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New method for virucide testing
Journal o f Virological Methods, 1 (1980) 27 - 31 ©Elsevier/North-Holland Biomedical Press
27
NEW METHOD FOR VIRUCIDE TESTING
G. VAN DER GROEN, A. A. EL MEKK1and S. R. PATTYN lnstituut voor Tropische Geneeskunde, Laboratory of Bacteriology and Virology, Nationalestraat 155, B- 2OOOAntwerpen, Belgium (Accepted 16 October 1979)
The virus virucide floating technique is a simple and rapid method of testing virucides in vitro. The method allows testing of the effect of high concentrations of disinfectants and the use of low titred viruses. Removal of excess disinfectant is rapid and simple and perfect control of contact time between virus and disinfectant is possible. The virus is attached to a surface, simulating as much as possible the in use conditions of the disinfectant. The method was used to study the effect of certain disinfectants on some arboviruses. INTRODUCTION The major problems for virucide testing are: rapid removal of excess disinfectant toxicity of disinfectant for tissue cultures necessity for high virus titers difficulties in detection o f low residual viral activity exact time of contact between virus and disinfectant. In order to solve these problems we developed the virucide floating technique. MATERIALS AND METHODS Cells and viruses Vero and BHK-21 cells were cultured in TC medium 199 supplemented with 7% and 10% inactivated calf serum respectively, buffered with 13 mM sodium bicarbonate. Virus stocks of vesicular stomatitis virus (VSV), chikungunya virus (CHIK), West Nile (WN) virus, Bangyi (BGN) virus, and a bunya-like virus (El Mekki, 1979) were prepared in Vero cells. AnY 1444, an orbivirus (El Mekki, 1979) was prepared in BHK-21 cells.
28
Virus assays All viruses, except AnY 1444, were assayed in Vero cells by the 50% end point method as previously described (Van der Groen, 1976). Serial 10-fold dilutions of AnY 1444 virus in TC medium 199 with 2% inactivated calf serum were inoculated into four wells of microtissue culture plates each well containing 5 X 103 BHK-21 cells per well.
Disinfectants Formaldehyde (FA) 35% (Merck) and glutaraldehyde (GA) 70% (Ladd) stock solutions were diluted in sterile distilled water to the appropriate rmal concentrations. A commercially available sodium hypochlorite solution (NaOC1) containing 1 6 0 - t 7 0 g of active chlorine/l was used undiluted. Uranylacetate (Fluka A.G.) was dissolved in water (1 g/100 ml) and used as 1% solution.
Virucide floating technique A block of 2% agarose (see Fig. 1) of about 1 cm 2 was cut and mounted on the edge of a glass slide. A known quantity of virus suspension was put on the surface of the agarose ~1 Drop Of virus suspension block
-r'277~7>~/TZT.71~z~/~2~
2°1~ 0 g o c o s e "~ glide
liaa. ~]lllll/lll/llfll}lllllll]J ,k
-II virus Ioye# (dry)
0
pzz
'.,-,' / 2 / ~
~ Drop of formvor Solution
~ Formotion of for-mvoF f~]m
~ Releose of viFus f i l m in dlslnfectont or" medium
p=
_ _~ _ _~ -:~-~-:-----Z=-::.-
L;
-~, . . . .
med~om
d~s~f . . . . . . . .
~1 Removal Of f l i m
D[SrmPetiiuO~mof virus f i l m
~
Fig. 1. Virucide floating technique.
hr
29 and allowed to diffuse and evaporate until the surface was completely dry; this usually took about 10 min. Two drops of 0.5% (w/v) formvar (BDH) dissolved in 1,2-diclaloroethane (Merck) were then added and left for a few seconds. Excess formvar was then removed by placing the slide vertically against blotting paper. When the fdm was dry, the edges of the block were cut with a sharp knife. By dipping the block gently into the disinfectant solution a film containing the virus was released and floated on the surface of the disinfectant solution; this moment of virus-disinfectant contact was noted. After a known time of contact the virus f'tim was removed from the disinfectant by picking up the film with a sealed Pasteur pipette. Any excess disinfectant still present was removed by gently touching the £dm against the side wall of a tube. The film was transferred into 1 ml tissue culture medium and disrupted with the sealed Pasteur pipette to release residual virus particles into suspension. This suspension was inoculated into tissue culture in order to determine residual infectious virus. In control experiments the disinfectant was replaced by tissue culture medium. RESULTS
The results of these inactivation studies are shown in Figs. 2 and 3. It is important to note that virus control titres remained unchanged during the selected contact times of these experiments and that there had been a rapid initial drop in titre during the first minute of virus-disinfectant contact. However, longer contact times produced a more gradual decline in virus titre. VSV was completely inactivated in 10 min contact with 4% FA and 2.5% GA. 20 min contact with 1% NaOC1 was necessary to produce the same effect. Virus strain AnY 1444 was found to be extremely resistant to 4% FA in l0 min contact. The total drop in titre was half a log compared to its control titre of 106 TCID so/ml.
6.4 5.
u
2,
1.
o
5
10
"~1 15 20 Contoct t~rne (mln)
Fig. 2. E f f e c t o f d i f f e r e n t d i s i n f e c t a n t s o n VSV. • . . . . o ....
o, 1% NaOC1.
• , c o n t r o l ; --
~, 4% F A a n d 2..~5% G A "
30
6.. 5. ¸
4..
1..
1.
5.
Contoct
time
(min)
10.
Fig. 3. Effect of 4% formaldehyde on some arboviruses, o, CHICK; o, BGN; % AnY 1444; xz, WN; - - , control; , test. -
CHIK virus was inactivated b y 4% F A in 5 min. The mode o f inactivation was more uniform and a linear relationship was observed. WN virus and BGN produced more or less the same inactivation curves and b o t h viruses showed no residual activity after 10 min contact with 4% FA. Since the inactivation technique described is very similar to the pseudoreplica technique (El Mekki, 1978), we investigated the residual virus activity after floating for 5 s in 1% uranylacetate as staining agent, in parallel with controls. The films were removed and allowed to stand at room temperature for 1 h. The residual virus activities are presented in Table 1. There was no significant change in titre o f the viruses studied. DISCUSSION The advantages of the virucide floating technique can be summarized as follows: The virus to be tested need not be a high titred one. High concentrations o f disinfectants highly toxic for tissue cultures can be used. Perfect control o f contact time between virus and disinfectant. Rapid and simple removal o f excess disinfectant. Possibility o f measuring very low residual viral activities. The method simulates as far as possible the in use conditions o f the disinfectant. The virus was inoculated onto a surface, allowed to dry, and then treated with the product, according to the directions for use. The technique is simple and easy to perform. The method is limited b y the chemical resistance o f the formvar sheet to the disinfectants. Ethanol can not be tested. The electron microscopist should be aware o f the fact that negative staining by 1%
31 TABLE 1 Effect of 1% uranylacetate on virus activity Virus VSV CHIK WN BGN AnY 1444
Control titre
Residual titre
10 s'7 105.7 104.7 10 s'2 106.0
105.2 104.7 104.7 104.7 106.0
Residual virus activities expressed as TCIDs0/ml after floating virus films for 5 s on 1% uranylacetate solution and standing for 1 h at room temperature.
u r a n y l a c e t a t e for a p e r i o d o f 3 - 5
s is n o t sufficient to inactivate viruses. U n d e r t h e
c o n d i t i o n s used, viruses m u s t b e i n a c t i v a t e d p r i o r t o t h e i r negative staining. REFERENCES El Mekki, A.A., Nieuwenhuysen, P., Van der Groen, G. and Pattyn, S.R., 1979, in: Proc. Belgian Biophysical Society 10, Universitaire Instelling Antwerpen, to be published in Arch. Intern. Biochim. Physiol. (in press). El Mekki, A.A., Van der Groen, G. and Pattyn, S.R., 1978, in: Ebola Virus Haemorrhagic Fever, Ed.: S.R. Pattyn (Elsevier/North-HoUand Biomedical Press, Amsterdam - New York) pp. 2 6 1 - 267. Van der Groen, G., Van den Berghe, D.A.R. and Pattyn, S.R., 1976, J. Gen. Virol., 3 4 , 3 5 3 - 3 6 1 .
27
NEW METHOD FOR VIRUCIDE TESTING
G. VAN DER GROEN, A. A. EL MEKK1and S. R. PATTYN lnstituut voor Tropische Geneeskunde, Laboratory of Bacteriology and Virology, Nationalestraat 155, B- 2OOOAntwerpen, Belgium (Accepted 16 October 1979)
The virus virucide floating technique is a simple and rapid method of testing virucides in vitro. The method allows testing of the effect of high concentrations of disinfectants and the use of low titred viruses. Removal of excess disinfectant is rapid and simple and perfect control of contact time between virus and disinfectant is possible. The virus is attached to a surface, simulating as much as possible the in use conditions of the disinfectant. The method was used to study the effect of certain disinfectants on some arboviruses. INTRODUCTION The major problems for virucide testing are: rapid removal of excess disinfectant toxicity of disinfectant for tissue cultures necessity for high virus titers difficulties in detection o f low residual viral activity exact time of contact between virus and disinfectant. In order to solve these problems we developed the virucide floating technique. MATERIALS AND METHODS Cells and viruses Vero and BHK-21 cells were cultured in TC medium 199 supplemented with 7% and 10% inactivated calf serum respectively, buffered with 13 mM sodium bicarbonate. Virus stocks of vesicular stomatitis virus (VSV), chikungunya virus (CHIK), West Nile (WN) virus, Bangyi (BGN) virus, and a bunya-like virus (El Mekki, 1979) were prepared in Vero cells. AnY 1444, an orbivirus (El Mekki, 1979) was prepared in BHK-21 cells.
28
Virus assays All viruses, except AnY 1444, were assayed in Vero cells by the 50% end point method as previously described (Van der Groen, 1976). Serial 10-fold dilutions of AnY 1444 virus in TC medium 199 with 2% inactivated calf serum were inoculated into four wells of microtissue culture plates each well containing 5 X 103 BHK-21 cells per well.
Disinfectants Formaldehyde (FA) 35% (Merck) and glutaraldehyde (GA) 70% (Ladd) stock solutions were diluted in sterile distilled water to the appropriate rmal concentrations. A commercially available sodium hypochlorite solution (NaOC1) containing 1 6 0 - t 7 0 g of active chlorine/l was used undiluted. Uranylacetate (Fluka A.G.) was dissolved in water (1 g/100 ml) and used as 1% solution.
Virucide floating technique A block of 2% agarose (see Fig. 1) of about 1 cm 2 was cut and mounted on the edge of a glass slide. A known quantity of virus suspension was put on the surface of the agarose ~1 Drop Of virus suspension block
-r'277~7>~/TZT.71~z~/~2~
2°1~ 0 g o c o s e "~ glide
liaa. ~]lllll/lll/llfll}lllllll]J ,k
-II virus Ioye# (dry)
0
pzz
'.,-,' / 2 / ~
~ Drop of formvor Solution
~ Formotion of for-mvoF f~]m
~ Releose of viFus f i l m in dlslnfectont or" medium
p=
_ _~ _ _~ -:~-~-:-----Z=-::.-
L;
-~, . . . .
med~om
d~s~f . . . . . . . .
~1 Removal Of f l i m
D[SrmPetiiuO~mof virus f i l m
~
Fig. 1. Virucide floating technique.
hr
29 and allowed to diffuse and evaporate until the surface was completely dry; this usually took about 10 min. Two drops of 0.5% (w/v) formvar (BDH) dissolved in 1,2-diclaloroethane (Merck) were then added and left for a few seconds. Excess formvar was then removed by placing the slide vertically against blotting paper. When the fdm was dry, the edges of the block were cut with a sharp knife. By dipping the block gently into the disinfectant solution a film containing the virus was released and floated on the surface of the disinfectant solution; this moment of virus-disinfectant contact was noted. After a known time of contact the virus f'tim was removed from the disinfectant by picking up the film with a sealed Pasteur pipette. Any excess disinfectant still present was removed by gently touching the £dm against the side wall of a tube. The film was transferred into 1 ml tissue culture medium and disrupted with the sealed Pasteur pipette to release residual virus particles into suspension. This suspension was inoculated into tissue culture in order to determine residual infectious virus. In control experiments the disinfectant was replaced by tissue culture medium. RESULTS
The results of these inactivation studies are shown in Figs. 2 and 3. It is important to note that virus control titres remained unchanged during the selected contact times of these experiments and that there had been a rapid initial drop in titre during the first minute of virus-disinfectant contact. However, longer contact times produced a more gradual decline in virus titre. VSV was completely inactivated in 10 min contact with 4% FA and 2.5% GA. 20 min contact with 1% NaOC1 was necessary to produce the same effect. Virus strain AnY 1444 was found to be extremely resistant to 4% FA in l0 min contact. The total drop in titre was half a log compared to its control titre of 106 TCID so/ml.
6.4 5.
u
2,
1.
o
5
10
"~1 15 20 Contoct t~rne (mln)
Fig. 2. E f f e c t o f d i f f e r e n t d i s i n f e c t a n t s o n VSV. • . . . . o ....
o, 1% NaOC1.
• , c o n t r o l ; --
~, 4% F A a n d 2..~5% G A "
30
6.. 5. ¸
4..
1..
1.
5.
Contoct
time
(min)
10.
Fig. 3. Effect of 4% formaldehyde on some arboviruses, o, CHICK; o, BGN; % AnY 1444; xz, WN; - - , control; , test. -
CHIK virus was inactivated b y 4% F A in 5 min. The mode o f inactivation was more uniform and a linear relationship was observed. WN virus and BGN produced more or less the same inactivation curves and b o t h viruses showed no residual activity after 10 min contact with 4% FA. Since the inactivation technique described is very similar to the pseudoreplica technique (El Mekki, 1978), we investigated the residual virus activity after floating for 5 s in 1% uranylacetate as staining agent, in parallel with controls. The films were removed and allowed to stand at room temperature for 1 h. The residual virus activities are presented in Table 1. There was no significant change in titre o f the viruses studied. DISCUSSION The advantages of the virucide floating technique can be summarized as follows: The virus to be tested need not be a high titred one. High concentrations o f disinfectants highly toxic for tissue cultures can be used. Perfect control o f contact time between virus and disinfectant. Rapid and simple removal o f excess disinfectant. Possibility o f measuring very low residual viral activities. The method simulates as far as possible the in use conditions o f the disinfectant. The virus was inoculated onto a surface, allowed to dry, and then treated with the product, according to the directions for use. The technique is simple and easy to perform. The method is limited b y the chemical resistance o f the formvar sheet to the disinfectants. Ethanol can not be tested. The electron microscopist should be aware o f the fact that negative staining by 1%
31 TABLE 1 Effect of 1% uranylacetate on virus activity Virus VSV CHIK WN BGN AnY 1444
Control titre
Residual titre
10 s'7 105.7 104.7 10 s'2 106.0
105.2 104.7 104.7 104.7 106.0
Residual virus activities expressed as TCIDs0/ml after floating virus films for 5 s on 1% uranylacetate solution and standing for 1 h at room temperature.
u r a n y l a c e t a t e for a p e r i o d o f 3 - 5
s is n o t sufficient to inactivate viruses. U n d e r t h e
c o n d i t i o n s used, viruses m u s t b e i n a c t i v a t e d p r i o r t o t h e i r negative staining. REFERENCES El Mekki, A.A., Nieuwenhuysen, P., Van der Groen, G. and Pattyn, S.R., 1979, in: Proc. Belgian Biophysical Society 10, Universitaire Instelling Antwerpen, to be published in Arch. Intern. Biochim. Physiol. (in press). El Mekki, A.A., Van der Groen, G. and Pattyn, S.R., 1978, in: Ebola Virus Haemorrhagic Fever, Ed.: S.R. Pattyn (Elsevier/North-HoUand Biomedical Press, Amsterdam - New York) pp. 2 6 1 - 267. Van der Groen, G., Van den Berghe, D.A.R. and Pattyn, S.R., 1976, J. Gen. Virol., 3 4 , 3 5 3 - 3 6 1 .