Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.5.

VIRAL HAEMORRHAGIC SEPTICAEMIA

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SUMMARY

Viral haemorrhagic septicaemia (VHS) is an infectious disease of rainbow trout (Oncorhynchus mykiss), brown trout (Salmo trutta), grayling (Thymallus thymallus), whitefish (Coregonus sp.), pike (Esox lucius), largemouth bass (Micropteus salmoides), Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus). The disease is caused by viral haemorrhagic septicaemia virus (VHSV, synonym: Egtved virus) (2), a virus belonging to the newly approved genus Novirhabdovirus within the family Rhabdoviridae (23).
 
Until the mid-1980s VHS was regarded as a disease affecting only rainbow trout and a few other freshwater fish species in aquaculture in continental Europe. In the past decades, however, VHSV has been isolated from a large range of free-living marine fish species in the North American part of the Pacific and Atlantic Oceans, in the North Atlantic, the North Sea and the Baltic Sea, and in the waters around Japan. The number of susceptible wild marine species is still growing and, to date, at least 45 different species (both freshwater and marine) have been found to test positive for the virus. Susceptible fish species are found among the Salmoniformes (seven species), Esociformes (one species), Clupeiformes (four species), Gadiformes (eleven species), Pleuronectiformes (seven species), Osmeriformes (three species), Perciformes (six species), Scorpaeniformes (two species), Anguilliformes (one species), Cyprinodontiformes (one species) and Gasterosteiformes (two species).
 
At present, the isolates from wild marine fish are not distinguishable from normal fresh water isolates by serological means. Isolations of VHSV from fish from the North American waters and Japan, however, have so far been associated with genetically characteristic virus strains (6, 8, 14). Four major genotypes have been identified (21): Genotype I - European freshwater VHSV isolates and a group of marine isolates from the Baltic Sea, Genotype II - a group of marine isolates circulating in the Baltic Sea, Genotype III - isolates from the North Sea, Skagerrak and Kattegat and Genotype IV - North American VHSV isolates. The genetic differences appear to be more strongly related to geographical location than to year of isolation or host species (1, 2, 21, 22). The genetic characteristics of VHSV isolates highly pathogenic for rainbow trout have not yet been defined. All examined isolates from wild marine fish have shown low or no mortality in infection trials by immersion with rainbow trout (3, 4, 24). Several marine isolates are pathogenic to turbot fry (9). Japanese flounder are highly susceptible to Japanese VHSV isolates (5). The Pacific isolates are highly pathogenic to Pacific herring (10. This newly discovered large host range and the significant differences in pathogenicity in different host species cause some problems for VHS control programmes, which are based mainly on the protection of the significant rainbow trout production industry. The main problem is whether the finding of marine VHSV in free-living fish in an approved VHSV-free area should lead to the withdrawal of that status. It has been observed in a large part of Europe that VHS in free-living fish in the marine environment affects an approved VHS-free status in only very few cases.
 
VHS occurs in continental Europe and is important because of its clinical and economic consequences to rainbow trout farming. For more detailed reviews of the condition, see Wolf (26) and Jorgensen (7). Natural infections with VHSV have caused significant mortality in turbot and Japanese flounder in aquaculture, and significant natural mortality in Pacific herring and pilchard along the Pacific coast of Alaska and Washington, United States of America (USA), and Canada.
 
The infection of susceptible fish species is often lethal, due to the impairment of the osmotic balance, and occurs within a clinical context of oedema and haemorrhages. Virus multiplication in endothelial cells of blood capillaries, leukocytes, haematopoietic tissues and nephron cells, underlies the clinical signs.
 
Three neutralising subtypes of VHSV have been recognised using a panel of polyclonal and monoclonal antibody preparations (20). Apart from the above variation, VHSV seems to share a VHS-group neutralising epitope, and several non-neutralising epitopes located on the viral glycoprotein (G protein). Variations in virus strain virulence have been recorded in both natural cases of disease and infection trials.
 
The reservoirs of VHSV are clinically infected fish and covert carriers among cultured, feral or wild fish. Virulent virus is primarily shed in the urine, whereas kidney, spleen, and heart are the sites in which virus is most abundant. Once VHSV is established in a farm stock and therefore in the water catchment system, the disease becomes enzootic due to the virus carrier fish.
 
Several factors influence susceptibility to VHS. Among each fish species, there is individual variability in susceptibility, and the age of the fish appears to be of some importance - the younger the fish the higher the susceptibility. In highly susceptible fish stocks, however, overt infection is seen in all sizes of fish.
 
Water temperature is an important environmental factor. Disease generally occurs at temperatures between 4°C and 14°C. Low water temperatures (1-5°C) generally result in an extended disease course with low daily mortality but high accumulated mortality. At high water temperatures (15-18°C), the disease generally takes a short course with a modest accumulated mortality. VHS outbreaks occur during all seasons, but are most common in spring when water temperatures are rising or fluctuating.
 
The screening procedure for VHS is based mainly on virus isolation in cell culture. Confirmatory testing is by immunological virus identification, e.g. neutralisation, immunofluorescence, enzyme-linked immuno-sorbent assay (ELISA), and immunoperoxidase staining or by reverse-transcription polymerase chain reaction (RT-PCR)-based techniques. However, more rapid diagnostic methods for presumptive evidence of viral antigen in infected organ imprints or homogenates (fluorescence, ELISA, immunohistochemistry, RT-PCR) may be suitable for fish with overt disease. Fish serology (neutralisation, ELISA) could be of importance to the detection of the carrier state among fish stocks, but has yet to be validated.
 
Control methods for VHS currently lie in official health surveillance schemes coupled with control policy measures, such as stamping-out and fallowing procedures, and have resulted in the eradication of the disease from several parts of Europe (2, 17). At present, genetic approaches using selection and intergeneric hybridisation and vaccination are both at experimental stages.
 

DIAGNOSTIC PROCEDURES

The standard screening method for viral haemorrhagic septicaemia (VHS) is based on direct methods, i.e. the isolation of VHS virus (VHSV) in cell culture followed by immunological identification or identification by reverse-transcription polymerase chain reaction (RT-PCR) (25). Due to low sensitivity the direct immunological demonstration of VHSV antigen in infected fish tissues can only be used when VHS infection is suspected. PCR-based technology using direct identification of the VHSV genome in fish tissue is currently being developed and validated. The technique can be used for confirmation of overt infection in fish, but has not yet been validated for use in direct surveillance programmes for obtaining approved VHS-free status.
 
Due to insufficient knowledge of the serology of virus infections of fish, the detection of fish antibodies to viruses has thus far not been accepted as a routine diagnostic method for assessing the virus status of fish populations (12).
 
Infected fish material suitable for virological examination is: whole alevin (body length < 4 cm), viscera including kidney (4 cm < body length < 6 cm) or, for larger size fish, kidney and spleen (heart and/or encephalon might be included), and ovarian fluid from broodfish at time of spawning.
 
Sampling procedures: see Chapter I.1. Section B.
 

1.	Standard Screening Methods for VHSV
	1.1.	Isolation of VHSV in cell culture
		Cell line to be used: BF-2, RTG-2
		Alternatively, EPC or FHM cells may be used, but are in general less susceptible than BF-2 and RTG-2 (11, 19).
		a)	Virus extraction
			Use the procedure described in Chapter I.1. (Section B.3.2.).
		b)	Inoculation of cell monolayers
			i)	Prior to inoculation of cells, the supernatant is mixed with equal parts of a suitably diluted pool of antisera to the indigenous serotypes of infectious pancreatic necrosis virus (IPNV) and incubated for a minimum of 1 hour at 15°C or for 12-18 hours at 4°C. The titre of the antiserum must be at least 1/2000 in a 50% plaque neutralisation test.
			ii)	Treatment of all inocula with antiserum to IPNV (a virus that in some parts of the world occurs in 50% of fish samples) aims at preventing cytopathic effect (CPE) due to IPNV from developing in inoculated cell cultures. This will reduce the duration of virological examinations as well as the number of cases in which occurrence of CPE would have to be considered potentially indicative of VHS.
			iii)	When samples come from production units that are considered to be free from IPNV, treatment of inocula with antiserum to IPNV may be omitted.
			iv)	Transfer organ homogenate supernatant on to 24-hour-old monolayers overlaid with cell culture medium containing 2-10% fetal calf serum (FCS) and suitable buffer (e.g. Tris or HEPES [N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid]) in two dilutions, i.e. the primary dilution and, in addition, a 1/10 dilution thereof, resulting in final dilutions of tissue material in cell culture medium of 1/100 and 1/1000, respectively (in order to prevent homologous interference). The ratio of inoculum size to volume of cell culture medium should be about 1:10.
			v)	For each dilution a minimum of about 2 cm2 cell area, corresponding to one well in a 24-well cell culture tray, must be used. Use of cell culture trays is recommended, but other units of similar or bigger growth area may be used instead.
		c)	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. If no positive controls are included, at least every six months or if decreased cell susceptibility is suspected, titration of frozen stocks of VHSV shall be performed to verify the susceptibility of the cell cultures to infection.
			ii)	Inoculated cell cultures shall be incubated at 15°C for 7-10 days and be inspected regularly (at least three times a week) for the occurrence of CPE at x40-100 magnification.
			iii)	Maintain the pH of the cell culture medium at between 7.3 and 7.6 during incubation. This is achieved by the addition of sterile bicarbonate buffer (for tightly closed cell culture flasks) or 2 M Tris buffer solution (for cell culture plates) to the cell culture medium or, preferably, by using HEPES-buffered medium.
			iv)	If CPE appears in cell cultures inoculated with dilutions of the homogenate, 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 it was 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 VHSV.
			v)	If no CPE has developed after the primary incubation for 7-10 days, subcultivation is performed to fresh cell cultures using a cell area similar to that of the primary culture.
		d)	Subcultivation procedures
			i)	Aliquots of medium (supernatant) from all cultures/wells constituting the primary culture are pooled according to cell line 7-10 days after inoculation.
			ii)	The pools are then inoculated into homologous cell cultures undiluted and diluted 1/10 (resulting in final dilutions of 1/10 and 1/100, respectively, of the supernatant) as previously described.
			iii)	The inoculation may be preceded by preincubation of the dilutions with IPN antiserum at appropriate dilution as described in Chapter I.1. Section B.1.
			iv)	Alternatively aliquots of 10% of the medium constituting the primary culture are inoculated directly into a well with fresh cell culture (well-to-well subcultivation).
			v)	The inoculated cultures are then incubated for 7 days with observation as described for the primary inoculation.
			vi)	If no CPE occurs the test may be declared negative.
	1.2.	Virus identification
		a)	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, or filter through a 45-nm pore membrane to remove cell debris.
			ii)	Dilute virus-containing medium from 10-2 to 10-4.
			iii)	Mix aliquots (for example 200 µl) of each dilution with equal volumes of a VHSV antibody solution, and likewise 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.)
			iv)	In parallel, other neutralisation tests must be performed against:
				.	a homologous virus strain (positive neutralisation test)
				.	a heterologous virus strain (negative neutralisation test).
			v)	If required, a similar neutralisation test may be performed using antibodies to IPNV.
			vi)	Incubate all the mixtures at 15°C for 1 hour.
			vii)	Transfer aliquots of each of the above mixtures on to 24-hour-old monolayers overlaid with cell culture medium containing 10% FCS (inoculate two wells per dilution) and incubate at 15°C; 24- or 12-well cell culture plates are suitable for this purpose, using a 50 µl inoculum.
			viii)	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 cell monolayers with a solution of 1% crystal violet in 20% ethanol.
			ix)	The tested virus is identified as VHSV when CPE is prevented or noticeably delayed in the cell cultures that received the virus suspension treated with the VHSV-specific antibody, whereas CPE is evident in all other cell cultures.
			x)	In the absence of any neutralisation by NAb to VHSV, it is mandatory to conduct an indirect fluorescent antibody test (IFAT), an immunoperoxidase test, an enzyme-linked immunosorbent assay (ELISA) or RT-PCR using the suspect sample. Some cases of antigenic drift of surface antigen have been observed, resulting in occasional failure of the neutralisation test using NAb to VHSV.
			Other neutralisation tests of proven efficiency may be used alternatively.
		b)	Indirect fluorescent antibody test
			i)	Prepare monolayers of cells in 2 cm2 wells of cell culture plastic plates or on cover-slips in order to reach around 80% confluency, which is usually achieved within 24 hours of incubation at 22°C (seed six cell monolayers per virus isolate to be identified, plus two for positive and two for negative controls). The FCS content of the cell culture medium can be reduced to 2-4%. If numerous virus isolates have to be identified, the use of Terasaki plates is strongly recommended.
			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 VHSV 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 a cold mixture of acetone 30%/ethanol 70% (v/v) (stored at -20°C).
			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.
			viii)	Prepare a solution of purified VHSV antibody or serum in 0.01 M PBS, pH 7.2, containing 0.05% Tween-80 (PBST), at the appropriate dilution (which has been established previously or is given by the reagent supplier).
			ix)	Rehydrate the dried cell monolayers by four rinsing steps with the PBST solution, and remove this buffer completely after the last rinsing.
			x)	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, e.g. by adding a piece of wet cotton to the humid chamber. The volume of solution to be used is 0.25 ml/2 cm2 well.
			xi)	Rinse four times with PBST as above.
			xii)	Treat the cell monolayers for 1 hour at 37°C with a solution of fluorescein isothiocyanate (FITC)- or tetramethylrhodamine-5-(and-6-)isothiocyanate (TRITC)-conjugated antibody to the immunoglobulin used in the first layer and prepared according to the instructions of the supplier. These conjugated antibodies are most often rabbit or goat antibodies.
			xiii)	Rinse four times with PBST.
			xiv)	Examine the treated cell monolayers on plastic plates immediately, or mount the cover-slips using, for example glycerol saline, pH 8.5 prior to microscopic observation.
			xv)	Examine under incident UV light using a microscope with x10 eye pieces and x20-40 objective lens having numerical aperture >0.65 and >1.3 respectively. Positive and negative controls must be found to give the expected results prior to any other observation.
			Other IFAT techniques of proven efficiency may be used alternatively.
		c)	Enzyme-linked immunosorbent assay
			i)	Coat the wells of microplates designed for ELISAs with appropriate dilutions of purified immunoglobulins (Ig) or serum specific for VHSV, in 0.01 M PBS, pH 7.2 (200 µl/well). Ig may be polyclonal or monoclonal originating most often from rabbit or mouse, respectively. For the identification of VHSV, monoclonal antibodies (MAbs) specific for certain domains of the nucleocapsid protein (N) is suitable (13).
			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.
			vi)	Add 2% Triton X-100 to the virus suspension to be identified.
			vii)	Dispense 100 µl/well of two- or four-step dilutions of the virus to be identified and of VHSV control virus, and negative control (e.g. infectious haematopoietic necrosis virus) and allow to react with the coated antibody to VHSV for 1 hour at 20°C.
			viii)	Rinse four times with PBST.
			ix)	Add to the wells either biotinylated polyclonal VHSV antiserum or MAb to N protein specific for a domain different from the one of the coating MAb and previously conjugated with biotin.
			x)	Incubate for 1 hour at 37°C.
			xi)	Rinse four times with PBST.
			xii)	Add streptavidin-conjugated horseradish peroxidase to those wells that have received the biotin-conjugated antibody, and incubate for 1 hour at 20°C.
			xiii)	Rinse four times with PBST. Add the substrate and chromogen. Stop the course of the test when positive controls react, and read the results.
			xiv)	Interpretations of the results is according to the optical absorbencies achieved by negative and positive controls and must follow the guidelines for each test, e.g. absorbency at 450 nm of positive control must be minimum 5-10 x A450 of negative control. Absorbencies > 3 x A450 of negative controls are regarded as positives.
			The above biotin-avidin-based ELISA version is given as an example (15). Other ELISA versions of proven efficiency may be used instead (16, 18).
		d)	Reverse-transcription polymerase chain reaction
			All work with RNA should be performed under a hood, using gloves.
			i)	Isolation of RNA: Total RNA from infected cells in suspension (cell culture medium) is extracted using the phenol-chloroform method or by RNA affinity spin columns, e.g. RNeasy Total RNA kit (Qiagen, Germany), according to the manufacturer's instructions. RNA must be resuspended in distilled RNAse-free water (e.g. water treated with 0.1% diethyl pyrocarbonate.
			ii)	RT-PCR: The RT-PCR amplification can be performed in one or two step(s) using a primer set that binds to a conserved region within the nucleocapsid (N): 5'-GGG-GAC-CCC-AGA-CTG-T-3' (forward primer) and 5'-TCT-CTG-TCA-CCT-TGA-TCC-3' (reverse primer). The resulting amplicon is 811  base pairs (bp).
			iii)	One-step RT-PCR: 50 µl single tube RT-PCR can be performed using Titan One Tube RT-PCR System (Roche, Germany) according to the manufacturer's instructions. Briefly, the PCR reaction mixture consists of: 5 µl of extracted viral RNA (approximately 0.5-2 µg), 50 pmol of each primer, 1 µl 10 mM dNTP, 10 µl 5 x RT-PCR reaction buffer (with 7.5 mM MgCl2 and dimethyl sulfoxide), 2.5 µl 100 mM DTT, and 1 µl enzyme mix. The following cycles are recommended: 50°C for 30 minutes, 94°C for 2 minutes, 35 cycles at 94°C for 30 seconds, 52°C for 30 seconds, 68°C for 60 seconds; the RNA reaction is finally held at 68°C for 7 minutes.
			iv)	Two-step RT-PCR: The cDNA mixture consists of 5 µl of the extracted viral RNA (approximately 0.5-2 µg), 50 pmol forward primer, 1 x reverse transcript buffer (with 50 mM Tris/HCl, pH 8.3, 3 mM MgCl2, 75 mM KCl, 10 mM), 1 mM mixed dNTP, 5 U reverse transcriptase (StrataScript RNase H- reverse transcriptase, Stratagene, USA) adjusted with H2O to a final volume of 20 ìl. Reverse transcription should be performed at 42°C for 30 minutes, 94°C for 5 minutes and cooled at 4-10°C for 3 minutes.
			v)	The master mixture for subsequent PCR amplification is prepared on 5 ìl of the cDNA reaction, 50 pmol of each primer, 1 x Taq polymerase buffer (with 10 mM Tris/HCl, pH 9, 1.5 mM MgCl2, 50 mM KCl, 0.01% gelatin, 0.1% Triton X-100), 1 mM mixed dNTP, 1.5 U Taq polymerase and adjusted with H2O up to final volume of 50 ìl. The subsequent PCR is performed by the following cycles: 94°C for 2 minutes, 35 cycles at 94°C for 30 seconds, 52°C for 30 seconds, 68°C for 60 seconds, and is finally held at 68°C for 7 minutes.
			vi)	Quantity and specificity of the RT-PCR reactions can be evaluated by 1% agarose gel electrophoresis of 1/10 reaction in 1.5% agarose gel with ethidium bromide and observed using UV transillumination.
			Please note: The thermal protocols might need optimisation, depending on the thermal cycler in use.
2.	Diagnostic Methods for VHSV
	2.1.	Virus isolation with subsequent identification
		As in Section 1.1. and 1.2.
	2.2.	Indirect fluorescent antibody test
		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.
		v)	Fix with acetone or ethanol/acetone and dry as indicated in Section 1.2.b. steps v-vii.
		vi)	Rehydrate the above preparations (see Section 1.2.b. step ix) and block with 5% skim milk or 1% bovine serum albumin, in PBST for 30 minutes at 37°C.
		vii)	Rinse four times with PBST.
		viii)	Treat the imprints with the solution of antibody to VHSV and rinse as indicated in Section 1.2.b.
		ix)	Block and rinse as described previously in steps vi and vii.
		x)	Reveal the reaction with suitable FITC-conjugated specific antibody, rinse and observe as indicated in Section 1.2.b. steps xii-xv.
		xi)	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 (16)
		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 of organ homogenates.
		c)	The enzyme-linked immunosorbent assay procedure
			i)	Microplate processing is described in Section 1.2.c. of this chapter up to step iv (inclusive).
			ii)	Set aside an aliquot of 1/4 dilution of each homogenate in case further virus isolation in cell culture is required.
			iii)	Treat the remaining part of the homogenate with 2% Triton X-100 as in Section 1.2.c. step vi, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
			iv)	Complete the other steps (vii-xiv) of the procedure described in Section 1.2.c.
			v)	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.
			The above biotin-avidin-based ELISA version is given as an example (7). Other ELISA versions of proven efficiency may be used instead (18).

REFERENCES

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