levels of infectivity were still detectable after 30 min exposure to 100 ppm of either disinfectant. Treatment with an acid peroxygen disinfectant rapidly reduced virus infectivity in both distilled
water and serum supplemented diluent within 5 min but infectious virus was not totally eliminated even after exposure for 30 min. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Disinfectant inactivation; Dicentrarchus labrax; Neuropathy nodavirus
1. Introduction
Nodavirus infection characterised by the development of a vacuolating encephalopa- thy and retinopathy with high mortality in larval and juvenile stages of affected fish was
Ž .
first recognised over 10 years ago in hatchery-reared sea bass Dicentrarchus labrax in Ž
. Martinique and the French Mediterranean Breuil et al., 1991 . The disease has since
been recorded in a wide variety of fish species from mariculture sites in Japan, southeast Asia, Australia, the south Pacific and Norway as well as the Mediterranean and is
probably the most serious disease threat to the marine finfish aquaculture industry at the present time.
In the development of effective control and eradication measures against virus diseases of international importance it is essential to establish the susceptibility of the
infectious agent to physical and chemical inactivation agents and evaluate the stability of the nodavirus under differing environmental conditions in order to implement rational
disinfection procedures. Although the virus could be classified as a member of the Nodaviridae following biochemical characterisation of the nucleic acid and structural
Ž .
proteins of virion material obtained directly from sea bass larvae Comps et al., 1994 , Ž
. and Arimoto et al.
1996 were able to assess a range of chemical and physical Ž
. treatments on virus purified directly from infected striped jack Pseudocaranx dentex
larvae, such studies have hitherto been hampered by the lack of an in vitro cell culture Ž
. system for piscine nodaviruses. The recently described snakehead cell line SSN-1
Ž .
culture system for the isolation and propagation of the nodavirus Frerichs et al., 1996 has now greatly facilitated the laboratory characterisation of this agent, and the present
in vitro study evaluates the efficacy of heat treatment, UV irradiation and chemical disinfectants for the inactivation of nodavirus isolated from Mediterranean sea bass and
the stability of the nodavirus under different environmental conditions.
2. Materials and methods
2.1. Virus preparations Two strains of piscine nodavirus recovered from diseased juvenile sea bass cultured
in the Mediterranean off Greece and Malta were used. The viruses were isolated in Ž
. striped snakehead cell line SSN-1 cultures using Leibovitz L-15 medium supplemented
Ž .
with 5 foetal bovine serum FBS and antibiotics at 208C as previously described Ž
. Frerichs et al., 1996 . Working preparations were obtained by further propagating each
strain on SSN-1 monolayer cultures at 258C and harvesting cell culture fluids when the infected monolayers had been completely destroyed 5–6 days later. Both virus isolates
were used for all inactivation and sensitivity tests.
2.2. Temperature stability Harvested cell culture fluids were clarified by centrifugation at 1000 g for 15 min and
Ž .
distributed in 1 ml aliquots in cryotubes Nunclon . Cryotube samples were held at y20, 4, 15, 25 and 378C and assayed for virus infectivity following storage for periods
between 1 day and 1 year.
2.3. pH sensitiÕity Five milliliters of a 1:100 dilution of each nodavirus preparation was made in aliquots
of sterile distilled water initially adjusted by the addition of HCl or NaOH to give a series of solutions at pH 2, 3, 5, 7, 9 and 11. Virus dilutions were held at 158C and
samples removed for infectivity assay at selected intervals between 1 and 42 days later.
2.4. Effect of electrolytes A 1:100 dilution of each clarified nodavirus cell culture harvest was made in the
following solutions:
2q 2q
Ž .
1. Hanks’ balanced salt solution without Ca and Mg
HBSS . 2. HBSS q 10 FBS.
3. De-chlorinated, Cu
2q
-free, tapwater. Ž
. 4. Artificial seawater, 37 salinity Instant Ocean; Aquarium Systems, France .
Ž 5. Estuarine water, 20 salinity Obtained from Fife Coast, Scotland, autoclave ster-
. ilised
All diluted virus preparations were held at 158C and samples removed for infectivity assay at selected intervals over a period of 6 months.
2.5. Heat treatment Ž
Five ml of a 1:100 dilution of each nodavirus preparation clarified at 1000 = g for .
Ž .
15 min was made both in Hanks’ balanced salt solution HBSS alone and HBSS q 10 Ž
. Ž
. foetal bovine serum FBS . Pre-treatment control samples 200 ml were removed before
Ž .
placing the diluted preparations in a water bath at 608C. Further samples 200 ml were taken after 30 min, 1 h, 2 h and 3 h treatment and immediately cooled and held at 158C
until all samples were available for infectivity assay.
2.6. UV irradiation Clarified culture fluid preparations for each virus strain were poured into petri dishes
Ž .
to a depth of 3–4 mm MacKelvie and Desautels, 1975 . The dishes were placed below Ž
. a UV lamp Mineralight UVGL 58, UVP with a wavelength of 254 nm at a distance set
to give a surface light intensity of 440 mWrcm
2
as measured by a digital radiometer Ž
. UVP . Samples were removed for infectivity assay at the start of the experiment and at
2, 4, 6, 8, and 10 min after UV light exposure. This study was performed at 208C. 2.7. Formalin treatment
Aliquots of clarified cell culture fluid for each virus strain were mixed with equal Ž
volumes of freshly prepared 4 and 0.05 solutions of commercial formalin 37 .
Ž .
formaldehyde in phosphate buffered saline PBS , pH 7.2, giving final concentrations of 2 and 0.025 formalin. Reaction mixtures were held at 158C and samples withdrawn
after 5 min, 30 min, 1 h and 6 h incubation. On the basis that 1 mole sodium Ž
. metabisulphite Na S O
dissociates in water to form 2 moles sodium bisulphite which
2 2
5
will then neutralize 2 moles formaldehyde, residual free formaldehyde in withdrawn samples was neutralized by the immediate addition of an equal volume of 4 or 0.04
Na S O in PBS as appropriate.
2 2
5
2.8. Chlorine inactiÕation Solutions containing 200, 100 and 50 ppm available chlorine in distilled water were
Ž prepared from commercially available dichloroisocyanurate disinfectant tablets Presept;
. Johnson Johnson, UK and added in equal volumes to clarified nodavirus culture fluids
diluted 1:100 in HBSS q 5 FBS and also in distilled water to provide reaction mixtures containing 100, 50 and 25 ppm available chlorine. Aliquots of each virus-disin-
fectant mixture were taken after 5, 15 and 30 min incubation at 158C and residual-free chlorine neutralized by the immediate addition of equal volumes of 0.00035 M sodium
thiosulphate.
Positive virus controls comprising diluted culture fluids mixed with equivalent volumes of distilled water or neutralized iodophore only were also prepared and held at
158C for 30 min. 2.9. Iodine inactiÕation
A buffered iodophore fish farming disinfectant containing 1–1.6 available iodine Ž
. Buffodine; Evans Vanodine International, UK was diluted in distilled water to provide
solutions containing not less than 200, 100, and 50 ppm available iodine. As for the chlorine inactivation procedure, the disinfectant solutions were added in equal volumes
to infective nodavirus fluids diluted in HBSS q 5 FBS and in distilled water to provide reaction mixtures containing 100, 50, and 25 ppm available iodine. Aliquots of each
virus-disinfectant mixture were similarly taken after 5, 15, and 30 min incubation at 158C and residual free iodine neutralized using 0.0008 M sodium thiosulphate.
2.10. Peroxygen treatment Clarified infective nodavirus culture fluids diluted 1:100 in HBSS q 5 FBS and in
Ž .
distilled water were mixed with equal volumes of 1:250, 1:500 and 1:1000 wrv Ž
. dilutions of a buffered acid peroxygen disinfectant Virkon; Antec International, UK .
Reaction mixtures were held at 158C for 5, 15 and 30 min when samples were taken and treated according to the manufacturer’s instructions with 9 vol. of neutralizer solution.
Positive virus controls of diluted culture fluids mixed with appropriate volumes of distilled water or neutralized disinfectant only and held at 158C for 30 min were
included in the test. 2.11. Virus assay
Virus infectivity assays were carried out by preparing serial 10-fold dilutions of the test sample across 96-well microtitration plates using Leibovitz L-15 q 5 FBS cell
culture growth medium as diluent. An equal volume of a suspension of SSN-1 cells in the same medium was added to each well and the plates incubated at 258C. Results were
