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
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 Ž
. mean tissue culture infective dose TCID
for the two isolates and recorded as a
50
combined value for clarity of presentation. 3.1. Temperature stability
The effect of storage of nodavirus at temperatures between y208C and 378C over a period of 1 year is summarised in Table 1. Rapid inactivation occurred at 378C with no
Table 1 Ž
. Infectivity log TCID
of sea bass nodavirus held in cell culture medium at different temperatures
10 50
nd s not done. Ž
. 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
37 4.0
Table 2 Ž
. Infectivity log TCID
of sea bass nodavirus held at 158C in distilled water at pH 2–11
10 50
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
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
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 at y208C appeared to be stable.
3.2. pH sensitiÕity Sea bass nodavirus appeared relatively resistant to changes in pH with no reduction in
Ž .
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
Ž .
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 Ž
. Infectivity log TCID
of sea bass nodavirus held at 158C in differing electrolyte solutions
10 50
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
Seawater 37‰ 6.5
6.5 7.0
6.0 5.5
4.5 Seawater 20‰
7.0 8.5
5.5 5.0
5.5 4.0
Fig. 1. Inactivation of sea bass nodavirus in culture fluids following UV irradiation at 440 mWrcm
2
.
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 10
7.0
TCID to zero value in HBSS medium alone
50
and from 10
8.0
to 10
1.5
TCID in serum-supplemented medium. No residual viable virus
50
was detected after 1 h treatment.
Table 4 Ž
. Infectivity log TCID
of sea bass nodavirus in cell culture fluid following treatment with formalin at 158C
10 50
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 6 h
4.5 6.5
Table 5 Ž
. Infectivity
log TCID of sea bass nodavirus in distilled water and HBSSqFBS following chlorine
10 50
treatment at 158C Ž
. Ž
. Exposure time
distilled water Cl ppm HBSSqFBS Cl ppm
2 2
100 50
25 100
50 25
Control 7.25
7.25 7.25
7.25 7.25
7.25 5 min
3.0 5.5
7.0 7.25
15 min 2.0
5.0 6.0
6.5 30 min
5.0 6.5
6.0
3.5. UV irradiation UV light irradiation of infective culture fluid at 440 mWrcm
2
resulted in a 3 log Ž
. 99.9 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
Ž .
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 Ž
. 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 = 10
3
mW srcm
2
. 3.6. Formalin treatment
A small reduction in virus titre of infective culture fluids was noted after 5 min Ž
. 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 Ž
. Infectivity log TCID
of sea bass nodavirus in distilled water and HBSSqFBS following iodine treatment
10 50
at 158C Ž
. Ž
. Exposure time
distilled water I ppm HBSSqFBS I ppm
2 2
100 50
25 100
50 25
Control 6.125
6.125 6.125
6.125 6.125
6.125 5 min
2.0 2.5
5.0 15 min
1.5 2.0
5.5 30 min
1.5 3.5
5.0
Table 7 Ž
. Infectivity log TCID
of sea bass nodavirus in distilled water and HBSSqFBS following peroxygen
10 50
treatment at 158C Ž
. Ž
. Exposure time
distilled water perO ppm HBSSqFBS perO ppm
2 2
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 HBSS q 5
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 HBSS q 5 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
