Manual of Diagnostic Tests for Aquatic Animals (2003)

CHAPTER 2.1.3.

ONCORHYNCHUS MASOU VIRUS DISEASE

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SUMMARY

Oncorhynchus masou virus disease (OMVD) is an oncogenic and skin ulcerative condition among salmonid fish in Japan, and probably in the coastal rivers of eastern Asia that harbour Pacific salmon. Oncorhynchus masou virus (OMV), the causative virus, is also known as Yamame tumour virus (YTV), Nerka virus Towada Lake, Akita and Amori prefecture (NeVTA), coho salmon tumour virus (CSTV), Oncorhynchus kisutch virus (OKV), coho salmon herpesvirus (CSHV), rainbow trout kidney virus (RKV), or rainbow trout herpesvirus (RHV). For a recent and more detailed review of the condition, see refs 4, 12 and 13.
 
Fish species that are susceptible to OMV include: kokanee (sockeye) salmon (Oncorhynchus nerka), masou salmon (O. masou), chum salmon (O. keta), coho salmon (O. kisutch) and rainbow trout (O. mykiss) (6).
 
Clinically, the initial infection by OMV (taxonomically known as Salmonid herpesvirus 2; SalHV-2) appears as a systemic and frequently lethal infection that is associated with oedema and haemorrhages. Virus multiplication in endothelial cells of blood capillaries, haematopoietic tissue and hepatocytes underlies the clinical signs (7, 11). Four months after this first clinical condition, a varying number of surviving fish exhibit epithelioma occurring mainly around the mouth (upper and lower jaw) and, to a lesser extent, on the caudal fin, operculum and body surface. These neoplasia may persist for up to 1 year post-infection. In the case of coho salmon, 1-year-old infected fish in particular show ulcers on the skin, white spots on the liver and neoplastic tissues around the mouth parts or body surface. In rainbow trout, the diseased fish exhibit almost no external signs, although some fish manifest ulcerative lesions on the skin. Internally, intestinal haemorrhage and white spots on the liver are observed (5, 15, 16).
 
Following the septicaemia phase of OMV infection, an immune response takes place that results in the synthesis of neutralising antibodies to OMV. A carrier state frequently occurs that leads to virus shedding via the sexual products at the time of spawning.
 
On the basis of antigenic studies conducted with neutralising polyclonal rabbit antisera, OMV differs from Salmonid herpesvirus 1(SalHV-1), which is present in the western United States of America and is only weakly pathogenic (3, 9, 12).
 
The reservoirs of OMV are clinically infected fish and covert carriers among groups of cultured, feral or wild fish. Infectious virus is shed via faeces, urine, sexual products and probably skin mucus, while the kidney, spleen, liver and tumours are the sites where virus is the most abundant during the course of overt infection. The transmission of OMV is horizontal and possibly 'egg-surface associated'. Horizontal transmission may be direct or vectorial, water being the major abiotic factor. Animate vectors and inanimate objects also act in OMV transmission. Disinfection of the eggs just after fertilisation and eyed stage is effective in preventing OMV infection. OMV disease was not reported in alevins originating from disinfected eggs that had been incubated and hatched in virus-free water (14).
 
Salmonids are the only fish species susceptible to OMV infection; the order of the fish species from the most to the least susceptible is: kokanee salmon, chum salmon, masou salmon, coho salmon and rainbow trout. The age of the fish is critical and 1-month-old alevins are the most susceptible target for virus infection (6). The main environmental factor favouring OMV infection is low water temperature (below 14°C) (10).
 
The screening procedures for OMV are based on direct isolation of the virus in cell culture and co-culture of neoplastic tissues with salmonid cell lines (8). Confirmatory testing is by immunological identification using neutralisation or immunofluorescence tests, and virus-specific gene detection using polymerase chain reaction.
 
Control methods currently rely on the implementation of avoidance and hygiene practices in the operating of salmonid husbandry. The thorough disinfection of fertilised eggs and the incubation of these eggs and rearing of fry and alevins in premises completely separated from those harbouring virus carriers and free from contact with inanimate objects are the key measures needed to decrease contamination of OMV in a defined fish production site (14).
 

DIAGNOSTIC PROCEDURES

The screening for and diagnosis of Oncorhynchus masou virus disease (OMVD) is based on direct methods, which are either the isolation of the Oncorhynchus masou virus (OMV) in cell culture followed by its immunological identification (conventional approach), or the immunological demonstration of OMV antigen in infected fish tissues (2, 5).
 
Due to insufficient knowledge of the serological responses of fish to virus infections, the detection of fish antibodies to viruses has not thus far been recognised as a valuable diagnostic method for assessing the viral status of fish populations. However, the validation of some serological techniques for diagnosis of certain fish virus infections could arise in the near future, rendering the use of fish serology more widely acceptable for diagnostic purposes.
 
Infected fish material suitable for virological examination is:
 

.	Clinically affected fish: Whole alevin (body length < or = 4 cm), viscera including kidney (4 cm < or = body length < or = 6 cm) or, for larger size fish, skin ulcerative lesions or neoplastic tissues, and kidney, spleen, liver and encephalon.
.	Asymptomatic fish (apparently healthy fish): Kidney, spleen and encephalon (any size fish) and/or ovarian fluid from broodfish at spawning time.

Sampling procedures: see Chapter I.1. Section B.
 

1.	Standard Screening Method for OMV
	1.1.	Isolation of OMV in cell culture
		Cell line to be used: RTG-2 or CHSE-214
		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 at 10-15°C and, without withdrawing the inoculate, add 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 10-15°C.
		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 14 days. The use of a phase-contrast microscope is recommended.
			ii)	Maintain the pH of the cell culture medium at between 7.3 and 7.6 during incubation. This can be achieved by the addition to the inoculated cell culture medium of sterile bicarbonate buffer (for tightly closed cell culture flasks) or Tris buffer solution (for cell culture plates) or, even better, by using HEPES-buffered medium (HEPES = N-2-hydroxyethyl-piperazine-N-2-ethanesulfonic acid).
			iii)	If a cytopathic effect (CPE) appears in those cell cultures inoculated with the dilutions of the tested homogenate supernatants, identification procedures have to be undertaken immediately (see Section 1.2. below).
				If a fish health surveillance/control programme is being implemented, provisions 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 virus-positive sample originated. The suspension of approved status will be maintained until it is demonstrated that the virus in question is not OMV.
			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 controls fail to develop CPE, the process should be repeated with fresh susceptible cells and new batches of samples.
		c)	Subcultivation procedures
			i)	Collect aliquots of cell culture medium from all monolayers inoculated with dilutions of each supernatant of organ homogenates.
			ii)	If required, repeat the neutralisation test to infectious pancreatic necrosis virus (IPNV) and/or infectious haematopoietic necrosis virus (IHNV) as previously described (see Chapter I.1. Section B.3.3.), with dilution of the above supernatant (1/1 to 1/100).
			iii)	Inoculate cell monolayers as described above in Section 1.1.a.
			iv)	Incubate and monitor as described above in Section 1.1.b.
			v)	If no CPE occurs, the test may be declared negative.
		d)	Isolation of OMV from cultures of neoplastic cells
			i)	Collect neoplastic tissues, disinfect with iodophore, 50 parts per million for 20 minutes, and wash three times with Hanks' balanced salt solution.
			ii)	The tissues are left overnight in 0.25% trypsin in phosphate buffered saline (PBS) at 5°C. Then, 3.5 x 105 neoplastic cells/ml are seeded in a tissue culture flask and incubated with culture medium containing 20% fetal bovine serum (FBS).
			iii)	Harvest the primary neoplastic cell culture and co-cultivate with RTG-2 or CHSE-214 cells.
			iv)	Incubate and monitor as described in Section 1.1.b.
	1.2.	Identification of OMV
		a)	Neutralisation test
			i)	Collect the culture medium of the cell monolayers exhibiting CPE and centrifuge at 2000 g for 15 minutes at 4°C to remove cell debris.
			ii)	Dilute the virus-containing medium from 10-2 to 10-4.
			iii)	Mix aliquots (for example 200 µl) of each virus dilution with equal volumes of an antibody solution specific for OMV, 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.)
			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, to ensure that no IPNV contaminant has escaped the first anti-IPNV test.
			vi)	Incubate all the mixtures at 15°C for 1 hour.
			vii)	Transfer aliquots of each of the above mixtures on to 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.
			viii)	When adsorption is completed, add cell culture medium supplemented with 2% FCS and buffered at pH 7.4-7.6 into each well and incubate at 10-15°C.
			ix)	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 20% ethanol.
			x)	The tested virus is identified as OMV when CPE is prevented or noticeably delayed in the cell cultures that had received the virus suspension treated with the OMV-specific antibody, whereas CPE is evident in all other cell cultures.
			xi)	In the absence of any neutralisation by NAb to OMV, it is mandatory to conduct an indirect fluorescent antibody test (IFAT) with the suspect sample.
		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 4 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 OMV 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 48 hours.
			v)	Remove the cell culture medium, rinse once with 0.01 M PBS, pH 7.2, then three times briefly with cold acetone (stored at -20°C) for cover-slips or a mixture of acetone 30%/ethanol 70%, 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.
			viii)	Prepare a solution of purified antibody or serum to OMV 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 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. 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)-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.
			xiii)	Rinse four times with PBST.
			xiv)	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.
			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.
		c)	Enzyme-linked immunosorbent assay
			i)	Coat the wells of microplates designed for enzyme-linked immunosorbent assays (ELISAs) with appropriate dilutions of monoclonal antibody or purified immunoglobulins (Ig) specific for OMV, in 0.01 M PBS, pH 7.2 (200 µl/well).
			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 a two- or four-step dilution of the virus to be identified and of OMV control virus, and allow to react with the coated antibody to OMV for 1 hour at 20°C.
			viii)	Rinse four times with PBST.
			ix)	Add to the wells, biotinylated polyclonal antibody to OMV.
			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.
			xiv)	Add the substrate and chromogen. Stop the course of the test when positive controls react, and monitor the results.
		d)	Polymerase chain reaction method
			i)	Extract nucleic acid from cells infected with OMV strains and SalHV-1 using the InstGene Matrix (Biorad).
			ii)	Pellet the virus-infected tissues or infected cultured cells by centrifugation at 19,000 g (14,800 rpm) for 15 minutes.
			iii)	Wash the pellets twice with 1 ml PBS and mix with 200 ìl of chelating resin (Sigma).
			iv)	Incubate the mixture at 56°C for 20 minutes in a water bath, vortex it, and then place it in a boiling water bath for 8 minutes.
			v)	Vortex the samples and centrifuge at 8200 g (10,000 rpm) for 90 seconds.
			vi)	Subject the supernatant to PCR.
			vii)	The forward primer (F10) is 5'-GTA-CCG-AAA-CTC-CCG-AGT-C-3', and the reverse primer (R5) is 5'-AAC-TTG-AAC-TAC-TCC-GGG-G-3'.
			viii)	Incubate the specimens, primer sets and reaction mixtures for 30 cycles in an automatic thermal cycler (GeneAmp PCR 9700, Applied Biosystems), with each cycle consisting of denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds and extension at 72°C for 30 seconds.
			ix)	Analyse the amplified product for size and purity by electrophoresis (100 V for 30 minutes) in 2% agarose gel and stain with ethidium bromide.
			x)	A PCR using these primer sets amplified a 439 base-pair segment of DNA from OMV strains isolated from masu salmon, coho salmon and rainbow trout, and liver, kidney, brain and nervous tissues, and an 800 base-pair segment of DNA from SalHV-1. SalHV-1 and SalHV-2 could be distinguished by agarose gel profile of this amplified DNA (1).
2.	Diagnostic Methods for OMV
	2.1.	Virus isolation with subsequent identification
		As in Sections 1.1. and 1.2.
	2.2.	Indirect fluorescent antibody test
		a)	Indirect fluorescent antibody test method
			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 Section B.3.1. in Chapter I.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 (BSA), in PBST for 30 minutes at 37°C.
			vii)	Rinse four times with PBST.
			viii)	Treat the imprints with the solution of antibody to OMV 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.
			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)	Microplate processing
			As described in Section 1.2.c. of this chapter up to step iv (inclusive).
		b)	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.
		c)	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.
		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 homogenate with 2% Triton X-100, as in Section 1.2.c. step vi, and 2 mM of phenyl methyl sulfonide fluoride; mix gently.
			iii)	Complete the other steps of the procedure described in Section 1.2.c.
			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 method
		a)	DNA extraction
			As described in Section 1.2.d. of this chapter up to step v (inclusive).
		b)	Primer set and PCR condition
			As described in Section 1.2.d of this chapter, steps vi-viii. PCR using these primer sets amplified a 439 base-pair segment of DNA from OMV. SalHV-1 and SalHV-2 could be distinguished by agarose gel profile of this amplified DNA (1).

REFERENCES

1.	Aso Y., Wani J., Klenner D.A.S. & Yoshimizu M. (2001). Detection and identification of Oncorhynchus masou virus (OMV) disease by polymerase chain reaction (PCR). Bull. Fish. Sci. Hokkaido Univ., 52, 111-116.
2.	Hayashi Y., Izawa H., Mikami T. & Kodama H. (1993). A monoclonal antibody cross-reactive with three salmonid herpesviruses. J. Fish Dis., 16, 479-486.
3.	Hedrick R.P., McDowell T.S., Eaton W., Kimura R. & Sano T. (1987). Serological relationships of five herpesvirus isolated from salmonid fishes. J. Ichthyol., 3, 87-92.
4.	Kimura T. & Yoshimisu M. (1989). Salmon herpesvirus: OMV, Oncorhynchus masou virus. In: Viruses of Lower Vertebrates, Ahne W. & Kurstak E., eds. Springer-Verlag, Berlin, Germany, 171-183.
5.	Kimura T., Yoshimisu M. & Tanaka M. (1981). Studies on a new virus (OMV) from Oncorhynchus masou II. Oncogenic nature. Fish Pathol., 15, 149-153.
6.	Kimura T., Yoshimisu M. & Tanaka M. (1983). Susceptibility of different fry stages of representative salmonid species to Oncorhynchus masou virus (OMV). Fish Pathol., 17, 251-258 (in Japanese).
7.	Kimura T., Yoshimisu M., Tanaka M. & Sannohe H. (1981). Studies on a new virus (OMV) from Oncorhynchus masou I. Characteristics and pathogenicity. Fish Pathol., 15, 143-147.
8.	Lannan C.N., Winton J.R. & Fryer J.L. (1984). Fish cells: Establishment and characterization of nine cell lines from salmonids. In Vitro, 20, 671-676.
9.	Roizman B. (1991). Family Herpesviridae. In: Classification and Nomenclature of Viruses, Francki R.I., Fauque C.M., Knudson D.L. & Brown F., eds. Arch. Virol., (Suppl. 2). Springer, New York, USA and Vienna, Austria, 103-110.
10.	Suzuki S., Kimura T. & Saneyoshi M. (1986). Characterization of DNA polymerase induced by salmon herpesvirus, Oncorhynchus masou virus. J. Gen. Virol., 67, 405-408.
11.	Tanaka M., Yoshimisu M. & Kimura T. (1984). Oncorhynchus masou virus: Pathological changes in masu salmon (Oncorhynchus masou), chum salmon (O. keta) and coho salmon (O. Kisutch) fry infected with OMV by immersion method. Bull. Jpn Soc. Scientif. Fisheries, 50, 431-437.
12.	Wolf K. (1988). Fish Viruses and Viral Diseases. Cornell University Press, Ithaca, New York, USA, 476 pp.
13.	Yoshimizu M., Fukuda H., Sano T. & Kimura T. (1995). Salmonid herpesvirus 2. Epizootiology and serological relationship. Vet. Res., 26, 486-492.
14.	Yoshimisu M., Nomura T., Ezura Y. & Kimura T. (1993). Surveillance and control of infectious hematopoietic necrosis virus (IHNV) and Oncorhynchus masou virus (OMV) of wild salmonid fish retruning to the northern part of Japan 1976-1991. Fisheries Res., 17, 163-173.
15.	Yoshimisu M., Tanaka M. & Kimura T. (1987). Oncorhynchus masou virus (OMV): Incidence of tumor development among experimentally infected representative salmonid species. Fish Pathol., 22, 7-10.
16.	Yoshimisu M., Tanaka M. & Kimura T. (1988). Histopathological study of tumours induced by Oncorhynchus masou virus (OMV) infection. Fish Pathol., 23, 133-138 (in Japanese).

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