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Temperature, pH and electrolyte sensitivity, and
heat, UV and disinfectant inactivation of sea bass
ž
Dicentrarchus labrax neuropathy nodavirus
/
G.N. Frerichs, A. Tweedie, W.G. Starkey
), R.H. Richards
Institute of Aquaculture, UniÕersity of Stirling, Stirling, Scotland, FK9 4LA, UK
Accepted 17 October 1999
Abstract
The effect of temperature, pH and electrolytes on the stability of two Mediterranean sea bass
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neuropathy nodavirus isolates SBNN and the inactivation of SBNN by heating, exposure to ultraviolet radiation and chemical disinfectants was investigated in vitro using a striped snakehead
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fish cell line SSN-1 for virus propagation and assay. The two nodavirus isolates showed no significant differences in response to the procedures examined. Nodavirus held in cell culture medium containing 5% foetal bovine serum was effectively inactivated within 4 days at 378C and 3 months at 258C but showed no significant loss of titre over 6 months at 158C and still retained a measurable level of infectivity after storage for 1 year at this temperature. Virus suspensions in distilled water tolerated exposure to pH 2–11 with no significant loss of titre over 24 h. Thereafter, a steady diminution in infectivity was noted at pH 11 from day 3 and at pH 2 from day 15 onwards, but no loss of titre was recorded between pH 3–7 over 6 weeks storage. Infectivity titres fell gradually at a very similar rate for virus held at 158C in balanced salt solution and full-and half-strength seawater over a 6-month test period. Virus held in freshwater, however, was markedly less stable and no viable virus could be detected after 6 months storage. SBNN was susceptible to heat treatment at 608C within 30 min with no viable virus detected after 1 h. UV irradiation at an intensity of 440mWrcm2 resulted in a 99.9% linear reduction in virus titre after 8 min exposure. Treatment with 2% formalin was not totally effective even after 6 hours exposure at 158C although virus titre was reduced. The effect of chlorine, iodine and peroxygen on SBNN
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at 158C was noticeably different when organic matter in the form of foetal bovine serum FBS was present in the virus diluent. Suspensions of virus in distilled water were completely inactivated within 5 min by 50 ppm chlorine and 25 ppm iodine. Virus in Hanks’ BSSqFBS, however, showed only a marginal loss in infectivity following similar treatments and significant
)Corresponding author. Tel.:q44-1786-4678-73.
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E-mail address: [email protected] W.G. Starkey .
0044-8486r00r$ - see front matterq2000 Elsevier Science B.V. All rights reserved. Ž .
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.q2000 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 retinopaencephalopa-thy with high mortality in larval and juvenile stages of affected fish was
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first recognised over 10 years ago in hatchery-reared sea bass Dicentrarchus labrax in
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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
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proteins of virion material obtained directly from sea bass larvae Comps et al., 1994 ,
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and Arimoto et al. 1996 were able to assess a range of chemical and physical
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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
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system for piscine nodaviruses. The recently described snakehead cell line SSN-1
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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
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with 5% foetal bovine serum FBS and antibiotics at 208C as previously described
ŽFrerichs 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
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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. HBSSq10% FBS.
3. De-chlorinated, Cu2q-free, tapwater.
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4. Artificial seawater, 37% salinity Instant Ocean; Aquarium Systems, France .
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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
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Five ml of a 1:100 dilution of each nodavirus preparation clarified at 1000=g for
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15 min was made both in Hanks’ balanced salt solution HBSS alone and HBSSq10%
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foetal bovine serum FBS . Pre-treatment control samples 200ml were removed before
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2.6. UV irradiation
Clarified culture fluid preparations for each virus strain were poured into petri dishes
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to a depth of 3–4 mm MacKelvie and Desautels, 1975 . The dishes were placed below
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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 mWrcm2 as measured by a digital radiometer
ŽUVP . 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
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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
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metabisulphite Na S O2 2 5 dissociates in water to form 2 moles sodium bisulphite which 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
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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 HBSSq5% 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
ŽBuffodine; Evans Vanodine International, UK was diluted in distilled water to provide.
2.10. Peroxygen treatment
Clarified infective nodavirus culture fluids diluted 1:100 in HBSSq5% FBS and in
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distilled water were mixed with equal volumes of 1:250, 1:500 and 1:1000 wrv
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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-15q5% 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
finally recorded after 10–14 days.
3. Results
There were no significant differences between the sensitivity of the Greek and Maltese nodavirus strains to temperature, pH and electrolyte treatments or any of the inactivation procedures used in the study. All virus titres were therefore calculated as the
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mean tissue culture infective dose TCID50 for the two isolates and recorded as a
combined value for clarity of presentation.
3.1. Temperature stability
The effect of storage of nodavirus at temperatures betweeny208C and 378C over a period of 1 year is summarised in Table 1. Rapid inactivation occurred at 378C with no
Table 1
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Infectivity log TCID10 50 of sea bass nodavirus held in cell culture medium at different temperatures ndsnot done.
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Storage temperature 8C Storage time
1 day 4 days 7 days 2 weeks 4 weeks 3 months 6 months 1 year
y20 nd nd nd nd 6.0 7.0 7.0 7.5
4 6.5 7.5 5.5 7.5 6.5 6.5 6.0 2.5
15 nd nd 7.0 7.0 6.5 6.5 6.0 4.0
25 nd 7.0 5.5 5.5 5.0 0
Table 2
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Infectivity log TCID10 50 of sea bass nodavirus held at 158C in distilled water at pH 2–11 pH Treatment time
1 h 1 day 3 days 7 days 15 days 21 days 42 days
2 6.5 6.5 6.5 6.5 4.5 1.5 0
3 6.5 6.5 6.5 6.5 6.0 6.5 6.0
7 6.5 6.5 6.5 6.5 6.0 6.5 6.0
9 6.5 6.5 6.5 6.5 6.0 6.5 6.0
11 6.5 5.5 4.0 4.0 0
viable virus detectable after 4 days at this temperature. A more gradual loss of
infectivity was noted at 258C with viable virus still present after 4 weeks but not
detectable at 3 months. No significant reduction in virus titre was recorded over 6 months at 158C or lower and the fluid preparations held at 48C or 158C still retained a measurable degree of infectivity after 1 year. Virus stored in the frozen state aty208C appeared to be stable.
3.2. pH sensitiÕity
Sea bass nodavirus appeared relatively resistant to changes in pH with no reduction in
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virus titre recorded after 1 h incubation at 158C across the pH range 2–11 Table 2 .
Inactivation was first noted at pH 11 after 24 h with a progressive reduction in titre to a
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zero value at 15 days. A 2 log 99% reduction in virus infectivity was also noted at pH 2 at day 15 with complete inactivation by day 42. No loss in infectivity was recorded over the pH range 3–9 during the 42-day test period.
3.3. Effect of electrolytes
The mean infectivity titres of the two strains of sea bass nodavirus following
incubation at 158C in a range of suspending solutions are shown in Table 3. Virus
maintained in a balanced salt solution with or without serum supplementation and
Table 3
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Infectivity log TCID10 50 of sea bass nodavirus held at 158C in differing electrolyte solutions Electrolyte solution Holding time
1 day 4 days 16 days 32 days 3 months 6 months
HBSS 8.0 8.0 7.5 7.0 6.5 4.5
HBSSqFBS 9.0 8.5 9.0 7.0 7.0 4.5
Freshwater 6.5 7.0 7.0 5.0 4.5 0
Seawater 37‰ 6.5 6.5 7.0 6.0 5.5 4.5
Fig. 1. Inactivation of sea bass nodavirus in culture fluids following UV irradiation at 440mWrcm2.
artificial seawater at 37‰ or 20‰ salinity all showed only a 1–2 log reduction in infectivity titre over 3 months with a further similar loss over the next 3 months. Virus held in freshwater, however, was markedly less stable with a 1–2 log titre reduction noted after 1 month progressing to no detectable viable virus at the end of the 6-month test period.
3.4. Heat treatment
Sea bass nodavirus was rapidly inactivated by heat treatment at 608C. Within 30 min
virus infectivity was reduced from 107.0TCID50 to zero value in HBSS medium alone
and from 108.0 to 101.5TCID in serum-supplemented medium. No residual viable virus
50 was detected after 1 h treatment.
Table 4
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Infectivity log TCID10 50 of sea bass nodavirus in cell culture fluid following treatment with formalin at 158C
Exposure time Control Treatment
2% formalin 0.025% formalin
5 min 8.75 8.5 8.75
30 min 7.0 7.5
1 h 6.0 7.0
Table 5
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Infectivity log TCID10 50 of sea bass nodavirus in distilled water and HBSSqFBS following chlorine treatment at 158C
Ž . Ž .
Exposure time distilled water Cl ppm2 HBSSqFBS Cl ppm2
100 50 25 100 50 25
Control 7.25 7.25 7.25 7.25 7.25 7.25
5 min 0 0 3.0 5.5 7.0 7.25
15 min 0 0 2.0 5.0 6.0 6.5
30 min 0 0 0 5.0 6.5 6.0
3.5. UV irradiation
UV light irradiation of infective culture fluid at 440 mWrcm2 resulted in a 3 log
Ž99.9% linear reduction in virus titre during an 8 min exposure time. Over the same.
time period there was a corresponding linear increase in concentration of interfering virus particles in the irradiated fluid, as shown by the increasing number of positive
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wells in which development of cpe was inhibited Fig. 1 . No positive virus samples were recorded following 10 min irradiation, but extrapolation of the increasing level of interference suggests that complete masking of infective viral particles would have
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occurred at this point in time. It can therefore only be inferred that a 4 log 99.99% loss
of infectivity was attained after UV exposure for 10 min equating to 290=103 mW
srcm2.
3.6. Formalin treatment
A small reduction in virus titre of infective culture fluids was noted after 5 min
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exposure at 158C to 2% and 0.025% formalin Table 4 . Even after 30 min exposure
virus infectivity was reduced by less than 2 logs for either treatment. Extended exposure for 6 h resulted in a 4 log fall in virus titre with 2% formalin but still only a 2 log reduction with the less concentrated solution.
Table 6
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Infectivity log TCID10 50 of sea bass nodavirus in distilled water and HBSSqFBS following iodine treatment at 158C
Ž . Ž .
Exposure time distilled water I ppm2 HBSSqFBS I ppm2
100 50 25 100 50 25
Control 6.125 6.125 6.125 6.125 6.125 6.125
5 min 0 0 0 2.0 2.5 5.0
15 min 0 0 0 1.5 2.0 5.5
Table 7
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Infectivity log TCID10 50 of sea bass nodavirus in distilled water and HBSSqFBS following peroxygen treatment at 158C
Ž . Ž .
Exposure time distilled water perO ppm2 HBSSqFBS perO ppm2
1:125 1:250 1:500 1:125 1:250 1:500
Control 6.375 6.375 6.375 6.375 6.375 6.375
5 min 2.5 2.0 6.0 3.5 5.5 6.0
15 min 4.0 3.0 3.0 3.5 4.5 6.0
30 min 3.0 2.0 3.0 3.5 4.0 5.5
3.7. Chlorine inactiÕation
The effect of chlorine on the survival of nodavirus in distilled water and in serum
supplemented HBSS at 158C is shown in Table 5. Virus in distilled water was
completely inactivated within 5 min of exposure to 50 ppm chlorine. Virus in HBSSq5%
FBS, however, showed only a marginal loss in infectivity with similar treatment and little more than a 2 log fall in virus titre after 30 min exposure to 100 ppm chlorine.
3.8. Iodine inactiÕation
The disinfectant activity of buffered iodophore against nodavirus in distilled water
and serum supplemented HBSS at 158C is shown in Table 6. Virus in distilled water
appeared to be completely inactivated within 5 min of exposure to 25 ppm available
iodine. As was noted with chlorine treatment, however, virus suspended in HBSSq5%
FBS showed only a small reduction in virus titre with a similar treatment and a residual level of infectious virus could still be detected after 30 min exposure to 100 ppm available iodine.
3.9. Peroxygen inactiÕation
The efficacy of a peroxygen disinfectant against nodavirus at 158C is shown in Table 7. At a 1:125 dilution, virus titres were rapidly reduced within 5 min by 3–4 logs in both distilled water and serum-supplemented HBSS but no further decrease in infectivity was noted up to 30 min exposure. A similar overall pattern of inactivation was recorded for 1:250 and 1:500 dilutions of disinfectant although the actual degree of inactivation was progressively less in the FBS-containing HBSS diluent.
4. Discussion
For logistical reasons, the assay of virus infectivity was performed using an end-point titration in the present study. This method was particularly suited to the large number of samples generated. Similar procedures have been used for infectivity assays of other
Ž . Ž .
and have been shown to be economical, accurate, and of comparable sensitivity to
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plaque assays Scotti, 1977 . We have occasionally observed inhibition of cytopathic
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effects in positive samples where virus titres are very high unpublished observations . This may be due to the presence of defective-interfering particles, which have been observed in many RNA virus families and whose amplification is favoured at high
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multiplicity of infection Holland et al., 1982 . Inhibition of cytopathic effects at high virus titre was more pronounced in UV-irradiated virus samples. This may have resulted from the effects of UV irradiation on nodavirus replication acting to increase the number of interfering particles. This phenomenon has previously been described for vesicular
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stomatitis virus VSV ; where UV-irradiated virus was found to inhibit virus replication
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to the same extent as VSV defective-interfering particles Miller and Lenard, 1982 .
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The rapid inactivation of sea bass neuropathy nodavirus SBNN at 608C over a
period of 30 min is comparable to the findings of Arimoto et al. and suggests that standard heat sterilisation procedures such as autoclaving at 1218C for 15 min at 15 psi
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would ensure total destruction of the virus Mahy and Kangro, 1996 . At a more practical level, it would also seem likely that treatment of non heat-labile equipment and
hatchery water by heating to 608C or above for not less than 30 min would render all
contaminating virus inactive.
UV irradiation treatment of hatchery water can be affected by the particulate nature
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of sea water inhibiting light penetration Arimoto et al., 1996 . This notwithstanding, Australian trials of UV sterilisation procedures on water-suspended model viruses concluded that viral inactivation under hatchery water-flow conditions was practicable
ŽMiocevic et al., 1993 . UV irradiation treatment of SBNN infected clarified culture.
fluid at 440 mWrcm2 reduced virus titre by 99.9%. The undetectable residual virus,
however, may still be sufficient to produce infection in vivo and it would be unwise to disregard this possibility when proposing UV irradiation as a sole means of disinfecting hatchery water supplies. It has been reported that UV-irradiated viable virus becomes
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more susceptible to other disinfectant methods MacKelvie and Desautels, 1975 , but this was not investigated in the present study.
Although 2% formalin has been shown to be effective against piscine viruses such as
Ž . Ž .
IPNV and Egtved virus Jorgensen, 1973 and snakehead rhabdovirus Frerichs 1990 , it
¨
is concluded that it would be of little value as a disinfectant against SBNN when a significant level of viable virus could still be detected after a 6-h treatment period. The
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concentrations of formalin Arimoto et al. 1996 tested had no effect and the lower concentration of 0.025% used in this study would be similarly ineffective in the treatment of fish stocks and water systems.
SBNN was completely inactivated by chlorine at a concentration of 50 ppm within 5
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min and by 25 ppm chlorine within 30 min when no organic material FBS was present. When the virus was diluted in HBSS containing 5% FBS the efficacy of the chlorine disinfectant was dramatically reduced, with less than a 2 log reduction in virus titre after 5 min contact with 100 ppm chlorine. Similarly, inactivation of the virus with iodine
was limited by FBSrHBSS although not to the same extent as with chlorine. As
previous studies have shown, the successful use of chlorine and iodine based disinfec-tants is greatly influenced by the presence of organic material and this must be allowed
Ž
.
MacKelvie 1975, Frerichs 1990 . For both these compounds, the period of contact with
Ž
the virus has been reported as an influencing factor in their efficacy Desautels and
.
MacKelvie 1975, Frerichs 1990 . However, this study shows that disinfectant concentra-tion, particularly for buffered iodophor, has the greatest effect on SBNN activity with the virus titre decreasing for each increasing concentration but then remaining constant over a 30-min contact period.
The buffered acid peroxygen disinfectant was effective in reducing but not eliminat-ing viable SBNN. Accordeliminat-ing to the summary of virucidal test results supplied by the manufacturer, an effective concentration would result in a 4 log reduction in the titre of the test virus in the presence of organic matter at 48C after a contact period of 30 min. This was achieved for the nodavirus in distilled water but not with 5% FBS in HBSS. The manufacturer’s test result summary also indicates that effective dilutions ranged
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from 1:50 for canine parvovirus to 1:2000 for HIV so it is possible that a higher concentration of disinfectant than presently selected would be more effective against SBNN. The application of this disinfectant would probably be restricted to equipment and water effluent.
Little information has so far become available on the survival of piscine nodaviruses under different environmental conditions due in large part to the absence of a laboratory cell culture system for the isolation, propagation and assay of the infectious agent. The
Ž .
recent development of a suitable cell culture procedure by Frerichs et al. 1996 has now overcome this limitation and facilitated the present study. The results essentially confirm
Ž .
the assertion of the OIE Fish Disease Commission 1995 that these small viruses might be quite resistant to environmental conditions and therefore readily spread by maricul-ture activities. Nodavirus disease of Mediterranean sea bass tends to be seasonal in
Ž
severity with the more acute form developing at higher temperatures Le Breton et al.,
.
1997 . Survival of the virus for at least 1 month at 258C would thus allow wide
distribution of the agent through the aquatic environment during the summer months when severe disease epizootics may be expected, and survival for a least 1 year at 158C would ensure natural carryover and dissemination of infectious virus through the marine environment during the winter.
Sea bass nodavirus appeared markedly tolerant to pH change as evidenced by the stability of infectivity over the pH range 3–9 during a 42 day test period. The degree of stability shown at pH 2 is also exceptional for the generality of animal viruses. The comparatively greater sensitivity to alkaline pH was also noted for striped jack nervous
Ž .
necrosis virus by Arimoto et al. 1996 who adopted the treatment of nets and equipment with an alkaline solution pH 12 as a disinfection procedure.
As might be expected for a small, unenveloped, icosahedral virus pathogen recovered from the marine environment, the sea bass nodavirus isolates proved equally stable in a range of salt solutions of different composition and tonicity. Nor did the addition of organic material in the form of bovine serum alter the stability profile. Suspension in salt-free tapwater, however, led to complete inactivation within six months suggesting that even if freshwater fish species may be inherently susceptible to infection, piscine nodavirus is unlikely to be found in this environment.
nodavirus to survive for extended periods of time under a wide range of environmental conditions strongly suggests that horizontal spread may be a significant factor in the dissemination of infection.
Acknowledgements
This study was supported and funded by the UK Overseas Development Administra-tion.
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