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Occurrence of reoviruses in environmental water samples
8)
Pergamon 0273-1223(95)00293-6
Waf. Sci. Toch. Vol. 31, No. 5-6. pp. 363-366,1995. Copyright 0 1995 IAWQ PriDtcd in Oreat Britam. All rights reselVed. 0273-122319559'50 +0-00
OCCURRENCE OF REOVIRUSES IN ENVIRONMENTAL WATER SAMPLES N. Milde. D. Tougianidou and K. Botzenhart Hygiene-lnstitut der Eberhard-Karls-Universitli~Abteilung Allgemeine Hygiene und Umwelthygiene. Wilhelmstr. 31. 72074 Tubingen, Germany
ABSTRACT Reoviruses are known to be present in water of different origin. The family of the Reoviridae includes viruses which are infecting not only humans but also animals and even plants. A differentiation between the reovirus types becomes possible by comparison of the electrophoresis pattern of their segmented dsRNA genome. The following presents the profiles of vtrUs isolates from different environmental water samples which have been concentrated and their nucleic acids purified and analysed by SDS-PAGE. Reoviruses were frequently found in different environmental water samples but also in drinking water samples. The most frequently isolated reovirus was the serotype I followed by the serotype 3. The serotype 2 was not found in the analysed samples.
KEYWORDS Reovirus; water samples; CsCI gradient; dsRNA isolation; SDS-PAGE; electrophoresis pattern. INTRODUCTION Conventional detection methods for enteric viruses are based on the multiplication of the virus on suitable cell culture systems leading to cytopathic effects (CPE) which indicates the growth of the virus. Enteroviral CPE's lead to very rapid and typical destruction of the cell culture. but the majority of CPE's from environmental water samples are very slow, incomplete and start very late after inoculation of the cell culture with the virus concentrates. Previous electron microscopical investigations on such slow and incomplete CPE's showed reovirus-like particles to be present. These observations lead to questions about the types of reoviruses present in different water samples. Viruses isolated from cell culture supernatants showing different types of CPE were concentrated, purified, and their dsRNA genomes were analysed by SDS-PAGE. MATERlALS AND METHODS Surface water, drinking water, water from karst springs, and treated raw water were analysed for the presence of enteric viruses. Water samples were concentrated by aluminium hydroxide flocculation (10 1) (Walter, 1981) and by filtration through Virosorb IMDS filter cartridges (300 I) followed by organic flocculation (Katzenelson, 1976). The concentrates were inoculated on MAI04 cells (Agbalika, 1984) and a mixed-cell culture system of BGM and RD (Tougianidou, 1989). The cells were observed for 3 and 2 weeks respectively followed by a second blind passage. Late occurring and incomplete cytopathic effects have been of main interest especially when inclusion bodies were microscopically visible in the cells. Viruses were 363
364
N. MIlDE tl al.
amplified by inoculating the MAI04 cell monolayer of a big cell culture flask with 0.5 ml of the virus supernatant. After 2 weeks incubation at 370C the cells were frozen and thawed three times and cell debris was pelleted by centrifugation. Viruses in the supernatant were pelleted by ultracentrifugation (SW28. 14-16 h. 22.000 rpm. 4OC) and the pellet resuspended in Iml PBS. A prefonned CsCI gradient consisting of a 131.6 mglml and a 588.4 mglml CsCI solution was overlaid with the virus concentrate. The gradient was fonned in a SW41 fixed angle rotor overnight at 40.000 rpm at 19OC. The gradient was collected in 0.5 ml aliquots and after measurement of the refractive indices the samples corresponding to a density of 1.36 g1cm 3 were pooled and dialysed overnight against 2 1PBS. The concentrated and purified virus was digested in a Proteinase K solution (final concentration of 2mglml; 60OC. 30 min). After phenol-chloroform extraction and ethanol precipitation the dsRNA was analysed by SDS-PAGE. 7.5% gels (ETC) were run at 600 V for 5 h in a horizontal electrophoresis system (Phannacia). The dsRNA segments were visualised by silver staining (Bassam. 1991) and the pattern was compared to ATCC reovirus strains type I (Lang). 2 (Jones). and 3 (Abney). RESULTS The cytopathic effects of many of the virus supernatants indicate the possible growth of reoviruses. 129 different virus supernatants were analysed as described for the presence of reoviruses. The nucleic acids derived from 41 samples have not been visible on the gel after silver staining. probably because the supernatant contains other viruses than reoviruses. which could not be concentrated in the range of the refractive index mentioned above. The nucleic acids of the remaining 88 virus supernatants showed a dsRNA pattern typical for reoviruses. A certain variability between the electrophoresis patterns could be observed (Fig. 2). Reovirus type I was the most frequently isolated strain (out of 51 virus supernatants). followed by reovirus type 3 (out of 15 supernatants). Reovirus type 2 was not isolated at all. and 22 virus supernatants showed a variable dsRNA pattern (Table I). Figure I shows a few electrophoresis patterns of different samples. The pattern of lane 4 corresponds to electropherotype B (see Fig. 2). whereas the patterns of lanes 5 and 9-12 show the presence of reovirus type I. and the patterns of lanes 6-8 reovirus type 3. A variability in the electrophoresis patterns could be observed for both virus types.
2
3
4
5
6
7
8
9
10
11
12
L
M
5
Figure I. E1ectropboresis patterns of several reoviral RNA and of viral RNA iso1aled out of different water samples on 7.5% polyacrylamide gels after silver staining. Lanes 1-3: Reovirus type 1.2, and 3 respectively. Lanes 4-12: virus supernatants isolated from different waler samples: Lanes 4-9: treated wastewater; Lanes 10. 11: surface water; Lane 12: water of a karst spring. lane 4 corresponds to the electropberotype B (see Fig. 2). Lanes 5 and 9-12 to reovirus type I and Lanes 6-8 to reovirus type 3.
Reoviroses in environmental water samples
365
Table I. Reoviruses isolated from water samples of different origin total amount of analYlCd ..moles
samples containing reoviru!IC!
Reovirus type!
Reovirus type 2
Reovirus type 3
variable dsRNA pattern
treated wlltewater
61
46
28
0
6
12
surface water
20
16
4
0
7
S
karst spring
40
23
18
0
2
3
dri.king water
8
3
I
0
0
2
total
129
88
SI
0
15
22
Some patterns show a mixed infection between two different reovirus types.
R1 R2 R3
-==
A
B
..... =
--C
- - -- .... --
D
~
- --
-
'NN
35
0
1
0
8 12
1
4
0 0
6
SW
0
2
0
KW
18
0
2
0
0
OW
2
0
0
1
0
E
59
0
1 8 21
1
4
3
0
Figure 2. Variable electrophoresis patterns of reovirus types. RI, R2 and R3: Reovirus type I (Lang), 2 (Jones) and 3 (Abney) respectively. A·O: variable electrophoresis patterns. WW: treated wastewater, SW: surface water, leW: water from a karst spring, OW: drinking water.l:: total of isolates of each elecuopberolype.
CONCLUSIONS Reoviruses could be demonstrated in almost every type of water including drinking water samples (Schwanzbrod. 1985; Tougianidou. 1994). The identification by the dsRNA electrophoresis pattern on polyacrylamide gels seems to be a sensitive method for the differentiation between the different virus types. As previously reported the electrophoresis patterns of the RNA segments can be of a great variability (Hrdy et al.• 1979). This pol ymorphism may be due to human and animal origin of the virus isolates. Matsuura et at. report after ecological and epidemiological studies of reoviruses in water and human feces that there was no diversity observed among their human isolates (1993). There was a similar variability observed in our findings. Therefore it was not possible to define the origin of the isolated reoviruses leading only to suggestions according to Matsuura's observations that the isolates with a constant electrophoresis pattern rna y be due to the same origin which can be a human one.
N. MllDE et al.
366
REFERENCES Agbalika, F.• Hartemann. P., Foliguet, J. M. (1984). Trypsin-treated Ma-I04: A sensitive cell line for isolating enteric viruses from environment al samples. Appl. Environ. Microbiol. 47: 378-380 Bassam, B.1.• Caetano·Annol~s, G.• Gresshoff. PM. (1991): Fast and sensitive staining of DNA on polyacrylamide gels. Altal. Biochem. 80: 81-84 Hrdy. D.B.• Rosen. L., Fields B.N. (1979): Polymorphism of the migration of double-stranded RNA genome segments of reovirus isolates from humans. cattle. and mice. J. Virol. 31: 104-111 Katzenelson. E.• Fattal, B.• Hostovesky T. (1976): Organic flocculation: An efficient second-step concentration method for the detection of viruses in tapwater. Appl. Environ. MicroblOl. 32: 638-639 Matsuura, K., Ishikura, M.• Nakayama, T•• Hasegawa, S., Morita, 0 .• Katori. K.• Uetake. H. (1993): Ecological studies of reovirus pollution of rivers in Toyama prefecture. II. Epidemiological study of reoviruses isolated from river water. Microbiol. lmmunol. 37: 305-310 Schwartzbrod, L., Finance. C.• Aymard, M., Brigaud, M.• Lucena, F. (1985): Recovery of reoviruses from tap water. Zbl. Bakt. Hyg.l. Abt. Orig. B. 181: 383-389 Tougianidou. D., Jacob, K., Herbold, K.• Hahn T.•Flehmig, B.• Botzenhart, K. (1989): Assessment of various cell lines (inclUding mixed-cell cu)tures)for the detection of enteric viruses in different water sources. Wat. Sci. Tech. 21: 311-314 Tougianidou, D.• Botzenhart K. (1994): Vorkommen und Nachweis von Viren im Trinkwasser.lmmun. lrifekt. 21: 122-125 Walter. R. and RUdiger S. (1981): Ein Zweistufenverfahren zur Virusanreicherung aus Ulsungen mit geringem Virustiter (z.B. Trinkwasser). J. Hyg. Epidemiol.lmmunol. 25: 71-81
Pergamon 0273-1223(95)00293-6
Waf. Sci. Toch. Vol. 31, No. 5-6. pp. 363-366,1995. Copyright 0 1995 IAWQ PriDtcd in Oreat Britam. All rights reselVed. 0273-122319559'50 +0-00
OCCURRENCE OF REOVIRUSES IN ENVIRONMENTAL WATER SAMPLES N. Milde. D. Tougianidou and K. Botzenhart Hygiene-lnstitut der Eberhard-Karls-Universitli~Abteilung Allgemeine Hygiene und Umwelthygiene. Wilhelmstr. 31. 72074 Tubingen, Germany
ABSTRACT Reoviruses are known to be present in water of different origin. The family of the Reoviridae includes viruses which are infecting not only humans but also animals and even plants. A differentiation between the reovirus types becomes possible by comparison of the electrophoresis pattern of their segmented dsRNA genome. The following presents the profiles of vtrUs isolates from different environmental water samples which have been concentrated and their nucleic acids purified and analysed by SDS-PAGE. Reoviruses were frequently found in different environmental water samples but also in drinking water samples. The most frequently isolated reovirus was the serotype I followed by the serotype 3. The serotype 2 was not found in the analysed samples.
KEYWORDS Reovirus; water samples; CsCI gradient; dsRNA isolation; SDS-PAGE; electrophoresis pattern. INTRODUCTION Conventional detection methods for enteric viruses are based on the multiplication of the virus on suitable cell culture systems leading to cytopathic effects (CPE) which indicates the growth of the virus. Enteroviral CPE's lead to very rapid and typical destruction of the cell culture. but the majority of CPE's from environmental water samples are very slow, incomplete and start very late after inoculation of the cell culture with the virus concentrates. Previous electron microscopical investigations on such slow and incomplete CPE's showed reovirus-like particles to be present. These observations lead to questions about the types of reoviruses present in different water samples. Viruses isolated from cell culture supernatants showing different types of CPE were concentrated, purified, and their dsRNA genomes were analysed by SDS-PAGE. MATERlALS AND METHODS Surface water, drinking water, water from karst springs, and treated raw water were analysed for the presence of enteric viruses. Water samples were concentrated by aluminium hydroxide flocculation (10 1) (Walter, 1981) and by filtration through Virosorb IMDS filter cartridges (300 I) followed by organic flocculation (Katzenelson, 1976). The concentrates were inoculated on MAI04 cells (Agbalika, 1984) and a mixed-cell culture system of BGM and RD (Tougianidou, 1989). The cells were observed for 3 and 2 weeks respectively followed by a second blind passage. Late occurring and incomplete cytopathic effects have been of main interest especially when inclusion bodies were microscopically visible in the cells. Viruses were 363
364
N. MIlDE tl al.
amplified by inoculating the MAI04 cell monolayer of a big cell culture flask with 0.5 ml of the virus supernatant. After 2 weeks incubation at 370C the cells were frozen and thawed three times and cell debris was pelleted by centrifugation. Viruses in the supernatant were pelleted by ultracentrifugation (SW28. 14-16 h. 22.000 rpm. 4OC) and the pellet resuspended in Iml PBS. A prefonned CsCI gradient consisting of a 131.6 mglml and a 588.4 mglml CsCI solution was overlaid with the virus concentrate. The gradient was fonned in a SW41 fixed angle rotor overnight at 40.000 rpm at 19OC. The gradient was collected in 0.5 ml aliquots and after measurement of the refractive indices the samples corresponding to a density of 1.36 g1cm 3 were pooled and dialysed overnight against 2 1PBS. The concentrated and purified virus was digested in a Proteinase K solution (final concentration of 2mglml; 60OC. 30 min). After phenol-chloroform extraction and ethanol precipitation the dsRNA was analysed by SDS-PAGE. 7.5% gels (ETC) were run at 600 V for 5 h in a horizontal electrophoresis system (Phannacia). The dsRNA segments were visualised by silver staining (Bassam. 1991) and the pattern was compared to ATCC reovirus strains type I (Lang). 2 (Jones). and 3 (Abney). RESULTS The cytopathic effects of many of the virus supernatants indicate the possible growth of reoviruses. 129 different virus supernatants were analysed as described for the presence of reoviruses. The nucleic acids derived from 41 samples have not been visible on the gel after silver staining. probably because the supernatant contains other viruses than reoviruses. which could not be concentrated in the range of the refractive index mentioned above. The nucleic acids of the remaining 88 virus supernatants showed a dsRNA pattern typical for reoviruses. A certain variability between the electrophoresis patterns could be observed (Fig. 2). Reovirus type I was the most frequently isolated strain (out of 51 virus supernatants). followed by reovirus type 3 (out of 15 supernatants). Reovirus type 2 was not isolated at all. and 22 virus supernatants showed a variable dsRNA pattern (Table I). Figure I shows a few electrophoresis patterns of different samples. The pattern of lane 4 corresponds to electropherotype B (see Fig. 2). whereas the patterns of lanes 5 and 9-12 show the presence of reovirus type I. and the patterns of lanes 6-8 reovirus type 3. A variability in the electrophoresis patterns could be observed for both virus types.
2
3
4
5
6
7
8
9
10
11
12
L
M
5
Figure I. E1ectropboresis patterns of several reoviral RNA and of viral RNA iso1aled out of different water samples on 7.5% polyacrylamide gels after silver staining. Lanes 1-3: Reovirus type 1.2, and 3 respectively. Lanes 4-12: virus supernatants isolated from different waler samples: Lanes 4-9: treated wastewater; Lanes 10. 11: surface water; Lane 12: water of a karst spring. lane 4 corresponds to the electropberotype B (see Fig. 2). Lanes 5 and 9-12 to reovirus type I and Lanes 6-8 to reovirus type 3.
Reoviroses in environmental water samples
365
Table I. Reoviruses isolated from water samples of different origin total amount of analYlCd ..moles
samples containing reoviru!IC!
Reovirus type!
Reovirus type 2
Reovirus type 3
variable dsRNA pattern
treated wlltewater
61
46
28
0
6
12
surface water
20
16
4
0
7
S
karst spring
40
23
18
0
2
3
dri.king water
8
3
I
0
0
2
total
129
88
SI
0
15
22
Some patterns show a mixed infection between two different reovirus types.
R1 R2 R3
-==
A
B
..... =
--C
- - -- .... --
D
~
- --
-
'NN
35
0
1
0
8 12
1
4
0 0
6
SW
0
2
0
KW
18
0
2
0
0
OW
2
0
0
1
0
E
59
0
1 8 21
1
4
3
0
Figure 2. Variable electrophoresis patterns of reovirus types. RI, R2 and R3: Reovirus type I (Lang), 2 (Jones) and 3 (Abney) respectively. A·O: variable electrophoresis patterns. WW: treated wastewater, SW: surface water, leW: water from a karst spring, OW: drinking water.l:: total of isolates of each elecuopberolype.
CONCLUSIONS Reoviruses could be demonstrated in almost every type of water including drinking water samples (Schwanzbrod. 1985; Tougianidou. 1994). The identification by the dsRNA electrophoresis pattern on polyacrylamide gels seems to be a sensitive method for the differentiation between the different virus types. As previously reported the electrophoresis patterns of the RNA segments can be of a great variability (Hrdy et al.• 1979). This pol ymorphism may be due to human and animal origin of the virus isolates. Matsuura et at. report after ecological and epidemiological studies of reoviruses in water and human feces that there was no diversity observed among their human isolates (1993). There was a similar variability observed in our findings. Therefore it was not possible to define the origin of the isolated reoviruses leading only to suggestions according to Matsuura's observations that the isolates with a constant electrophoresis pattern rna y be due to the same origin which can be a human one.
N. MllDE et al.
366
REFERENCES Agbalika, F.• Hartemann. P., Foliguet, J. M. (1984). Trypsin-treated Ma-I04: A sensitive cell line for isolating enteric viruses from environment al samples. Appl. Environ. Microbiol. 47: 378-380 Bassam, B.1.• Caetano·Annol~s, G.• Gresshoff. PM. (1991): Fast and sensitive staining of DNA on polyacrylamide gels. Altal. Biochem. 80: 81-84 Hrdy. D.B.• Rosen. L., Fields B.N. (1979): Polymorphism of the migration of double-stranded RNA genome segments of reovirus isolates from humans. cattle. and mice. J. Virol. 31: 104-111 Katzenelson. E.• Fattal, B.• Hostovesky T. (1976): Organic flocculation: An efficient second-step concentration method for the detection of viruses in tapwater. Appl. Environ. MicroblOl. 32: 638-639 Matsuura, K., Ishikura, M.• Nakayama, T•• Hasegawa, S., Morita, 0 .• Katori. K.• Uetake. H. (1993): Ecological studies of reovirus pollution of rivers in Toyama prefecture. II. Epidemiological study of reoviruses isolated from river water. Microbiol. lmmunol. 37: 305-310 Schwartzbrod, L., Finance. C.• Aymard, M., Brigaud, M.• Lucena, F. (1985): Recovery of reoviruses from tap water. Zbl. Bakt. Hyg.l. Abt. Orig. B. 181: 383-389 Tougianidou. D., Jacob, K., Herbold, K.• Hahn T.•Flehmig, B.• Botzenhart, K. (1989): Assessment of various cell lines (inclUding mixed-cell cu)tures)for the detection of enteric viruses in different water sources. Wat. Sci. Tech. 21: 311-314 Tougianidou, D.• Botzenhart K. (1994): Vorkommen und Nachweis von Viren im Trinkwasser.lmmun. lrifekt. 21: 122-125 Walter. R. and RUdiger S. (1981): Ein Zweistufenverfahren zur Virusanreicherung aus Ulsungen mit geringem Virustiter (z.B. Trinkwasser). J. Hyg. Epidemiol.lmmunol. 25: 71-81