BRIEF COMMUNICATIONS
Effect of temperature and relative humidity on variola virus in crusts * FARIDA HUQ 1
Abstract
0
The viability of variola virus in crusts under different conditions of temperature and relative humidity was studied for 16 weeks. At the ambient temperature of 25.8-26.4°C and 85-90°/ relative humidity, the virus survived only 8 weeks but at lower temperatures and relative humidities the survival time was considerably prolonged.
2os
05
3
3S°c3 5
Materials and methods Crusts were collected from a single smallpox patient in a sterile bottle and were divided that day into batches and stored under the following conditions: at incubator temperature (35°C); at room temperature; at room temperature in a desiccator; at refrigerator temperature (4°C); at refrigerator temperature (4°C) in a desiccator; and at deep-freeze temperature (-20°C). The bottle caps were loosely screwed so that the RH would remain the same outside and inside the bottle. The temperature and RH of the room, incubator, and refrigerator were noted each day and the weekly average calculated. The RH inside the desiccator was not measured owing to difficulties in * This work was supported principally by a research grant from the World Health Organization. I Head, Microbiological Laboratory and in charge, Smallpox Diagnostic Laboratory, Institute of Public Health, Mohakhali, Dacca-12, Bangladesh.
3571
-
710
\
6
ROOM
%\.
....
NROOM TEMPERATURE \\'%
IN DESICCATOR
TEMPERATUREU
7 8 0
The ability of variola virus to remain viable and infective under certain conditions for long periods is well known (1, 2). The viability of the virus in crusts will depend on various factors such as temperature and relative humidity (RH). In countries such as Bangladesh, where the confirmation of smallpox diagnosis is by isolation of virus and samples may take several days to reach the laboratory, knowledge of the effect of various temperatures and RH on the viability of variola virus is important.
v o ;.......... a
1
2
3
4
5
6
7 8 9 Time in weeks
10
11
12
13
14
15 HO
16 ,u,,
Fig. 1. Fall in log titre of variola virus with time under different environmental conditions.
putting the hygrometer inside, but it was assumed to be less than 1%O. Each week, three crusts were taken from each bottle and were titrated on egg chorioallantoic membrane (CAM) and the average titre noted. Gel precipitation was also done each week with each batch of crusts. The experiments in each group were terminated when for two consecutive weeks no virus could be grown and there was no evidence of gel precipitation. The titre of virus at the end of each week was calculated and plotted as log Po/P, where PO is the original titre of the virus and P is the titre of the crust at any given week. Results The results of the titrations are shown in Fig. 1 and those of the gel precipitation test in Table 1. The average initial titres of virus in the crusts were approximately 2.2 x 108. At 350C and with an average RH of 65-68%, viable virus was isolated for 3 weeks. The gel precipitation test was positive for 4 weeks. The average ambient room temperature remained between 25.80 and 26.4°C throughout the experiment and the RH between 85%4 and 90/0. At this temperature and RH, no viable virus could be isolated after 8 weeks. The gel precipitation test was positive for up to 10 weeks. However, viable virus was BULL. WORLD HEALTH ORGAN., Vol. 54, 1976
711
BRIEF COMMUNICATIONS
Table 1. Viability of variola virus in crusts in successive weeks under different environmental conditions Week
At 35tC and 65-68 % RH a
At room temperature Desiccator 85-90 % RH
At 4C 60-62 % RH
Desiccator
Vb
GPc
v
GP
v
GP
v
GP
v
GP
1
+
+
+
+
+
+
+
+
+
2
+
+
+
+
+
+
+
+
+
3
+
+
+
+
+
+
+
+
+
+
At -20'C V
GP
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
5
-+
+
+
+
+
+
+
+
+
+
6
-+
+
+
+
+
+
+
+
+
+
+
+
4
7
+
+
+
+
+
+
+
+
8
+
+
+
+
+
+
+
+
+
+
9
-
+
+
+
+
+
+
+
+
+
10
-
+
+
+
+
+
+
+
+
+
11
_
_
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
13
-
+
+
+
+
+
+
+
14
-
12
+
+
+
+
+
+
+
15
-
+
+
+
+
+
+
16
-
+
+
+
+
+
+
a
RH = relative humidity.
b V = viability of variola virus as determined c G P = gel precipitation test.
by titration on the chorioallantoic membrane of chicken egg.
found for up to 12 weeks in crusts kept in a desiccator at this temperature and the gel precipitation test was positive for 14 weeks. At 4°C and with a RH of 60-62%/, virus could be isolated for 16 weeks but the titre decreased to 1.2 x 104. The gel precipitation test was positive throughout this period. The results obtained when crusts were kept in the desiccator at this same temperature were essentially the same. The experiment could not be carried beyond 16 weeks as the stock of crusts was exhausted. At -20°C, the titre at the end of 16 weeks was 3.1 x 105 and the gel precipitation test remained positive throughout this
period. Discussion The viability of virus in crusts differed markedly under different conditions of temperature and RH. At 35°C and with 65-68% RH, viable virus was
found for only 3 weeks. This is higher than the average ambient temperature but the RH was lower. Notably, the antigenicity of the crust persisted even after no viable virus could be detected. At ambient temperature and RH, the crusts showed viable virus for 2 months. The experiment was conducted during May, June, and July, when the average temperature varied between 25.8°C and 26.4°C and the RH between 85% and 90X0. Results obtained by MacCallum & McDonald (3) were similar to our own. They found that virus in crusts kept at a temperature of 30°C and at a RH of 84% could be detected for only 2-3 months. At a lower RH, virus survival was somewhat longer. Unfortunately, our experiments could not be repeated during the colder months of December, January, and February. MacCallum & McDonald (3), however, showed that at ambient temperatures of 200-240C and 55-75Y% RH, virus in crusts survived for up to
712
BRIEF COMMUNICATIONS
18 months. This corresponds roughly with the temperature and humidity in Bangladesh during the winter months. At 4°C, the virus is viable for a considerably longer time. In our experiment, studies could be carried out for only 4 months, but it is assumed that virus would remain viable in crusts for several years although with declining titre. Not surprisingly, the virus titres of scabs kept in the deep freeze were highest. The decline in titres of scabs kept at 4°C and -20°C appears somewhat surprising since virus kept at this temperature is usually very stable. A possible reason for this decrease is that each week the same bottle was removed from storage and three
crusts were removed. In this way, the crusts were thawed and thus exposed to room temperature and RH once each week. REFERENCES
1. DIXON, C. W. Smallpox. Boston, Little, Brown & Co., 1962. 2. RHODES, A. J. & VAN ROOYEN, C. E. Textbook of virology. Baltimore, Williams & Wilkins, 1968, pp. 318-331. 3. MACCALLUM, F. 0. & MCDONALD, J. R. Survival of variola virus in raw cotton. Bulletin of the World Health Organization, 16: 247-254 (1957).
Effect of temperature and relative humidity on variola virus in crusts * FARIDA HUQ 1
Abstract
0
The viability of variola virus in crusts under different conditions of temperature and relative humidity was studied for 16 weeks. At the ambient temperature of 25.8-26.4°C and 85-90°/ relative humidity, the virus survived only 8 weeks but at lower temperatures and relative humidities the survival time was considerably prolonged.
2os
05
3
3S°c3 5
Materials and methods Crusts were collected from a single smallpox patient in a sterile bottle and were divided that day into batches and stored under the following conditions: at incubator temperature (35°C); at room temperature; at room temperature in a desiccator; at refrigerator temperature (4°C); at refrigerator temperature (4°C) in a desiccator; and at deep-freeze temperature (-20°C). The bottle caps were loosely screwed so that the RH would remain the same outside and inside the bottle. The temperature and RH of the room, incubator, and refrigerator were noted each day and the weekly average calculated. The RH inside the desiccator was not measured owing to difficulties in * This work was supported principally by a research grant from the World Health Organization. I Head, Microbiological Laboratory and in charge, Smallpox Diagnostic Laboratory, Institute of Public Health, Mohakhali, Dacca-12, Bangladesh.
3571
-
710
\
6
ROOM
%\.
....
NROOM TEMPERATURE \\'%
IN DESICCATOR
TEMPERATUREU
7 8 0
The ability of variola virus to remain viable and infective under certain conditions for long periods is well known (1, 2). The viability of the virus in crusts will depend on various factors such as temperature and relative humidity (RH). In countries such as Bangladesh, where the confirmation of smallpox diagnosis is by isolation of virus and samples may take several days to reach the laboratory, knowledge of the effect of various temperatures and RH on the viability of variola virus is important.
v o ;.......... a
1
2
3
4
5
6
7 8 9 Time in weeks
10
11
12
13
14
15 HO
16 ,u,,
Fig. 1. Fall in log titre of variola virus with time under different environmental conditions.
putting the hygrometer inside, but it was assumed to be less than 1%O. Each week, three crusts were taken from each bottle and were titrated on egg chorioallantoic membrane (CAM) and the average titre noted. Gel precipitation was also done each week with each batch of crusts. The experiments in each group were terminated when for two consecutive weeks no virus could be grown and there was no evidence of gel precipitation. The titre of virus at the end of each week was calculated and plotted as log Po/P, where PO is the original titre of the virus and P is the titre of the crust at any given week. Results The results of the titrations are shown in Fig. 1 and those of the gel precipitation test in Table 1. The average initial titres of virus in the crusts were approximately 2.2 x 108. At 350C and with an average RH of 65-68%, viable virus was isolated for 3 weeks. The gel precipitation test was positive for 4 weeks. The average ambient room temperature remained between 25.80 and 26.4°C throughout the experiment and the RH between 85%4 and 90/0. At this temperature and RH, no viable virus could be isolated after 8 weeks. The gel precipitation test was positive for up to 10 weeks. However, viable virus was BULL. WORLD HEALTH ORGAN., Vol. 54, 1976
711
BRIEF COMMUNICATIONS
Table 1. Viability of variola virus in crusts in successive weeks under different environmental conditions Week
At 35tC and 65-68 % RH a
At room temperature Desiccator 85-90 % RH
At 4C 60-62 % RH
Desiccator
Vb
GPc
v
GP
v
GP
v
GP
v
GP
1
+
+
+
+
+
+
+
+
+
2
+
+
+
+
+
+
+
+
+
3
+
+
+
+
+
+
+
+
+
+
At -20'C V
GP
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
5
-+
+
+
+
+
+
+
+
+
+
6
-+
+
+
+
+
+
+
+
+
+
+
+
4
7
+
+
+
+
+
+
+
+
8
+
+
+
+
+
+
+
+
+
+
9
-
+
+
+
+
+
+
+
+
+
10
-
+
+
+
+
+
+
+
+
+
11
_
_
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
13
-
+
+
+
+
+
+
+
14
-
12
+
+
+
+
+
+
+
15
-
+
+
+
+
+
+
16
-
+
+
+
+
+
+
a
RH = relative humidity.
b V = viability of variola virus as determined c G P = gel precipitation test.
by titration on the chorioallantoic membrane of chicken egg.
found for up to 12 weeks in crusts kept in a desiccator at this temperature and the gel precipitation test was positive for 14 weeks. At 4°C and with a RH of 60-62%/, virus could be isolated for 16 weeks but the titre decreased to 1.2 x 104. The gel precipitation test was positive throughout this period. The results obtained when crusts were kept in the desiccator at this same temperature were essentially the same. The experiment could not be carried beyond 16 weeks as the stock of crusts was exhausted. At -20°C, the titre at the end of 16 weeks was 3.1 x 105 and the gel precipitation test remained positive throughout this
period. Discussion The viability of virus in crusts differed markedly under different conditions of temperature and RH. At 35°C and with 65-68% RH, viable virus was
found for only 3 weeks. This is higher than the average ambient temperature but the RH was lower. Notably, the antigenicity of the crust persisted even after no viable virus could be detected. At ambient temperature and RH, the crusts showed viable virus for 2 months. The experiment was conducted during May, June, and July, when the average temperature varied between 25.8°C and 26.4°C and the RH between 85% and 90X0. Results obtained by MacCallum & McDonald (3) were similar to our own. They found that virus in crusts kept at a temperature of 30°C and at a RH of 84% could be detected for only 2-3 months. At a lower RH, virus survival was somewhat longer. Unfortunately, our experiments could not be repeated during the colder months of December, January, and February. MacCallum & McDonald (3), however, showed that at ambient temperatures of 200-240C and 55-75Y% RH, virus in crusts survived for up to
712
BRIEF COMMUNICATIONS
18 months. This corresponds roughly with the temperature and humidity in Bangladesh during the winter months. At 4°C, the virus is viable for a considerably longer time. In our experiment, studies could be carried out for only 4 months, but it is assumed that virus would remain viable in crusts for several years although with declining titre. Not surprisingly, the virus titres of scabs kept in the deep freeze were highest. The decline in titres of scabs kept at 4°C and -20°C appears somewhat surprising since virus kept at this temperature is usually very stable. A possible reason for this decrease is that each week the same bottle was removed from storage and three
crusts were removed. In this way, the crusts were thawed and thus exposed to room temperature and RH once each week. REFERENCES
1. DIXON, C. W. Smallpox. Boston, Little, Brown & Co., 1962. 2. RHODES, A. J. & VAN ROOYEN, C. E. Textbook of virology. Baltimore, Williams & Wilkins, 1968, pp. 318-331. 3. MACCALLUM, F. 0. & MCDONALD, J. R. Survival of variola virus in raw cotton. Bulletin of the World Health Organization, 16: 247-254 (1957).
