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Concentration of rotavirus by ultrafiltration
)Vat. Res. Vol. 20, No. I, pp. 79-83, 1986 Printed in Great Britain. All rights reserved
0043-1354/86 $3.00+0.00 Copyright © 1986Pergamon Press Ltd
CONCENTRATION OF ROTAVIRUS BY ULTRAFILTRATION JANIS JANSONS and MARION R. BUCENS Virology Section, Combined Clinical Microbiology Service, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009 (Received June 1985)
Abstract--A hollow fibre ultrafiltration unit was evaluated for its capacity to concentrate human rotavirus from experimentally contaminated groundwater. The results of these experiments suggest that virus recovery is greatly improved by utilizing either a protein wash with beef extract or by backflushing the hollow fibres with phosphate buffered saline. An enzyme-linkedimmunosorbent assay (ELISA) was used to determine rotavirus recoveries after concentration. Key words--virus concentration, ultrafiltration, groundwater, rotavirus
rotaviruses and adenoviruses. The reliability of poliovirus as an indicator of faecally contaminated water has also been questioned (Katzenelson and Kedmi, 1979). These factors have created a need for the development of concentration techniques that would enable more reliable detection of a broader range of enteric viruses from contaminated water. Ultrafiltration is an alternative concentration technique and overcomes many of the drawbacks of filter adsorption-elution methodology. Concentration is carried out at ambient pH and addition of polycationic salts and acidification of the sample is not required. Ultrafiltration techniques should not be affected by competing organic compounds such as humic acids since virus particles are retained on the basis of size. In this study a number of parameters were varied in an attempt to optimize recoveries during ultrafiltration.
INTRODUCTION The use of wastewater for the recharge of groundwater aquifers offers a practical and economic method for its disposal. In order to overcome the problem of contamination of the aquifer with human pathogens which include enteric viruses, careful studies of the survival and movement of these agents must be performed. One such investigation is being carried out at the Canning Vale Groundwater Recharge Scheme, at Canning Vale, Western Australia. The currently accepted procedure for concentrating viruses from large volumes of water is the Viradel method which utilizes the filter adsorption-elution principle, with secondary concentration usually being carried out by membrane filtration, aluminium hydroxide precipitation or organic flocculation. This technique was largely developed using poliovirus as a model and very little is known about its efficiency at recoverying other enteric viruses from water. A recent study (Melnick et al., 1984) has revealed that virus recoveries using the Viradei method are prone to considerable variation and this is thought to be related to the chemical quality of sample water. Organic compounds such as humic acids are often found in natural waters (Packham, 1964) and have been shown to adsorb to membrane filters (Farrah et al., 1976). The role and effect of these compounds in the Viradel procedure is poorly understood and their presence in sample water may result in unreliable detection of virus. In our experience, filter adsorption-elution methodology is tedious and time consuming. It exposes viruses to high and low pH during adsorption and elution. Laboratory grown strains of poliovirus appear to survive this treatment, but field isolates of other enteric viruses may not. In recent times, poliovirus has become less significant as a waterborne human pathogen in most Western countries compared with other enteric viruses such as hepatitis A virus, Norwalk-like agents,
MATERIALS AND
Preparation o f virus inoculum
A 10% faecal extract was prepared from pooled rotavirus positive faeces (previously detected by electron microscopy) and suspended in Hanks BSS. To ensure that working dilutions of the rotavirus inoculum had a linear absorbance in response to dilution in the ELISA assay, it was necessary to further dilute the faecal extract by 1/100. Rotavirus recovery studies
Five ml of rotavirus inoculum were mixed with 21. of either phosphate buffered saline (PBS) or groundwater (GW). The latter was collected at the Canning Vale site. A typical chemical analysis of the groundwater is given in Table 1. After inoculation, the sample was processed by ultrafiltration using an Amicon DC2 hollow fibre ultrafiltration unit (Amicon Corporation, Danvers, Mass.) with twin H 1P100-20cartridges which have a nominal cutoff of 100,000 mol. wt and a total surface area of ll00cm 2. Concentration procedure using the Amicon DC2
After the inoculated sample was placed in the DC2 reservoir, the restriction valve (CD valve) was pushed into the concentration mode and turned back to zero. The DC2 79
WK
20iI--F
METHODS
80
JAN1S JANSONS and MARION R. BUCENS
Table 1 pH 6.3 Turbidity 4 (NTU) Conductivity 59 (mS m ~) Alkalinity (CaCO~) 12 (mg I-~) Colour 100 (Hazen) Anions C1 NO 3 SO4 PO4
(mg 1-]) 95 4.9 30 1.8
Cations Ca Mg Na K Fe Mn
(mg 1-I) 14 6 95 5.5 <0.05 < 0.05
was set into operation with the variable speed knob turned to approximately half speed. The CD valve was gradually restricted until the inlet pressure reached 10 psi. After completion of a run, the concentrate was collected, and the volume was measured and recorded. In some o f the experiments, the DC2 was backflushed prior to sample collection. A control sample not having undergone ultrafiltration was set up in parallel. Two-fold dilutions ranging from undiluted to 1/32 o f the concentrate and the control were then prepared in either PBS, GW or beef extract. These dilutions were assayed for rotavirus using the Rotazyme test.
The colour intensity developed by the substrate should be proportional to the concentration of rotavirus antigen in the sample. In each experiment it was necessary to set up a separate control since the sample volume collected after concentration was variable.
Integrity testing of hollow fibres A 0.1~o solution of blue dextran 2000 (Pharmacia, Uppsala, Sweden) was processed in the normal way as described in the concentration section. Blue dextran has a molecular weight of approx. 2,000,000 and should be retained by hollow fibre cartridges with a 100,000 mol. wt. cut-off. During the concentration procedure the filtrate was collected and examined visually for traces o f blue dextran. RESULTS R o t a v i r u s c o u l d n o t be d e t e c t e d in the c o n c e n t r a t e s following n o r m a l c o n c e n t r a t i o n using either P B S o r g r o u n d w a t e r as p r o c e s s fluids, with optical d e n s i t y (O.D.) values falling b e l o w the positive cut-off.
Effect of backflushing B a c k f l u s h i n g o f t h e fibres resulted in c o n s i d e r a b l e i m p r o v e m e n t in recovery with PBS (Fig. 1) a n d with
Backflushing procedure The ultrafiltrate effluent line was clamped off and the CD valve was turned to zero. About 30 ml o f either filtrate or PBS were forced back across the fibres through one of the filtrate ports situated on the cartridge holder. After backflushing, a rapid forward recirculation was carried out for 30s and the sample was collected. This manoeuvre assisted in the removal o f any non-specifically adsorbed virus by washing the membrane surface o f the fibre lumen. In one experiment using groundwater, the backflush was tested for rotavirus in parallel with the concentrate and the control. To evaluate overall recovery from this experiment, the backflush was pooled with the concentrate and the control volume adjusted with PBS so that it had the same parameters as the combined concentrate and backflush. These were tested for rotavirus.
0.5
=E 0.4 e~ O~
~
_•
Rotavirus assay The rotavirus assay was carried out according to the manufacturer's instructions using a commercially available ELISA test (Rotazyme, Abbott Laboratories, North Chicago, I11.). In brief, the sample was incubated with an anti-rotavirus guinea pig antibody coated polystyrene bead for 3 h at 45°C. The beads were washed with distilled water and then incubated with rabbit anti-rotavirus horseradish peroxidase conjugated antiserum for 1 h at 45°C. After this incubation the beads were washed again and transferred to a box of reaction tubes and the substrate, OPD (ophenylenediamine.2HC1) in citrate phosphate buffer, was added. The tubes were incubated in the dark for a further 15 min. The enzyme reaction was stopped by adding 1 N sulphuric acid. The photometric reading of the results was performed on an Abbott Quantum reader using the Rotazyme protocol.
Control
•
Concentrate
0.3
_= c5 (5 0.2 0.1
Beef extract washing procedure Five hundred ml of 10% beef extract were added to the DC2 reservoir after approx. 1 1. of sample had been processed. The concentration was then allowed to proceed normally and the concentrate collected as already described. In another experiment 500 ml of 3% beef extract were concentrated by ultrafiltration. The concentrate was collected and inoculated with 5 ml of rotavirus extract. A control was also tested using unconcentrated 3~o beef extract and an identical inoculum in the same volume as the concentrate. These were tested using the Rotazyme assay.
Exp.A •
I I
Rotovirus
I
t
I
J
2
3
4
5
inoculum dilution(log;z)
0.5 Exp, B Control
~ t~
0.4
~ $ 3 ~ "; 0
0.3
Concentrate
0.2 '\\ Positive c u t - o f f
0.I
I
0
t
I
I
1 2 3 4 Rotovirus inoculum dilution ( log 2 )
J
5
Fig. 1. Recovery of rotavirus from phosphate buffered saline as measured by the Rotazyme assay. Exp. A was conducted without backfiushing, whereas in Exp. B, hollow fibres were backflushed.
Concentration of rotavirus by ultraflltration 0.5 -
=E
0.4
-
• •
Exp. A Control Concentrate
o
Back flush
81
0.5 r
~N ~-
0.4
Exp. A
L~
•
e
n
Control
t
rate
(M
,¢
0.3
'6
0.3 -
v
O
> 0.2 c~ d 0.1
°Oo )"z
0
,
,
e%
Positive
cut-off
_.= >~ 0.2 d d
~
Positive cut-off
o.1
" " "°O
I 1
I 2
I 3
I 4
I 5
-.
I I
0
Rotovirus inoc.ulurn dilution (log z)
--_--
•
I 2
I 3
I 4
I 5
Rotavirus inoculum dilution ( loge)
0.5 0.5
Exp. B 0.4
•
Exp. B • •
Control
• Concentrate
E
\
Control Concentrate
0.4
C C
0.3
N
'¢
0.3
O B
0.2 [
c~
"~[
.
0.1
O
Positive cut-off
(?.2
-.. ~-• I
o
I
1
I
2
I
3
I
4
I
5
Rotovirus inoculum dilution (log 2) Fig. 2. Recovery of rotavirus from groundwater. In Exp A, backflush was individually tested. In Exp. B, backflush was combined with concentrate, and the control diluted to a similar extent.
groundwater [Fig. 2(B)]. In this experiment the backflush was tested and compared with the concentrate and the control. It was found that the backflush contained a greater proportion of virus inoculum than the concentrate [Fig. 2(A)]. When the backflush was combined with the concentrate and compared to a control with identical parameters almost complete virus recovery was achieved [Fig. 2(B)].
/ 0
Positive cut-off
I 1
I 2
I 3
I 4
I 5
Rotovirus inoculum dilution (log z) Fig. 3. Recovery of rotavirus from groundwater. Exp. A used the normal concentration method. Exp. B was recovery after 10% beef extract wash.
virus inoculum in concentrated beef extract were reduced when compared with unconcentrated beef extract. Alkaline beef extract pH 9.5 did not improve virus recovery compared to neutral beef extract. After a beef extract wash backflushing appeared to have little effect on recovery rates.
Effect o f beef extract washing on virus recovery
Beef extract washing of the hollow fibres also improved recovery of rotavirus (Fig. 3). Virus recovery was found to be proportional to the concentration of beef extract although improvement was only minimal at concentrations above 5%. Washing of the hollow fibres with beef extract at concentrations greater than 10% caused excessive foaming. However, when concentrated beef extract was tested for any inhibitory effect on the Rotazyme assay, it was found that absorbance values of rota-
DISCUSSION There has been an increasing number of reports of waterborne outbreaks of virus infections by a wide range of enteric viruses other than polioviruses. Waterborne outbreaks of hepatitis A virus (Morse et al., 1972; Hejkal et al., 1982), gastroenteritis attributed to a Norwalk-like agent (Taylor, et al., 1981) and rotavirus (Sutmoller et al., 1982), have been reported, as well as an outbreak of swimming pool
82
JANIS JANSONSand MARIONR. BUCENS
conjunctivitis caused by adenovirus type 7 (Caldwell et aL, 1974). Murphy et al. (1983) recently described an outbreak of infectious gastroenteritis on Norfolk Island. They detected rotaviruses, small round viruses and adenoviruses by electron microscopy, as well as isolating polioviruses and adenoviruses in cell culture from groundwater which was used to supplement the island's water supply. Water has also been implicated in viral disease outbreaks by contamination of shellfish with agents such as hepatitis A virus (Portnoy et al., 1975) and small round structured viruses (Gill et al., 1983). An extensive waterborne hepatitis outbreak in India has been recently described (Sreenivasan et al., 1984) where the aetiological agent is still unknown. The epidemiological data from this outbreak suggested hepatitis A virus as the aetiological agent but serological evidence indicated non-A non-B hepatitis. An earlier study (Wong et al., 1980) also suggested that the 1955 waterborne hepatitis epidemic in Delhi. India, where 29,300 cases were reported, may have been due to a similar agent. There is a need for the development of more reliable techniques for the recovery of a greater range of enteric viruses from contaminated water. in this study, ultrafiltration was examined as an alternative method for virus concentration because of unsatisfactory performance with the filter adsorption-elution technique when concentrating viruses from Canning Vale groundwater. The difficulties experienced with recovery of virus from groundwater were thought to be caused by the presence of interfering compounds. Rotavirus was selected as a model for this study since it has been associated with outbreaks of gastroenteritis in infants (Anon., 1980) and adults (von Bonsdorff et a l , 1978). Until recently, attempts to grow rotavirus in cell cultures had been unsuccessful. and the only two practical methods available to detect these viruses were electron microscopy and immunoassay. Steinmann (1981) described an ELISA technique in which he concentrated domestic sewage on to Seitz filters using an adsorption-elution technique followed by ultracentrifugation. Using this method he was able to detect rotavirus in 25% of the samples. In view of this finding, an ELISA technique was chosen to determine virus recoveries after ultrafiltration. A similar immunoassay could be developed to test water concentrates for the presence of hepatitis A virus and other enteric viruses. The introduction of hollow fibre ultrafiltration technology has resulted in high filtration rates and the use of this technique as a practical alternative to filter adsorption-elution methodology for concentration of viruses from large volumes of water has become feasible. It has been successfully used to recover viruses from effluent (Ratnamohan et al., 1980) and from groundwater (Murphy et al., 1983).
By using some variations of the basic ultrafiltration technique we have shown that recovery of rotavirus is greatly enhanced by PBS backflushing, which supports the findings of Belfort et al. (1974) who reported that 13-54°,o of poliovirus was recovered after backflushing. A beef extract wash also appears to have an effect on rotavirus recovery. Similar findings have previously been reported by Berman et aL (1980) who described a 5-fold increase in poliovirus recovery after pretreatment of ultrafiltration membranes with flocculated beef extract. Poorer recoveries of rotavirus after beef extract washes compared with PBS backflushing may be due to the assay. This could result in an underestimation of true recovery when an immunoassay is used and may be due to beef extract protein non-specifically binding to the antibody coated polystyrene beads, thus blocking the antigenic sites for virus. In view of this finding, further investigations of virus recovery after beef extract washing may be warranted, although an alternative method of virus assay may have to be evaluated. We recommend frequent integrity testing of the hollow fibre cartridges with dextran blue. Although the cartridges have an estimated life span of approx. 2 years we experienced periodic failure. On most occasions, poor recoveries after concentration were traced back to broken tibres. Although the sample size in these experiments was only 21., a larger ultrafiltration unit (Amicon DC30) is available, which is capable of filtration rates of approx. 3001h t. We are currently evaluating a DC30 for its capability to recover enteric viruses from groundwater. Acknowledgement--The authors acknowledge the financial assistance of the Metropolitan Water Authority of Western Australia. REFERENCES
Anon. (1980) Editorial. Rotavirus infections in infancy. Br. reed. J. 281, 1162-1163. Belfort G.. Rotem Y. and Katzenelson E. (1974) Virus concentration using hollow fiber membranes. Water Res. 9, 79-85. Berman D., Rohr M-E. and Safferman R. S. (1980) Concentration of poliovirus in water by molecular filtration. Appl. envir. Microbiol. 40, 426-428. Bonsdorff C. H. yon, Hovi T., Makela P. and Morttinen A. (1978) Rotavirus infections in adults in association with acute gastroenteritis. J. med. Virol. 2, 21-28. Caldwell G. G., Lindsey N. J., Wulff H., Donnelly D. D. and Bohl F. N. (1974) Epidemic of adenovirus type 7 acute conjunctivitis in swimmers. Am. J. Epid. 99, 230--234. Farrah S. R., Goyal S. M., Gerba C. P., Wallis C. and Shaffer P. T. B. (1976) Characteristics of humic acid and organic compounds concentrated from tapwater using the Aquella virus concentrator. Water Res. I0, 897-901. Gill O. N., Cubitt W. D., McSwiggan D. A., Watney B. M. and Bartlett C. L. R. (1983) Epidemic of gastroenteritis caused by oysters contaminated with small round structured viruses. Br. reed. J. 287, 1532-1534. Hejkal T. W., Keswick B., LaBelle R. L., Gerba C. P.,
Concentration of rotavirus by ultrafiltration Sanchez Y., Dreesman G., Hafkin B. and Melnick J. L. (1982) Viruses in a community water supply associated with an outbreak of gastroenteritis and infectious hepatitis. J. Am. Wat. Wks Ass. 74, 318-321. Katzenelson E. and Kedmi S. (1979) Unsuitability of polioviruses as indicators of virological quality of water. Appl. envir. Microbiol. 37, 343-344. Melnick J. L., Safferman R., Rao V. C., Goyal S., Berg G., Dahling D. R., Wright B. A., Akin E., Stetler R., Sorber C., Moore B., Sobsey M. D., Moore R., Lewis A. L. and Wellings F. M. (1984) Round robin investigation of methods for the recovery of poliovirus from drinking water. Appl. envir. Microbiol. 47, 144-150. Morse L. J., Bryan J. A., Hurley J. P., Murphy J. F., O'Brien T. F. and Wacker W. E. C. (1972) The Holy Cross College football team hepatitis outbreak. J. Am. med. Ass. 219, 706-708. Murphy A. M., Grohmann G. S. and Sexton M. F. H. (1983) Infectious gastroenteritis in Norfolk Island and recovery of viruses from drinking water. J. Hyg., Camb. 91, 13%146. Packham R. F. (1964) Studies of organic colour in natural water. Proc. Soc. Wat. Treat. Exam. 13, 316-320. Portnoy B. L., Mackowiak P. A., Caraway C. T., Walker
83
J. A., McKinley T. W. and Klein C. A. Jr (1975) Oyster-associated hepatitis. J. Am. reed. Ass. 233, 1065-1068. Ratnamohan N., Garretson R. and Irving L. G. (1980) An ultrafiltration system for the recovery of viruses in effluent. Aust. J. reed. lab. Sci. 1, 149-154. Sreenivasan M. A., Sehgal A., Prasad S. R. and Dhorje S. (1984) A sero-epidemiologic study of a water-borne epidemic of viral hepatitis in Kolhapur City, India. J. Hyg., Camb. 93, 113-122. Steinmann J. (1981) Detection of rotavirus in sewage. Appl. envir. Microbiol. 41, 1043-1045. Sutmoller F., Azeredo R. S., Lacerda M. D., Barth O. M., Pereira H. G., Hoffer E. and Schatzmayr H. G. (1982) An outbreak of gastroenteritis caused by both rotavirus and Shigella sonnei in a private school in Rio de Janeiro. J. Hyg., Camb. 88, 285-293. Taylor J. W., Gary G. W. and Greenberg H. B. (1981) Norwalk-related viral gastroenteritis due to contaminated drinking water. Am. J. Epid. 114, 584-592. Wong D. C., Purcell R. H., Sreenivasan M. A., Prasad S. R. and Pavri K. M. (1980) Epidemic and endemic hepatitis in India: evidence for a non-A non-B hepatitis virus aetiology. Lancet ii, 876-879.
0043-1354/86 $3.00+0.00 Copyright © 1986Pergamon Press Ltd
CONCENTRATION OF ROTAVIRUS BY ULTRAFILTRATION JANIS JANSONS and MARION R. BUCENS Virology Section, Combined Clinical Microbiology Service, Queen Elizabeth II Medical Centre, Nedlands, Western Australia 6009 (Received June 1985)
Abstract--A hollow fibre ultrafiltration unit was evaluated for its capacity to concentrate human rotavirus from experimentally contaminated groundwater. The results of these experiments suggest that virus recovery is greatly improved by utilizing either a protein wash with beef extract or by backflushing the hollow fibres with phosphate buffered saline. An enzyme-linkedimmunosorbent assay (ELISA) was used to determine rotavirus recoveries after concentration. Key words--virus concentration, ultrafiltration, groundwater, rotavirus
rotaviruses and adenoviruses. The reliability of poliovirus as an indicator of faecally contaminated water has also been questioned (Katzenelson and Kedmi, 1979). These factors have created a need for the development of concentration techniques that would enable more reliable detection of a broader range of enteric viruses from contaminated water. Ultrafiltration is an alternative concentration technique and overcomes many of the drawbacks of filter adsorption-elution methodology. Concentration is carried out at ambient pH and addition of polycationic salts and acidification of the sample is not required. Ultrafiltration techniques should not be affected by competing organic compounds such as humic acids since virus particles are retained on the basis of size. In this study a number of parameters were varied in an attempt to optimize recoveries during ultrafiltration.
INTRODUCTION The use of wastewater for the recharge of groundwater aquifers offers a practical and economic method for its disposal. In order to overcome the problem of contamination of the aquifer with human pathogens which include enteric viruses, careful studies of the survival and movement of these agents must be performed. One such investigation is being carried out at the Canning Vale Groundwater Recharge Scheme, at Canning Vale, Western Australia. The currently accepted procedure for concentrating viruses from large volumes of water is the Viradel method which utilizes the filter adsorption-elution principle, with secondary concentration usually being carried out by membrane filtration, aluminium hydroxide precipitation or organic flocculation. This technique was largely developed using poliovirus as a model and very little is known about its efficiency at recoverying other enteric viruses from water. A recent study (Melnick et al., 1984) has revealed that virus recoveries using the Viradei method are prone to considerable variation and this is thought to be related to the chemical quality of sample water. Organic compounds such as humic acids are often found in natural waters (Packham, 1964) and have been shown to adsorb to membrane filters (Farrah et al., 1976). The role and effect of these compounds in the Viradel procedure is poorly understood and their presence in sample water may result in unreliable detection of virus. In our experience, filter adsorption-elution methodology is tedious and time consuming. It exposes viruses to high and low pH during adsorption and elution. Laboratory grown strains of poliovirus appear to survive this treatment, but field isolates of other enteric viruses may not. In recent times, poliovirus has become less significant as a waterborne human pathogen in most Western countries compared with other enteric viruses such as hepatitis A virus, Norwalk-like agents,
MATERIALS AND
Preparation o f virus inoculum
A 10% faecal extract was prepared from pooled rotavirus positive faeces (previously detected by electron microscopy) and suspended in Hanks BSS. To ensure that working dilutions of the rotavirus inoculum had a linear absorbance in response to dilution in the ELISA assay, it was necessary to further dilute the faecal extract by 1/100. Rotavirus recovery studies
Five ml of rotavirus inoculum were mixed with 21. of either phosphate buffered saline (PBS) or groundwater (GW). The latter was collected at the Canning Vale site. A typical chemical analysis of the groundwater is given in Table 1. After inoculation, the sample was processed by ultrafiltration using an Amicon DC2 hollow fibre ultrafiltration unit (Amicon Corporation, Danvers, Mass.) with twin H 1P100-20cartridges which have a nominal cutoff of 100,000 mol. wt and a total surface area of ll00cm 2. Concentration procedure using the Amicon DC2
After the inoculated sample was placed in the DC2 reservoir, the restriction valve (CD valve) was pushed into the concentration mode and turned back to zero. The DC2 79
WK
20iI--F
METHODS
80
JAN1S JANSONS and MARION R. BUCENS
Table 1 pH 6.3 Turbidity 4 (NTU) Conductivity 59 (mS m ~) Alkalinity (CaCO~) 12 (mg I-~) Colour 100 (Hazen) Anions C1 NO 3 SO4 PO4
(mg 1-]) 95 4.9 30 1.8
Cations Ca Mg Na K Fe Mn
(mg 1-I) 14 6 95 5.5 <0.05 < 0.05
was set into operation with the variable speed knob turned to approximately half speed. The CD valve was gradually restricted until the inlet pressure reached 10 psi. After completion of a run, the concentrate was collected, and the volume was measured and recorded. In some o f the experiments, the DC2 was backflushed prior to sample collection. A control sample not having undergone ultrafiltration was set up in parallel. Two-fold dilutions ranging from undiluted to 1/32 o f the concentrate and the control were then prepared in either PBS, GW or beef extract. These dilutions were assayed for rotavirus using the Rotazyme test.
The colour intensity developed by the substrate should be proportional to the concentration of rotavirus antigen in the sample. In each experiment it was necessary to set up a separate control since the sample volume collected after concentration was variable.
Integrity testing of hollow fibres A 0.1~o solution of blue dextran 2000 (Pharmacia, Uppsala, Sweden) was processed in the normal way as described in the concentration section. Blue dextran has a molecular weight of approx. 2,000,000 and should be retained by hollow fibre cartridges with a 100,000 mol. wt. cut-off. During the concentration procedure the filtrate was collected and examined visually for traces o f blue dextran. RESULTS R o t a v i r u s c o u l d n o t be d e t e c t e d in the c o n c e n t r a t e s following n o r m a l c o n c e n t r a t i o n using either P B S o r g r o u n d w a t e r as p r o c e s s fluids, with optical d e n s i t y (O.D.) values falling b e l o w the positive cut-off.
Effect of backflushing B a c k f l u s h i n g o f t h e fibres resulted in c o n s i d e r a b l e i m p r o v e m e n t in recovery with PBS (Fig. 1) a n d with
Backflushing procedure The ultrafiltrate effluent line was clamped off and the CD valve was turned to zero. About 30 ml o f either filtrate or PBS were forced back across the fibres through one of the filtrate ports situated on the cartridge holder. After backflushing, a rapid forward recirculation was carried out for 30s and the sample was collected. This manoeuvre assisted in the removal o f any non-specifically adsorbed virus by washing the membrane surface o f the fibre lumen. In one experiment using groundwater, the backflush was tested for rotavirus in parallel with the concentrate and the control. To evaluate overall recovery from this experiment, the backflush was pooled with the concentrate and the control volume adjusted with PBS so that it had the same parameters as the combined concentrate and backflush. These were tested for rotavirus.
0.5
=E 0.4 e~ O~
~
_•
Rotavirus assay The rotavirus assay was carried out according to the manufacturer's instructions using a commercially available ELISA test (Rotazyme, Abbott Laboratories, North Chicago, I11.). In brief, the sample was incubated with an anti-rotavirus guinea pig antibody coated polystyrene bead for 3 h at 45°C. The beads were washed with distilled water and then incubated with rabbit anti-rotavirus horseradish peroxidase conjugated antiserum for 1 h at 45°C. After this incubation the beads were washed again and transferred to a box of reaction tubes and the substrate, OPD (ophenylenediamine.2HC1) in citrate phosphate buffer, was added. The tubes were incubated in the dark for a further 15 min. The enzyme reaction was stopped by adding 1 N sulphuric acid. The photometric reading of the results was performed on an Abbott Quantum reader using the Rotazyme protocol.
Control
•
Concentrate
0.3
_= c5 (5 0.2 0.1
Beef extract washing procedure Five hundred ml of 10% beef extract were added to the DC2 reservoir after approx. 1 1. of sample had been processed. The concentration was then allowed to proceed normally and the concentrate collected as already described. In another experiment 500 ml of 3% beef extract were concentrated by ultrafiltration. The concentrate was collected and inoculated with 5 ml of rotavirus extract. A control was also tested using unconcentrated 3~o beef extract and an identical inoculum in the same volume as the concentrate. These were tested using the Rotazyme assay.
Exp.A •
I I
Rotovirus
I
t
I
J
2
3
4
5
inoculum dilution(log;z)
0.5 Exp, B Control
~ t~
0.4
~ $ 3 ~ "; 0
0.3
Concentrate
0.2 '\\ Positive c u t - o f f
0.I
I
0
t
I
I
1 2 3 4 Rotovirus inoculum dilution ( log 2 )
J
5
Fig. 1. Recovery of rotavirus from phosphate buffered saline as measured by the Rotazyme assay. Exp. A was conducted without backfiushing, whereas in Exp. B, hollow fibres were backflushed.
Concentration of rotavirus by ultraflltration 0.5 -
=E
0.4
-
• •
Exp. A Control Concentrate
o
Back flush
81
0.5 r
~N ~-
0.4
Exp. A
L~
•
e
n
Control
t
rate
(M
,¢
0.3
'6
0.3 -
v
O
> 0.2 c~ d 0.1
°Oo )"z
0
,
,
e%
Positive
cut-off
_.= >~ 0.2 d d
~
Positive cut-off
o.1
" " "°O
I 1
I 2
I 3
I 4
I 5
-.
I I
0
Rotovirus inoc.ulurn dilution (log z)
--_--
•
I 2
I 3
I 4
I 5
Rotavirus inoculum dilution ( loge)
0.5 0.5
Exp. B 0.4
•
Exp. B • •
Control
• Concentrate
E
\
Control Concentrate
0.4
C C
0.3
N
'¢
0.3
O B
0.2 [
c~
"~[
.
0.1
O
Positive cut-off
(?.2
-.. ~-• I
o
I
1
I
2
I
3
I
4
I
5
Rotovirus inoculum dilution (log 2) Fig. 2. Recovery of rotavirus from groundwater. In Exp A, backflush was individually tested. In Exp. B, backflush was combined with concentrate, and the control diluted to a similar extent.
groundwater [Fig. 2(B)]. In this experiment the backflush was tested and compared with the concentrate and the control. It was found that the backflush contained a greater proportion of virus inoculum than the concentrate [Fig. 2(A)]. When the backflush was combined with the concentrate and compared to a control with identical parameters almost complete virus recovery was achieved [Fig. 2(B)].
/ 0
Positive cut-off
I 1
I 2
I 3
I 4
I 5
Rotovirus inoculum dilution (log z) Fig. 3. Recovery of rotavirus from groundwater. Exp. A used the normal concentration method. Exp. B was recovery after 10% beef extract wash.
virus inoculum in concentrated beef extract were reduced when compared with unconcentrated beef extract. Alkaline beef extract pH 9.5 did not improve virus recovery compared to neutral beef extract. After a beef extract wash backflushing appeared to have little effect on recovery rates.
Effect o f beef extract washing on virus recovery
Beef extract washing of the hollow fibres also improved recovery of rotavirus (Fig. 3). Virus recovery was found to be proportional to the concentration of beef extract although improvement was only minimal at concentrations above 5%. Washing of the hollow fibres with beef extract at concentrations greater than 10% caused excessive foaming. However, when concentrated beef extract was tested for any inhibitory effect on the Rotazyme assay, it was found that absorbance values of rota-
DISCUSSION There has been an increasing number of reports of waterborne outbreaks of virus infections by a wide range of enteric viruses other than polioviruses. Waterborne outbreaks of hepatitis A virus (Morse et al., 1972; Hejkal et al., 1982), gastroenteritis attributed to a Norwalk-like agent (Taylor, et al., 1981) and rotavirus (Sutmoller et al., 1982), have been reported, as well as an outbreak of swimming pool
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JANIS JANSONSand MARIONR. BUCENS
conjunctivitis caused by adenovirus type 7 (Caldwell et aL, 1974). Murphy et al. (1983) recently described an outbreak of infectious gastroenteritis on Norfolk Island. They detected rotaviruses, small round viruses and adenoviruses by electron microscopy, as well as isolating polioviruses and adenoviruses in cell culture from groundwater which was used to supplement the island's water supply. Water has also been implicated in viral disease outbreaks by contamination of shellfish with agents such as hepatitis A virus (Portnoy et al., 1975) and small round structured viruses (Gill et al., 1983). An extensive waterborne hepatitis outbreak in India has been recently described (Sreenivasan et al., 1984) where the aetiological agent is still unknown. The epidemiological data from this outbreak suggested hepatitis A virus as the aetiological agent but serological evidence indicated non-A non-B hepatitis. An earlier study (Wong et al., 1980) also suggested that the 1955 waterborne hepatitis epidemic in Delhi. India, where 29,300 cases were reported, may have been due to a similar agent. There is a need for the development of more reliable techniques for the recovery of a greater range of enteric viruses from contaminated water. in this study, ultrafiltration was examined as an alternative method for virus concentration because of unsatisfactory performance with the filter adsorption-elution technique when concentrating viruses from Canning Vale groundwater. The difficulties experienced with recovery of virus from groundwater were thought to be caused by the presence of interfering compounds. Rotavirus was selected as a model for this study since it has been associated with outbreaks of gastroenteritis in infants (Anon., 1980) and adults (von Bonsdorff et a l , 1978). Until recently, attempts to grow rotavirus in cell cultures had been unsuccessful. and the only two practical methods available to detect these viruses were electron microscopy and immunoassay. Steinmann (1981) described an ELISA technique in which he concentrated domestic sewage on to Seitz filters using an adsorption-elution technique followed by ultracentrifugation. Using this method he was able to detect rotavirus in 25% of the samples. In view of this finding, an ELISA technique was chosen to determine virus recoveries after ultrafiltration. A similar immunoassay could be developed to test water concentrates for the presence of hepatitis A virus and other enteric viruses. The introduction of hollow fibre ultrafiltration technology has resulted in high filtration rates and the use of this technique as a practical alternative to filter adsorption-elution methodology for concentration of viruses from large volumes of water has become feasible. It has been successfully used to recover viruses from effluent (Ratnamohan et al., 1980) and from groundwater (Murphy et al., 1983).
By using some variations of the basic ultrafiltration technique we have shown that recovery of rotavirus is greatly enhanced by PBS backflushing, which supports the findings of Belfort et al. (1974) who reported that 13-54°,o of poliovirus was recovered after backflushing. A beef extract wash also appears to have an effect on rotavirus recovery. Similar findings have previously been reported by Berman et aL (1980) who described a 5-fold increase in poliovirus recovery after pretreatment of ultrafiltration membranes with flocculated beef extract. Poorer recoveries of rotavirus after beef extract washes compared with PBS backflushing may be due to the assay. This could result in an underestimation of true recovery when an immunoassay is used and may be due to beef extract protein non-specifically binding to the antibody coated polystyrene beads, thus blocking the antigenic sites for virus. In view of this finding, further investigations of virus recovery after beef extract washing may be warranted, although an alternative method of virus assay may have to be evaluated. We recommend frequent integrity testing of the hollow fibre cartridges with dextran blue. Although the cartridges have an estimated life span of approx. 2 years we experienced periodic failure. On most occasions, poor recoveries after concentration were traced back to broken tibres. Although the sample size in these experiments was only 21., a larger ultrafiltration unit (Amicon DC30) is available, which is capable of filtration rates of approx. 3001h t. We are currently evaluating a DC30 for its capability to recover enteric viruses from groundwater. Acknowledgement--The authors acknowledge the financial assistance of the Metropolitan Water Authority of Western Australia. REFERENCES
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