Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.2.

INFECTIOUS HAEMATOPOIETIC NECROSIS

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SUMMARY

Infectious haematopoietic necrosis (IHN) is an infectious disease of rainbow or steelhead trout (Oncorhynchus mykiss), Pacific salmon including chinook (O. tshawytscha), sockeye (O. nerka), chum (O. keta), masou (O. masou), and coho (O. kisutch), and Atlantic salmon (Salmo salar). Historically, the geographical range of IHN was limited to the western parts of North America, but the disease, caused by a rhabdovirus (IHNV), has spread to continental Europe and the Far East via the importations of infected fish and eggs. For more detailed reviews of the disease, see Bootland & Leong (3) or Wolf (19).
 
The principal clinical and economic consequences of IHN occur at farms rearing rainbow trout in freshwater; however, both Pacific and Atlantic salmon reared in freshwater or seawater can be severely affected. Large mortalities have also been recorded among some wild stocks of Pacific salmon. Infection is often lethal due to the impairment of osmotic balance, and occurs within a clinical context of oedema and haemorrhage. Virus multiplication in endothelial cells of blood capillaries, haematopoietic tissues and nephron cells, underlies the clinical signs. High levels of virus are shed from infected juvenile fish. Older fish are increasingly resistant to infection, but adult fish at spawning may shed virus in sexual products. Survivors of IHNV infection demonstrate a strong protective immunity with the synthesis of circulating antibodies to the virus (12) and, in certain individuals, a covert carrier state (6).
 
On the basis of antigenic studies conducted with neutralising polyclonal rabbit antisera, IHNV isolates form a single serogroup (7). However, mouse monoclonal antibodies have revealed a number of neutralising epitopes on the glycoprotein (8, 14, 17), as well as the existence of a non-neutralising, group epitope borne by the nucleoprotein (13). Variations in the virulence of IHNV strains have been recorded during both natural cases of disease and in experimental infections (10).
 
The reservoirs of IHNV are clinically infected fish and covert carriers among cultured, feral or wild fish. Virus is shed via faeces, urine, sexual fluids and external mucus, whereas kidney, spleen, encephalon and the digestive tract are the sites in which virus is most abundant during the course of overt infection. The transmission of IHNV between fish is primarily horizontal; however, cases of vertical or 'egg-associated' transmission have been recorded. Horizontal transmission is typically by direct exposure, but invertebrate vectors have been proposed to play a role in some cases. Egg-associated transmission is significantly reduced by the now common practice of surface disinfection of eggs with an iodophor solution, but is the only mechanism accounting for the appearance of IHN in new geographical locations among alevins originating from eggs that were incubated and hatched in virus-free water. Once IHNV is established in a farmed stock or in a watershed, the disease may become established among carrier fish.
 
Among individuals of each fish species, there is a high degree of variation in susceptibility to IHNV. The age of the fish appears to be extremely important: the younger the fish, the more susceptible to disease. As with viral haemorrhagic septicaemia virus, good overall fish health condition seems to decrease the susceptibility to overt IHN, while co-infections with bacterial diseases (e.g. bacterial coldwater disease), handling and other types of stress frequently cause subclinical infections to become overt.
 
The most prominent environmental factor affecting IHN is water temperature. Clinical disease occurs between 8°C and 15°C under natural conditions.
 
The screening procedure for IHNV is based on virus isolation in cell culture. Confirmatory identification may be achieved by use of immunological (neutralisation, indirect fluorescent antibody test or enzyme-linked immunosorbent assay), or molecular (DNA probe or polymerase chain reaction) methods (1, 2, 4, 5, 9, 11, 18).
 
Control methods for IHN currently rely on avoidance of exposure to the virus through the implementation of strict control policies and sound hygiene practices (15). The thorough disinfection of fertilised eggs and the incubation of eggs and rearing of fry and alevins on virus-free water supplies in premises completely separated from those harbouring possible virus carriers and free from possible contact with inanimate objects, are critical for preventing the occurrence of IHNV in a defined fish production site. At present, vaccination is at an experimental stage; however, several new vaccine preparations have shown substantial promise in both laboratory and field trials (16).
 

DIAGNOSTIC PROCEDURES

The standard screening method for infectious haematopoietic necrosis (IHN) is based on the isolation of IHN virus (IHNV) in cell culture followed by its immunological or molecular identification as described in Section 1. Diagnosis of IHN during outbreaks of disease may be made via this conventional approach or by more rapid immunological or molecular methods that detect IHNV antigens or nucleic acids in infected fish tissues as described in Section 2. While the diagnostic methods listed in Section 2 can be used for confirmation of overt infections in fish, they are not approved for use as screening methods for obtaining approved IHN-free status.
 
Due to substantial variation in the serological responses of fish to virus infections, the detection of fish antibodies to viruses has not thus far been accepted as a routine diagnostic method for assessing the viral status of fish populations. In the future, validation of serological techniques for diagnosis of fish virus infections could render the use of fish serology more widely acceptable for diagnostic purposes.
 
For general information, sampling procedures, specimen preparation, and materials required for virological assays: see Chapter I.1.
 

1.	Standard Screening Method for IHN
	1.1.	Isolation of IHNV in cell culture
		Cell lines to be used: EPC and BF-2
		a)	Inoculation of cell monolayers
			i)	Make an additional tenfold dilution of the 1/10 organ homogenate supernatants and transfer an appropriate volume of each of the two dilutions on to 24-hour-old cell monolayers. Inoculate at least 2 cm2 of drained cell monolayer with 100 µl of each dilution.
			ii)	Allow to adsorb for 0.5-1 hour and, without withdrawing the inoculate, add the cell culture medium buffered at pH 7.6 and supplemented with 2% fetal calf serum (FCS) (1 ml/well for 24-well cell culture plates), and incubate at 15°C.
			iii)	If required, the inoculate may be pre-incubated with neutralising antiserum against infectious pancreatic necrosis virus (IPNV) or other enzootic viruses as previously described (see Chapter I.1. Section B.3.3.).
		b)	Monitoring incubation
			i)	Follow the course of infection in positive controls and other inoculated cell cultures by daily microscopic examination at x40-100 magnification for 7 days. The use of a phase-contrast microscope is recommended.
			ii)	Maintain the pH of the cell culture medium between 7.3 and 7.6 during incubation. This can be achieved, especially in open plates, by using cell culture medium containing sodium bicarbonate that is further buffered by addition of Tris or HEPES (see Chapter I.1.C).
			iii)	If a cytopathic effect (CPE) appears in cell cultures inoculated with dilutions of the fluids or homogenates, identification procedures must be undertaken immediately (see Section 1.2. below).
				If a fish health surveillance/control programme is being implemented, steps may have to be taken to suspend the approved health status of the production unit and/or the zone (if approved previously) from which the suspected virus-positive sample originated. The suspension of approved status will be maintained until it is demonstrated that the virus in question is not IHNV.
			iv)	If no CPE develops in the inoculated cultures (despite normal progression of CPE in the virus controls), the inoculated cultures should be subcultured for a further 7 days. Should the virus control fail to develop CPE, the process should be repeated with fresh cell cultures and new batches of samples.
		c)	Subcultivation procedures
			i)	Collect aliquots of cell culture medium from all monolayers inoculated with various dilutions of fluid or organ homogenate samples.
			ii)	If required, repeat the neutralisation test to IPNV or other enzootic viruses as previously described (see Section 1.1.a).
			iii)	Inoculate cell monolayers as described above in Section 1.1.a.
			iv)	Incubate and monitor as described in Section 1.1.b.
			v)	If no CPE occurs, the test may be declared negative.
	1.2.	Identification of IHNV
		.	1.2.1. Neutralisation test
			i)	Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge it at 2000 g for 15 minutes at 4°C to remove cell debris.
			ii)	In parallel, other neutralisation tests must be performed against:
				.	a known isolate of IHNV (positive neutralisation test).
				.	a heterologous virus (negative neutralisation test).
			iii)	If required, a similar neutralisation test may be performed using antibodies to IPNV or other enzootic viruses to ensure that no contaminant has escaped the first assay.
		a)	The neutralisation test procedure
			i)	Dilute the virus-containing medium from 10-2 to 10-4.
			ii)	Mix aliquots (for example 200 µl) of each virus dilution with equal volumes of rabbit polyclonal or mouse monoclonal antibody (MAb) against IHNV, and similarly treat aliquots of each virus dilution with cell culture medium. (The neutralising antibody [Nab] solution must have a 50% plaque reduction titre of at least 2000.)
			iii)	Incubate all the mixtures at 15°C for 1 hour.
			iv)	Transfer aliquots of each of the above mixtures on to drained cell monolayers (inoculate two cell cultures per dilution) and allow adsorption to occur for 0.5-1 hour at 15°C; 24- or 12-well cell culture plates are suitable for this purpose, using a 50 µl inoculum.
			v)	When adsorption is complete, add cell culture medium, supplemented with 2% FCS and buffered at pH 7.4-7.6, to each well and incubate at 15°C.
			vi)	Check the cell cultures for the onset of CPE and read the results as soon as it occurs in non-neutralised controls (cell monolayers being protected in positive neutralisation controls). Results are recorded either after a simple microscopic examination (phase-contrast preferable) or after discarding the cell culture medium and staining the cell monolayers with a solution of 1% crystal violet in 10% buffered formalin.
			vii)	The tested virus is identified as IHNV when CPE is prevented or noticeably delayed in the cell cultures that received the virus suspension treated with the IHNV-specific antibody, whereas CPE is present in the untreated sample.
			In the absence of significant neutralisation when IHNV is suspected, it is advisable to conduct an indirect fluorescent antibody test (IFAT) as antigenic drift has been observed in the neutralising epitopes on the IHNV glycoprotein, resulting in occasional failures of the neutralisation test for certain strains of the virus.
		.	1.2.2. Indirect fluorescent antibody test
		a)	Preparation of monolayers
			i)	Prepare monolayers of cells in 2 cm2 wells of plastic cell culture plates or on glass cover-slips to reach around 80% confluency within 24 hours of incubation at 22°C. Seed six cell monolayers per virus isolate to be tested, plus two for positive and two for negative controls. The FCS content of the cell culture medium can be reduced to 2-4%.
			ii)	When the cell monolayers are ready for infection (i.e. on the same day or on the day after seeding), inoculate the virus suspensions to be identified by making tenfold dilution steps directly in the cell culture wells or flasks.
			iii)	Dilute the control virus suspension of IHNV in a similar way, in order to obtain a virus titre of about 5000-10,000 plaque-forming units (PFU)/ml in the cell culture medium.
			iv)	Incubate at 15°C for 24 hours.
			v)	Remove the cell culture medium, rinse once with 0.01 M phosphate buffered saline (PBS), pH 7.2, then three times briefly with cold acetone (stored at -20°C) for glass cover-slips or a mixture of 30% acetone and 70% ethanol, also at -20°C, for plastic wells.
			vi)	Let the fixative act for 15 minutes. A volume of 0.5 ml is adequate for 2 cm2 of cell monolayer.
			vii)	Allow the cell monolayers to air-dry for at least 30 minutes and process immediately or freeze at -20°C.
		b)	The indirect fluorescent antibody procedure
			i)	Prepare a solution of antibody against IHNV in PBS containing 0.05% Tween 80 (PBST) at the appropriate dilution (established previously or given by the reagent supplier). The antibody used should be able to bind to all isolates of the virus.
			ii)	Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, and remove this buffer completely after the last rinsing.
			iii)	Treat the cell monolayers with the antibody solution for 1 hour at 37°C in a humid chamber and do not allow evaporation to occur. The volume of solution to be used is 0.25 ml/2 cm2 well.)
			iv)	Rinse four times with PBST as above.
			v)	Treat the cell monolayers for 1 hour at 37°C with a solution of fluorescein isothiocyanate (FITC)-conjugated antibody to the immunoglobulin used in the first layer and prepared according to the instructions of the supplier. These FITC antibodies are most often rabbit or goat antibodies.
			vi)	Rinse four times with PBST.
			vii)	Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips using glycerol saline at pH 8.5 prior to microscopic observation.
			viii)	Examine under incident UV light using a microscope with a x20-40 objective lens having a high numerical aperture. Positive and negative controls must be found to give the expected results prior to any other observation.
		.	1.2.3. Enzyme-linked immunosorbent assay
		a)	Preparation of microplates
			i)	Coat the wells of microplates designed for enzyme-linked immunosorbent assays (ELISAs) with appropriate dilutions of immunoglobulins (Ig) specific for IHNV, in 0.01 M PBS, pH 7.2 (200 µl/well). The Ig may be polyclonal or monoclonal, most often from rabbit or mouse. For the identification of IHNV, MAbs specific for certain domains of the nucleocapsid (N) protein are suitable.
			ii)	Incubate overnight at 4°C.
			iii)	Rinse four times with 0.01 M PBS containing 0.05% Tween 20 (PBST).
			iv)	Block with skim milk (5% in PBST) or other blocking solution for 1 hour at 37°C (200 µl/well).
			v)	Rinse four times with PBST.
		b)	The enzyme-linked immunosorbent assay procedure
			i)	Add 2% Triton X-100 to the virus suspension to be identified.
			ii)	Dispense 100 µl/well of two- or four-step dilutions of the virus to be identified and of IHNV control virus, and allow to react with the coated antibody to IHNV for 1 hour at 20°C.
			iii)	Rinse four times with PBST.
			iv)	Add to the wells either biotinylated polyclonal rabbit antiserum to IHNV or biotinylated mouse MAb to an N protein epitope different from the one recognised by the coating MAb.
			v)	Incubate for 1 hour at 37°C.
			vi)	Rinse four times with PBST.
			vii)	Add streptavidin-conjugated horseradish peroxidase to those wells that have received the biotin-conjugated antibody, and incubate for 1 hour at 20°C.
			viii)	Rinse four times with PBST.
			ix)	Add the substrate and chromogen. Stop the course of the test when positive controls react, and read the results.
		.	1.2.4. DNA probe
		a)	Infect cells with virus isolates
			i)	Trypsinise EPC (or other) cells and resuspend to 1.0 x 106 cells per ml with MEM-5-T (5% FBS [fetal bovine serum], Tris buffer). Transfer 1 ml of cell suspension to each well of a 24-well tissue culture plate and incubate the plate at an appropriate temperature.
			ii)	On the day after seeding cells, remove the fluid from each cell monolayer. Infect cells with positive control (IHNV) and with the unknown fish virus isolates in 1 ml total volume to provide a multiplicity of infection (m.o.i.) of 10 PFU per cell. Negative control cells receive 1 ml MEM-5-T. Incubate for 2 hours at 15-20°C on a rocker platform.
			iii)	Incubate infected and control cells at 15°C overnight. The desired product is the N gene mRNA, and maximum production occurs at about 12 hours post-infection when inoculated at high m.o.i. Thus, mRNA production is optimum when infected cells are at an early stage of virus replication, and RNA can be extracted from cells at the first sign of any CPE. Alternatively, use recently infected cells from the virus isolation procedure.
		b)	Make or purchase the following solutions
			i)	Prehybridisation buffer

Deionised water (DEPC [diethyl pyrocarbonate]-treated; autoclaved)	69.5 ml
10 x Denhardt's solution	10 ml of 100 x stock (step iv)
2 x SSC (standard saline citrate)	10 ml of 20 x stock (step vi)
1% SDS (sodium dodecyl sulfate)	10 ml of 10% stock (step viii)
0.1 mg/ml SSS (sonicated salmon sperm) DNA	0.5 ml stock (step v)

 

ii)

Hybridisation solution

Prehybridisation buffer	10 ml (step i)
Biotinylated DNA probe	100 ng/ml
(Store at -20°C; may re-use up to five times)

 

iii)

Post-hybridisation solution

2 x SSC	50 ml of 20 x stock (step vi)
0.1% SDS	5 ml of 10% stock (step viii)
Deionised water (DEPC-treated; autoclaved)	up to 500 ml

 

iv)

100 x Denhardt's solution

Bovine serum albumin	50 g
Polyvinylpyrrolidone 360	50 g
Ficoll 400	50 g
Deionised water (DEPC-treated; autoclaved)	up to 500 ml

 

v)

Sonicated salmon sperm DNA (SSS DNA at 20 mg/ml)

Transfer 0.5 ml of SSS DNA (20 mg/ml) into vials and place into boiling water for 10 minutes. Cool vials in crushed ice and store at -20°C. When needed, add 0.5 ml to prehybridisation buffer (see step i).

 

vi)

20 x standard saline citrate (20 x SSC)

NaCl	87.65 g
Citric acid	44.11 g
Deionised water (DEPC-treated; autoclaved)	up to 500 ml
(Adjust to pH 7.0 with HCl, autoclaved)

 

vii)

10 x standard saline citrate (10 x SSC)

NaCl	43.82 g
Citric acid	22.05 g
Deionised water (DEPC-treated; autoclaved)	up to 500 ml
(Adjust to pH 7.0 with HCl, autoclaved OR dilute 1/2 from 20 x SSC using deionised water, autoclaved)

 

viii)

10% sodium dodecyl sulfate (10% SDS)

Lauryl sulfate sodium salt	10.0 g
Deionised water (DEPC-treated; autoclaved)	up to 100 ml
(Adjust to pH 7.2. Do not autoclave this solution!)

 

ix)

Streptavidin/alkaline phosphate conjugate (SA/AP)

0.1 µg/ml streptavidin/alkaline phosphatase conjugate. Prepare by diluting SA/AP 1/1000 in buffer A (step x). (May re-use this solution up to five times, store at 4°C.)

 

x)

Buffer A

0.1 M Tris, pH 7.5	50 ml of 1 M stock (step xiv)
0.1 M NaCl	10 ml of 5 M stock (step xii)
2 mM MgCl2	1 ml of 1 M stock
0.05% Triton X-100	0.25 ml
Deionised water (DEPC-treated; autoclaved)	up to 500 ml

 

xi)

Buffer B

0.1 M Tris, pH 9.5	50 ml of 1 M stock (step xiii)
0.1 M NaCl	10 ml of 5 M stock (step xii)
50 mM MgCl2	25 ml of 1 M stock
Deionised water (DEPC-treated; autoclaved)	up to 500 ml

 

xii)

5 M NaCl

NaCl	146.1 g
Deionised water (DEPC-treated; autoclaved)	up to 500 ml
(autoclave this solution)

 

xiii)

1 M Tris buffer, pH 9.5

Tris base	54.7 g
Tris HCl	7.6 g
Deionised water (DEPC-treated; autoclaved)	up to 500 ml
(Adjust to pH 9.5, then autoclave)

 

xiv)

1 M Tris buffer, pH 7.5

Tris base	11.8 g
Tris HCl	63.5 g
Deionised water (DEPC-treated; autoclaved)	up to 500 ml
(Adjust to pH 7.5, then autoclave)

 

xv)

Chloroform

Chloroform (Store at -20°C until needed)

 

xvi)

Isopropyl alcohol

2 propanol (isopropyl alcohol). Use undiluted for precipitation of RNA.

 

xvii)

Trizol

RNA isolation reagent, store at 2-8°C in the dark. Invitrogen Catalog No. 15596-026. Caution: Contains guanidinium isothiocyanate and phenol.

 

xviii)

Alkaline phosphatase conjugate substrate kit

(NOTE: This product contains dimethylformamide. Use in area with good ventilation.)

Dissolve alkaline phosphatase (AP) colour development buffer in 1 litre volume of distilled deionised water. Filter-sterilise then store at 4°C. Immediately before use, add 0.4 ml of AP colour reagent A and 0.4 ml AP colour reagent B to 39.2 ml colour development buffer at room temperature.

 

		c)	Prepare biotinylated oligonucleotide probe
			i)	The biotinylated probe (5'-CTT-GTT-TTG-GCA-GTA-TGT-GGC-CAT-CTT-GTC-3') is made using the 30-nucleotide sequence identified by Deering et al. (4); however, three nucleotides containing biotin can be conveniently added to the 5' end during DNA synthesis rather than by a subsequent terminal transferase reaction at the 3' end. This antisense probe is complementary to a conserved region in the middle of the IHNV nucleoprotein gene mRNA. It should react only with IHNV and should recognise all isolates of the virus. The final concentration of the biotinylated probe will be 0.1 µg/ml in hybridisation solution.
		d)	Extraction of mRNA from infected cells
			i)	Always wear protective gloves to avoid contaminating solutions with RNase from skin and to avoid injury. Provide adequate ventilation, especially for RNA extraction steps with Trizol and for dimethylformamide in colour development solutions.
			ii)	Remove culture medium from infected cells and add 1.0 ml Trizol to each well. Replace lid and place plates on a rocker platform for 5-10 minutes at room temperature to digest the cells. During incubation, load 100 µl chloroform into siliconised 1.7 ml centrifuge tubes, keep on ice.
			iii)	Triturate cell debris by pipetting with a sterile 1 ml pipette (5x), then transfer solution from each well into a separate tube. Vortex the tubes (about 3 seconds each), store on crushed ice for 5 minutes to allow phase separation.
			iv)	Centrifuge the suspension at 12,500 g for 15 minutes at 4°C. The RNA will remain in the clear aqueous phase while the DNA and protein will be left in the lower red phenol phase.
			v)	During step iv, load another set of tubes with 0.5 ml of absolute isopropyl alcohol, store on ice.
			vi)	Carefully transfer the upper aqueous phase, which contains the RNA (about 0.5 ml), to a tube that contains an equal volume (0.5 ml) of absolute isopropyl alcohol. Vortex tubes for 1 second, and chill tubes on ice for 15 minutes to precipitate RNA.
			vii)	Centrifuge the mixture at 12,500 g for 15 minutes at 4°C. Remove as much fluid from the pellet as possible. Partially dry the pellet following the manufacturer's instructions.
			viii)	Prepare a nitrocellulose membrane (0.45 µm pore size): wet in distilled deionised water for 1 minute, pour water off, then soak for at least 5 minutes in 10 x SSC.
			ix)	Warm the prehybridisation buffer to 55°C in a water bath.
			x)	Add 170 µl distilled deionised, ribonuclease-free (DEPC-treated and autoclaved) or molecular-biology grade water to the RNA pellets. Mix the contents and warm the tubes in a heat block at 65°C for 15-20 minutes (RNA pellets should dissolve).
			xi)	Install the membrane in 96-well vacuum blotting device, attach vacuum pump hoses and add 200 µl of 10 x SSC to each well to insure that the membrane is not dry when the RNA is added.
			xii)	Heat IHNV PCR products (if used as positive control instead of RNA from IHNV-infected cells) to 95°C for 5 minutes to denature the double-stranded DNA, then put tubes directly on ice.
			xiii)	Add 170 µl of 20 x SSC into microcentrifuge tubes containing the dissolved pellets of RNA in 170 µl of water (tubes now contain 340 µl of 10 x SSC). Store on ice.
			xiv)	Add 100 µl of each RNA solution to wells of a blotting device that contain 200 µl of 10 x SSC. Blot positive controls last. Apply vacuum for about 1 minute until fluid is pulled through the membrane. Remove membrane and transfer to thick filter paper wetted with 10 x SSC.
			xv)	Cut the membrane into sections if required and label the membranes in one corner.
			xvi)	Place the membrane(s) between dry sheets of blotting paper and microwave for 60 seconds on high power to attach nucleic acids to the membrane. Include a beaker of water in the microwave. (Ultraviolet radiation or other suitable methods may be used.)
		e)	Hybridisation of probe to RNA on nitrocellulose membrane
			i)	Place the membranes (spots up) in a hybridisation pouch, bottle or plastic bag. Add 10 ml prehybridisation buffer to each membrane. Prehybridise for 1 hour at 55°C in a shaker water bath.
			ii)	Thaw the probe solution and add 100 µl to prehybridisation buffer. React the membranes in probe solution for 1 hour in shaker water bath at 55°C.
			iii)	Remove probe solutions and store in tubes at -20°C for re-use up to five times.
			iv)	Rinse the membranes with 40 ml of post-hybridisation solution. Discard the solution then add a fresh 40 ml of post-hybridisation solution. Wash for 15 minutes on a rocker platform at room temperature. Wash two more times (40 ml) for 15 minutes each at room temperature on a rocker platform. Put the container with the membranes into a 55°C water bath with pre-warmed post-hybridisation solution (55°C) for 15 minutes.
			v)	Rinse the membranes briefly with 40 ml of buffer A.
		f)	Colour development of biotinylated probe
			i)	Incubate the membranes in a solution containing 40 µl streptavidin/alkaline phosphatase in 40 ml buffer A for 30 minutes at room temperature on a rocker platform. This solution can be re-used at least five times.
			ii)	Rinse the membranes briefly (40 ml) then wash twice using 40 ml buffer A incubated for 7 minutes at room temperature on a rocker platform.
			iii)	Wash twice in 40 ml buffer B incubated for 7 minutes at room temperature on a rocker platform.
			iv)	Warm the colour development buffer prepared according to the manufacturer's protocol. Buffer should be filter-sterilised through a 0.2 µm membrane and stored at 4°C. Immediately before use, add 0.4 ml alkaline phosphatase (AP) colour reagent A and 0.4 ml AP colour reagent B to 39.2 ml AP colour development buffer at room temperature. Add 40 ml colour development solution containing nitro-blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to each container with the membranes. Incubate at room temperature on a rocker platform and observe for colour development (up to 15 minutes).
			v)	Wash the membranes in distilled deionised water for 10 minutes, change the water at least once to remove excess colour development solution. Store membranes in distilled deionised water until photographs can be taken (if desired).
		.	1.2.5. Polymerase chain reaction
		a)	Viral RNA preparation
			i)	Collect aliquots of culture medium from cell monolayers exhibiting CPE and centrifuge at 2000 g for 15 minutes at 4°C to remove cell debris.
			ii)	Release RNA from virus solution by diluting the sample 1/20 in sterile deionised water, heating the tube at 95°C for 2 minutes, then storing on ice. This simple procedure is especially suitable for virus grown in cell culture where few PCR-inhibiting substances are present. Alternatively, RNA can be extracted from a pellet of infected cells using methods described in Section 1.2.4. or by use of other commercially available kits (18).
		b)	Reverse-transcription and first round PCR protocol
			i)	Prepare a master mix for the number of samples to be analysed. Work under a hood and wear gloves.
			ii)	The master mix for one 50 µl reverse-transcription PCR is prepared as follows: 23.75 µl ribonuclease-free (DEPC-treated) or molecular-biology grade water; 5 µl 10 x buffer; 5 µl 25 mM MgCl2; 5 µl 2 mM dNTP; 2.5 µl (20 pmoles/µl) Upstream Primer 5'-TCA-AGG-GGG-GAG-TCC-TCG-A-3'; 2.5 µl (20 pmoles/µl) Downstream Primer 5'-CAC-CGT-ACT-TTG-CTG-CTA-C-3'; 0.5 µl Taq polymerase (5 U/µl); 0.5 µl AMV reverse transcriptase (9 U/µl); 0.25 µl RNasin (39 U/µl).
			iii)	Centrifuge the tubes briefly (10 seconds) to make sure the contents are at the bottom.
			iv)	Place the tubes in the thermal cycler and start the following cycles - 1 cycle: 50°C for 15 minutes; 1 cycle: 95°C for 2 minutes; 25 cycles: 95°C for 30 seconds, 50°C for 30 seconds, 72°C for 60 seconds; 1 cycle: 72°C for 7 minutes and soak at 4°C.
			vi)	Visualise the 786 bp PCR amplicon by electrophoresis of the product in 1.5% agarose gel with ethidium bromide and observe using UV transillumination.
		c)	Second round PCR protocol
			i)	If the first round PCR provides insufficient amplified product, an internally nested set of primers is used for additional DNA amplification. Prepare a master mix for the number of samples to be analysed. Work under a hood and wear gloves.
			ii)	The master mix for one 50 µl second round PCR is prepared as follows: 27.5 µl molecular-biology grade water; 5 µl 10 x buffer; 5 µl 25 mM MgCl ; 5 µl 2 mM dNTP; 2.5 µl (20 pmoles/µl) Upstream Primer 5'-TTC-GCA-GAT-CCC-AAC-AAC-AA-3'; 2.5 µl (20 pmoles/µl) Downstream Primer 5'-GCG-CAC-AGT-GCC-TTG-GCT-3'; 0.5 µl Taq polymerase (5 U/µl).
			iii)	Dispense 48 µl of the master mix into each tube and add 2 µl of first round PCR template.
			iv)	Centrifuge the tubes briefly (10 seconds) to make sure the contents are at the bottom.
			v)	Place the tubes in a thermal cycler and start the following cycles - 1 cycle: 95°C for 2 minutes; 25 cycles: 95°C for 30 seconds, 50°C for 30 seconds, 72°C for 60 seconds; 1 cycle: 72°C for 7 minutes and soak at 4°C.
			vi)	Visualise the 323 bp PCR amplicon by electrophoresis of the product in 1.5% agarose gel with ethidium bromide and observe using UV transillumination.
		The DNA probe procedure (Section 1.2.4. of this chapter) can be used to further confirm that the amplicon is from IHNV. Alternatively, the amplicon can be sequenced (18).
		NOTE: Other primer sets have been used for the successful amplification of a portion of the N gene of IHNV (18); however, the primer sequences used here have been shown to be conserved among all known isolates of IHNV and are not present in the N gene of the related fish rhabdoviruses, viral hemorrhagic septicemia virus or hirame rhabdovirus.
2.	Diagnostic Methods for IHN
	2.1.	Virus isolation with subsequent identification
		a)	Sampling procedures
			See the following sections in Chapter I.1.:
			B.1. for the selection of fish specimens
			B.2. for the selection of materials sampled.
		b)	Processing of organ samples
			See the following sections in Chapter I.1.:
			B.3.1. for transportation
			B.3.2. for virus extraction and obtaining organ homogenates.
		c) Virus isolation and identification
			As in Sections 1.1. and 1.2.
	2.2.	Indirect fluorescent antibody test
		a)	Processing of organ samples
			i)	Bleed the fish thoroughly.
			ii)	Make kidney imprints on cleaned glass slides or at the bottom of the wells of a plastic cell culture plate.
			iii)	Store the kidney pieces (as indicated in Chapter I.1. Section B.3.1.) together with the other organs required for virus isolation in case this becomes necessary later.
			iv)	Allow the imprint to air-dry for 20 minutes.
		b)	Indirect fluorescent antibody procedure
			i)	Fix with acetone or ethanol/acetone and dry as indicated in Section 1.2.2. of this chapter.
			ii)	Rehydrate the above preparations and block with 5% skim milk or 1% bovine serum albumin, in phosphate buffered saline containing 0.05% Tween 80 (PBST) for 30 minutes at 37°C.
			iii)	Rinse four times with PBST.
			iv)	Treat the imprints with the solution of antibody to IHNV and rinse as indicated in Section 1.2.2.
			v)	Block and rinse as described previously.
			vi)	Reveal the reaction with suitable FITC-conjugated specific antibody, rinse and observe as indicated in Section 1.2.2.
			If the test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as described in Section 1.1.
	2.3.	Enzyme-linked immunosorbent assay
		a)	Sampling procedures
			See the following sections in Chapter I.1.:
			B.1. for the selection of fish specimens
			B.2. for the selection of materials sampled.
		b)	Processing of organ samples
			See the following sections in Chapter I.1.:
			B.3.1. for transportation
			B.3.2. for virus extraction and obtaining organ homogenates.
		c)	Microplate processing
			As described in Section 1.2.3. of this chapter.
		d)	The enzyme-linked immunosorbent assay procedure
			i)	Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell culture is required.
			ii)	Treat the remaining part of the homogenate with 2% Triton X-100, as in Section 1.2.3. of this chapter, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
			iii)	Complete the other steps of the procedure described in Section 1.2.3.
			If the test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as described in Section 1.1.
	2.4.	Polymerase chain reaction
		a)	Sampling procedures
			See the following sections in Chapter I.1.:
			B.1. for the selection of fish specimens
			B.2. for the selection of materials sampled.
		b)	Processing of organ samples
			See the following sections in Chapter I.1.:
			B.3.1. for transportation
			B.3.2. for virus extraction and obtaining organ homogenates.
		c)	Viral RNA preparation
			i)	Set aside an aliquot of 1/4 of each homogenate in case further virus isolation in cell culture is required.
			ii)	Release RNA from tissue homogenate by diluting sample 1/20 in sterile deionised water, heating the tube at 95°C for 2 minutes, then storing on ice. Alternatively, RNA can be extracted from tissue homogenates using methods described in Section 1.2.4. or by use of other commercially available kits (18).
		d)	The polymerase chain reaction procedure
			Perform the remaining steps of the PCR procedure described in Section 1.2.5.
			If the test is negative, process the organ samples stored at 4°C for virus isolation in cell culture as described in Section 1.1.

REFERENCES

1.	Arakawa C.K., Deering R.E., Higman K.H., Oshima K.H., O'Hara P.J. & Winton J.R. (1990). Polymerase chain reaction (PCR) amplification of a nucleoprotein gene sequence of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 8, 165-170.
2.	Arnzen J.M., Ristow S.S., Hesson C.P. & Lientz J. (1991). Rapid fluorescent antibody tests for infectious hematopoietic necrosis virus (IHNV) utilizing monoclonal antibodies to the nucleoprotein and glycoprotein. J. Aquat. Anim. Health, 3, 109-113.
3.	Bootland L.M. & Leong J.C. (1999). Infectious hematopoietic necrosis virus. In: Fish Diseases and Disorders, Volume 3: Viral, Bacterial and Fungal Infections, Woo P.T.K. & Bruno D.W., eds. CAB International, Oxon, UK, 57-121.
4.	Deering R.E., Arakawa C.K., Oshima K.H., O'Hara P.J., Landolt M.L. & Winton J.R. (1991). Development of a biotinylated DNA probe for detection and identification of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 11, 57-65.
5.	Dixon P.F. & Hill B.J. (1984). Rapid detection of fish rhabdoviruses by the enzyme-linked immunosorbent assay (ELISA). Aquaculture, 42, 1-12.
6.	Drolet B.S., Chiou P.P., Heidel J. & Leong J.C. (1995). Detection of truncated virus particles in a persistent RNA virus infection in vivo. J. Virol., 69, 2140-2147.
7.	Engelking H.M., Harry J.B. & Leong J.C. (1991). Comparison of representative strains of infectious hematopoietic necrosis virus by serological neutralization and cross-protection assays. Appl. Environ. Microbiol., 57, 1372-1378.
8.	Huang C., Chien M-S., Landolt M. & Winton J.R. (1994). Characterization of the infectious hematopoietic necrosis virus glycoprotein using neutralizing monoclonal antibodies. Dis. Aquat. Org., 18, 29-35.
9.	Jorgensen P.E.V., Olesen N.J., Lorenzen N., Winton J.R. & Ristow S.S. (1991). Infectious hematopoietic necrosis (IHN) and viral hemorrhagic septicemia (VHS): detection of trout antibodies to the causative viruses by means of plaque neutralization, immunofluorescence, and enzyme-linked immunosorbent assay. J. Aquat. Anim. Health, 3, 100-108.
10.	LaPatra S.E., Fryer J.L. & Rohovec J.S. (1993). Virulence comparison of different electropherotypes of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 16, 115-120.
11.	LaPatra S.E., Roberti K.A., Rohovec J.S. & Fryer J.L. (1989). Fluorescent antibody test for the rapid diagnosis of infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 1, 29-36.
12.	LaPatra S.E., Turner T., Lauda K.A., Jones G.R. & Walker S. (1993). Characterization of the humoral response of rainbow trout to infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 5, 165-171.
13.	Ristow S.S. & Arnzen J.M. (1989). Development of monoclonal antibodies that recognize a type 2 specific and a common epitope on the nucleoprotein of infectious hematopoietic necrosis virus. J. Aquat. Anim. Health, 1, 119-125.
14.	Ristow S.S. & Arnzen De Avila J.M. (1991). Monoclonal antibodies to the glycoprotein and nucleoprotein of infectious hematopoietic necrosis virus (IHNV) reveal differences among isolates of the virus by fluorescence, neutralization and electrophoresis. Dis. Aquat. Org., 11, 105-115.
15.	Winton J.R. (1991). Recent advances in the detection and control of infectious hematopoietic necrosis virus (IHNV) in aquaculture. Ann. Rev. Fish Dis., 1, 83-93.
16.	Winton J.R. (1997). Immunization with viral antigens: Infectious haematopoietic necrosis. Dev. Biol. Stand., 90, 211-220.
17.	Winton J.R., Arakawa C.K., Lannan C.N. & Fryer J.L. (1988). Neutralizing monoclonal antibodies recognize antigenic variants among isolates of infectious hematopoietic necrosis virus. Dis. Aquat. Org., 4, 199-204.
18.	Winton J.R. & Einer-Jensen K. (2002). Molecular diagnosis of infectious hematopoietic necrosis and viral hemorrhagic septicemia. In: Molecular Diagnosis of Salmonid Diseases. Cunningham C.O., ed. Kluwer, Dordrecht, The Netherlands. pp. 49-79.
19.	Wolf K. (1988). Infectious hematopoietic necrosis. In: Fish Viruses and Fish Viral Diseases. Cornell University Press, Ithaca, New York, USA. pp. 83-114.

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