISAV SYBR Green real-time RT–PCR

Reagent	Concentration
5× RT–PCR reaction mix SYBR Green Ia	4.0 μl
5× resolution solutiona	3.0 μl
MgCl2a	5.0 mM
Forward primer-F5 (5′-GAA GAG TCA GGA TGC CAA GAC G-3′)b	0.3 μM
Reverse primer-R5 (5′-GAA GTC GAT GAT CTG CAG CGA-3′)b	0.3 μM
RT–PCR enzyme mixa	0.4 μl
Viral RNA template	≥0.006 ng in 1.0 μl
Sterile PCR-grade water	9.4 μl

a: Reagents are part of the RNA Amplification Kit SYBR Green I (Roche Diagnostics GmbH).
b: Primers have been previously used in ISAV RT–PCR assays ( [Devold et al., 2000] and [Kibenge et al., 2000]).

Table 2. Thermal profile for ISAV SYBR Green real-time RT–PCR

Step	Temperature (°C)	Time (s)
1 cycle of reverse transcription	55	1800
Pre-denaturation	95	30
50 cycles of
Denaturation	95	5
Annealing	59	10
Elongation	72	10
Fluorescence acquisition	80	2

Melting curve was performed from 70 to 95 °C in 0.1 °C/s increments.

The tissue samples from fish that survived an experimental ISAV infection were tested to demonstrate the use of SYBR Green real-time RT–PCR assay for fish tissues with different amounts of ISAV RNA.
Both conventional one-tube RT–PCR and SYBR Green-based real-time RT–PCR assays were compared on these samples to determine which of these assays work best in detecting ISAV RNA.

The real-time RT–PCR assay detected ISAV RNA in more tissues that the conventional one-tube RT–PCR assay.This is expected of the result as real-time RT PCR shows that it is 100 times more sensitive than the conventional one-tube RT-PCR. Real-time RT-PCR assay also proves that it is able to obtain a relative amount of viral RNA in the sample from the Ct values. Since all the fish tested were survivors of a lethal experimental ISAV challenge, the results demonstrate that the real-time RT–PCR assay would be useful in detecting subclinical ISAV infections in fish.

Table 3. Detection of ISAV RNA segment 8 by conventional RT–PCR and SYBR Green real-time RT–PCR in different tissues of fish at 76 days post-ISAV challenge

Method	Fish species	Fish tissues
		Liver	Spleen	Kidney	Heart	Caeca	Gill
Conventional RT–PCR	Atlantic salmon	+a	−b	+	+	−	+
	Rainbow trout	+	+	−	−	+	−
Real-time RT–PCR	Atlantic salmon	+ (36.44,c0.0022d)	+ (37.92, 0.001)	+ (37.71, 0.001)	+ (35.61, 0.0036)	+ (35.99, 0.0029)	+ (39.71, NCe)
	Rainbow trout	+ (35.75, 0.0033)	+ (37.98, 0.001)	−	+ (36.61, 0.0020)	+ (36.43, 0.0022)	+ (38.04, NC)

a: + Denotes positive by conventional one-tube RT–PCR or real-time RT–PCR detection format.
b: − Denotes negative by either agarose gel electrophoresis for conventional one-tube RT–PCR or real-time RT–PCR detection format.
c: Ct value.
d: ISAV RNA concentration (ng/μl) extrapolated from the standard curve
e: NC denotes not calculated

Treatment for ISAV

Infectious Salmon Anemia Virus(ISAV) can be inactivated by using a variety of disinfectants that includes sodium hypochlorite, chloramines-T, chlorine dioxide, iodophors, sodium hydroxide, formic acid, formaldehyde and potassium peroxymonosulfate.

The virus is vulnerable towards ozonated seawater, temperatures above 55°C (131°F) for more than 5 minutes or highly acidic or basic pH (e.g., pH 4 or pH 12 for 24 hours), and also ultraviolet irradiation.

Vaccines for ISAV

Oral vaccination against Infectious Salmon Anemia Virus.

Using ISAV-derived antigens expressed in yeast cells that will then be encapsulated and consumed by the fish through its fish feed. Fish fed with these encapsulated ISAV vaccine has shown an increase in Anti-ISAV antibody (IgM) titers.

Vaccine though injection in freshwater

Vaccine through injection may induce stress-related effects on fish, such as immunosuppression, reduction in feeding rate and mortality.

Distribution

ISAV outbreaks occur in Norway, Canada, Scotland, Chile, as well as in a limited region of North America shared by the US and Canada. In North America, it only affects the small areas of the Bay of Fundy in New Brunswick, the Cobscook Bay area of Maine, and Passamaquoddy Bay, which is shared by Maine and New Brunswick.

ISAV replicates in Atlantic salmon, which forming a marine reservoir for the virus. Their migration allows distant distribution of the disease to other regions.

The virus can survive in seawater and it is mainly transmitted via the water, virus being shed into it in mucus, faeces, urine, gonadal fluids. It then enters vulnerable fish via the gills or broken skin. It can also be transmitted via crustacean vector.

Transmission of the virus can be done by contact with infected fish or their secretions. Contact with equipment or people who have handled infected fish can also transmit the virus.

ISAV replicates best at the cold temperatures where salmon thrive (5-15°C). The optimal growth temperature for the virus is 10-15°C and it does not replicate in 25°C or higher.

The virus also replicates in other fish species, e.g. rainbow trout, Atlantic cod, chum, coho and Chinook salmon, Arctic Char, but no disease has been observed in these species under aquaculture conditions. However, these species may serve as reservoir hosts for the virus.

Symptoms

Unlike mammals, red blood cells of fish have DNA and causes them to be infected with viruses. ISAV causes severe anemia, resulting pale gills of infected fish. They may swim close to the water surface, gulping for air. Fish may develop a “pop-eye” appearance (exopthalmos). Other symptoms may include lethargy, leukopenia, ascites, darkened skin and increased mortality. Hemorrhages may also be found in the anterior chamber of eye. In Chile, Jaundice on the ventral portion of the body has been reported among Atlantic salmon.

The disease can also develop without the fish showing any external signs of illness. The fish maintain a normal appetite and then they suddenly die. The disease can progress slowly throughout an infected farm and the death rates may approach 100%.

Post-mortem examination of the fish has shown a wide range of causes of death. The gills are pale and the skin can the darkened and Exophthalmia may also be seen. Yellow- or blood-tinged fluid may be found in the peritoneal and pericardial cavities. Petechiae, which may be extensive, can usually be found on various organs and tissues, including the eye, the internal organs, the visceral fat and the skeletal muscles.

The liver and spleen may be swollen, congested, necrosed or partially dead. In some cases, the spleen may turned dark brown or even black in colour, and it may be covered with a layer of fibrin. The kidney may also be swollen and dark, blood and liquid may exuded from the cut surface. The gastrointestinal tract may also be congested, but blood is not usually found in the intestinal lumen if the carcass is fresh.

The circulatory system may stop working, and the blood may be contaminated with lots of dead blood cells. Red blood cells which are still present in the circulatory system often burst easily and the numbers of immature and damaged blood cells increased.

Control Strategy

In the case of an outbreak of ISAV,
there would be several options to carry out at once, which are:

a) Destruction of the diseased fish
- Removing and disposing of the infected fish. This is of considerably high priority so as to keep the spreading and causing agents of the disease under control.

b) Quarantine
- Setting restrictions on the movement of fishes, materials, personnel and various equipments involved. This would thus require the affected area to be identified and disinfection of the personnel, equipments and discharged waste to be done.
c) Treatment of the affected fish population

Approaches to preventing spread and elimination of the pathogens:

Quarantine & movement controls

When implemented, practices to be considered includes

- live salmon transportation between and within freshwater and marine operations

- salmon harvesting and transportation to processing plants

- the discharge of salmon processing-plant wastes

- transportation of ready salmon consumer products

- the disposal of dead salmon

Establishing of quarantine areas

- Infected premises (where ISAV infection were identified)

- Restricted areas (in the vicinity of infected premises)

- Control area (the buffer between restricted premises and free areas)

- Free areas (areas not affected by the disease or unassessed yet)

After the quarantine areas are declared, factors to be accounted are the freshwater

and marine phases of salmon production, movement of infected wild populations of rainbow trout and brown trout, the possibility of the existence or presence of an unknown susceptible species of wild fish that could be a carrier.

Movement controls include

- banning movement of live salmon out of affected areas

- banning movement of live salmon into areas free of the disease

- bans on releasing salmon into rivers or marine locations

- bans on movement of salmon between different river systems, between marine farm locations and freshwater farm locations

- bans on usage or movement of equipment between river systems and marine farms

Tracing of the disease

This is crucial in determining locations and causative agents of the disease and information derived will help to select the most appropriate response action to take. The following should be traced

- salmonids such as broodstock, smolts (a young salmon in the stage of its first migration to the sea), etc

- salmon products for human consumption

- water (inputs and outputs)

- vehicles, materials and personnel involved when the outbreak of disease occurred

Surveillance is a must to find out the extent of the infection, detect new outbreaks, and also to monitor the progress and the successfulness of the strategy employed in eradicating the disease after which decontamination will then take place.

Disposal of diseased salmon

The fishes must be disposed of safely and effectively. The best practice is to bury slaughtered animals on site but this is definitely not possible for diseased fish. Therefore, culled fish have to be transported to the site of burial or incineration safely without spreading the disease to other sites.

⚠ Public awareness

People should be informed that:

humans will not be infected by ISAV
consuming fishes with exposure to ISAV will not impose any health risks
salmon or fishes that have died from the disease mustn’t be used as bait or made into feed for other aquatic animals

Diagnosis

Listed below are signs the diagnosis of ISAV is based on initially, before further isolation of the causative agent.

Clinical signs:

pale gills (sign of anaemia)

exophthalmia (protusion of eyeballs)

enlarged abdomen

the presence of skin haemorrhages (discharge of blood), especially at the abdomen

scale pocket oedema (swellings due to accumulation of fluid)

blood present in eye chamber

Behavioural changes to affected salmon

may appear lethargic and keep close to walls/ sides of the pen

loss of appetite

found gasping at surface of the water

Gross Pathology

petechiae (pinpoint haemorrhages) in the skeletal muscles

oedema of the swim bladder (swelling)

accumulation of fluid in body cavity, could be bloody/ yellowish

ascites (enlarged abdomen due to fluids in peritoneal cavities)

swollen, dark red liver with possible thin fibrin layer

swollen, edges rounded, dark red spleen

congested, dark red kidney, with blood& liquid exuding out from cuts

dark redness in the mucosa of the intestinal walls, with absence of blood in fresher specimens of the diseased fish

anaemia (deficiency of red blood cells)

References:

For Introduction, Life Cycle & Key Proteins:
Berit Lyng Schiøtz, Sven Martin Jørgensen, Caird Rexroad, Tor Gjøen, Aleksei Krasnov, June 2008. Transcriptomic analysis of responses to infectious salmon anemia virus infection in macrophage-like cells, Virus Research 136, pp. 65–74. Available through: Science Direct[Accessed on 22/01/2012]

Esther Garc´ıa-Rosado, Turhan Markussen, Øyvind Kileng, Espen S. Baekkevold, Børre Robertsen, Siri Mjaaland, Espen Rimstad, March 2008. Molecular and functional characterization of two infectious salmon anaemia virus (ISAV) proteins with type I interferon antagonizing activity, Virus Research 133, pp. 228–238. Available through: Science Direct[Accessed on: 14/01/2012]

Anita Müller, Turhan Markussen, Finn Drabløs, Tor Gjøen, Trond Ø. Jørgensen, Stein Tore Solem, Siri Mjaaland, April 2010. Structural and functional analysis of the hemagglutinin-esterase of infectious salmon anaemia virus, Virus Research 151, pp. 131–141. Available through Science Direct[ Accessed on 10/01/2012]

Knut Falk, Ellen Namork, Espen Rimstad, Siri Mjaaland, Birgit H. Dannevig, December 1997. Characterization of Infectious Salmon Anemia Virus, an Orthomyxo-Like Virus Isolated from Atlantic Salmon (Salmo salar L.), Journal of Virology, vol.71[12], pp. 9016-9023. Available through: NCBI[Accessed on 10/01/2012]

College of Veterinary Medicine Iowa State University, March 2010. Infectious Salmon Anemia(Hemorrhagic Kidney Syndrome). [Accessed on 22/01/2012]

Rahul Hate(Human Biology 115A), August 1999. Replication: Influenza Replication in Greater Detail. Available through: http://www.stanford.edu/group/virus/1999/rahul23/orthomyxoviridae.html [Accessed on 14/01/2012]

Picture for Life Cycle of Virus segment: From Robert A. Lamb and Robert M. Krug’s “Orthomyxoviridae: The Viruses and Their Replication,”in Fields Virology, Lippincott-Raven Publishers. 1996. P 1370. http://www.stanford.edu/group/virus/1999/rahul23/replication.html[Accessed on 14/01/2012]

Picture for Introduction segment: Swiss Institute of Bioinformatics, ViralZone 2009. Isavirus: Molecular Biology, Virion. Available through: http://viralzone.expasy.org/all_by_species/95.html[ Accessed on 22/01/2012]

Cartoon of ISAV: Gery Hummel, November 2011. Cartoon: ISAvirus Salmon. Available through: http://thecanadian.org/k2/item/1140-cartoon-isav-salmon[ Accessed on 22/01/2012]

Picture of Infected Salmon: Department of Inland Fisheries and Wildlife State of Maine, Fish Health Laboratory, November 2002. Infectious Salmon Anemia, volume 3[Issue 3]. Available through: http://www.maine.gov/ifw/fishing/health/vol3issue3.htm[Accessed on 22/01/2012]

Picture of Electron Microscopy of ISAV: Department of Inland Fisheries and Wildlife State of Maine, Fish Health Laboratory, November 2002. Infectious Salmon Anemia, volume 3[Issue 3]. Available through: http://www.maine.gov/ifw/fishing/health/vol3issue3.htm[Accessed on 22/01/2012]

Illustration of Atlantic Salmon Fish: Nicole D. Jamieson,Ted Walke, April 2007. Infectious Salmon Anemia Virus(ISAV). Available through: http://homepage.usask.ca/~vim458/virology/studpages2007/Nicole/isav.html, http://www.fish.state.pa.us/pafish/atlantic_salmon.jpg[Accessed on 22/01/2012]

For Distribution & Symptoms:
-5 January 2012, Infectious salmon anemia virus, UK. Retrieved 21 January 2012. From http://en.wikipedia.org/wiki/Infectious_salmon_anemia_virus
-8 December 2012, Infectious Salmon Anaemia, UK. Retrieved 21 January 2012. From http://en.wikivet.net/Infectious_Salmon_Anaemia
-The Centre for Food Security & Public Health, Institute For International Cooperation In Animal Biologics (March 2010), Infectious Salmon Anemia-Hemorrhagic Kidney Syndrome, Iowa State University, US. Retrieved 21 January 2012. From http://www.cfsph.iastate.edu/Factsheets/pdfs/infectious_salmon_anemia.pdf

for Control Strategy & Diagnosis:
I) Fisheries Research Services, (2009). Diagnosis of Infectious Salmon Anaemia (ISA), UK, Retrieved 22 Jan 2012, from http://www.scotland.gov.uk/Resource/Doc/1062/0076334.pdf
II) Department of Agriculture, Fisheries and Forestry (March 2009), Infectious Salmon Anaemia (Version 1.0). In: Australian Aquatic Veterinary Emergency Plan (AQUAVETPLAN), Australian Government Department of Agriculture, Fisheries and Forestry, Canberra, ACT, Retrieved 22 Jan 2012, from , Retrieved 22 Jan 2012, from http://www.daff.gov.au/__data/assets/pdf_file/0004/1038676/infectious-salmon-anaemia.pdf
III) World Organisation for Animal Health, (2009), Chapter 2.3.5. Infectious Salmon Anaemia. In: Manual of Diagnostic Tests for Aquatic Animals 2009, France, Retrieved 22 Jan 2012, from http://www.oie.int/fileadmin/Home/eng/Health_standards/aahm/2010/2.3.05_ISA%20.pdf

For Detection,vaccination & treatment of ISAV:

Khalid Munir, Frederick S.B Kibenge, April 2004.
Journal of Virological Methods (2004) Detection of infectious salmon anaemia virus by real-time RT–PCR
Available though: Science Direct [Accessed on 14/01/2012]

Jaime A. Tobar1*, Francisco C. Contreras1, Yelena Betz2, Catalina Bravo1, Mario Caruffo1, SofJerez1, Thomas Goodrich2, and Arun K. Dhar2. Oral vacciantion against Infectious Salmon Anemia in Atlantic Salmon (Salmon Salar) induces specific immunity and provides protection against infectious SAlmon Anemia Virus Challenge.
https://www.was.org/wasmeetings/meetings/ShowAbstract.aspx?Id=20064