A comparison of current methods of poliovirus concentration from tap water

Water Res. ~,ol. 19. No. I. pp. 85--88. 1985 Pnnted in Great Britain. All rights reserved

0043-1354;85 5300*0.00 Copyright ~ 1985 Pergamon Press Ltd

A COMPARISON OF C U R R E N T METHODS OF POLIOVIRUS C O N C E N T R A T I O N FROM TAP WATER NAOMI GUTTMAN-BAsS, TOVA HOSTOVSKY, MARGALITH LUGTEN and ROBERT ARMON Environmental Health Laboratory, Hebrew University--Hadassah Medical School. P.O. Box 1172, Jerusalem, Israel (Received Norember 1983)

Abstract--The efficiency of concentration of poliovirus from Jerusalem tap water was investigated for several types of "'electronegatively-charged'" and "electropositively-charged" microporous filters. In addition, the efficiency of organic fiocculation as a procedure for reconcentrating poliovirus from filter eluates was investigated. The Balston and Cox filters had similar recovery efficiencies from tap water, with recovery of approx. 90% of the input virus, both after filtration and after organic flocculation. Decreasing the concentration of beef extract in the eluent from 3 to 1% did not negatively influence virus recovery. However. recovery of low virus numbers from large volume samples by Cox filters was variable. Balston filters were used in a series of high volume experiments to test the efficacy of the tentative standard method for virus recovery using a proportioner pump with two additive pumps. The method was inefficient without a simple modification, the addition of a second mixing chamber, which increased the virus recovery to an acceptable level. The Zeta Plus eleetropositively-charged type of filter had a high efficiency of virus recovery in the eluate, but approximately half of the virus was lost during organic flocculation. This may indicate the need for modification of the organic fiocculation method when used with filters. Key words--virus concentration, poliovirus, tap water, microporous filters, organic flocculation

INTRODUCTION

Virus concentration

Jerusalem tap water dechlorinated by the addition of 10 rag l-t sodium thiosulfate was seeded with poliovirus, in the amounts indicated in the text. The pH of the sample was adjusted to the desired level and the sample was filtered. The filters used were: Balston 17.8-cm long, 8-gin nominal porosity fiberglass--epoxy filter tube (grade C, Balston, Inc., Lexington, MA); Cox 142 and 267-mm dia, 0.45-gin nominal porosity fiberglass-asbestos--epoxy filter (Type M-780, series AA, Cox Instrument Div., Lynch Corp., Detroit, MI) with a Sartorius SM 13430 fiberglass prefilter (Sartorius, W. Germany); and Zeta Plus 60S 47- and 142-ram dia, 0.45-#m nominal porosity cellulose--diatomaceous earth"charge-modified" resin filter (AMF, Cuno Div., Meriden, CT). Eluents were beef extract (Lab-Lemco, Oxoid, Ltd, Long, England); tryptose phosphate broth (Difco, Detroit, MI) and 0.05 M glycine-NaOH, pH 9.9. The virus was eluted from the filters and reconcentrated by organic flocculation as described (Katzenelson et al., 1976). In some cases the virus was concentrated by a portable apparatus described by Jakubowski et al. (1978). In these experiments, the tap water was dechlorinated, virus was added, and the pH of the water adjusted by the addition of HCI through a proportioner pump (Johanson & Son Machine Corp., Clifton N J). Modifications of the original arrangement of the equipment are as described in the text.

The potential danger of viral contamination of water, which might lead to large-scale disease outbreaks, has been a subject of much controversy. This is in part due to the lack of reliable and efficient techniques of virus recovery from water, and the lack of convenient methods of detection of some of the suspected waterborne agents. A number of approaches have been used in the development of methods for the concentration of viruses from water and several recent reviews have appeared in the literature (Belfort and Dziewulski, 1982; Guttman-Bass, 1983). One type of methodology which has been investigated extensively and recommended for use with large volumes of water ( A P H A , 1981) is adsorption to and elution from microporous filters. However, the methods which have been developed using various types of filters and eluents have not proved to be as reliable as required, and similar methods used by different laboratories have been found to vary widely in their efficiency of virus recovery. In this paper we report the results of comparative studies of poliovirus concentration from tap water using several methods in current use, in an attempt to compare the techniques and evaluate their efficiency for virus concentration from tap water.

RESULTS AND DISCUSSION Virus concentration by electronegative filters

The efficiency of recovery of poliovirus from two negatively-charged microporous filters which have been recommended for virus concentration from tap water ( A P H A , 1981) was explored. Cox filters, which have been reported to recover poliovirus from tap water with high ( > 70%; Sobsey et al., 1973; Katzenelson et al., 1976), intermediate (39%; Jakubowski et

MATERIALS AND METHODS Virus and t'irus assay

Poliovirus type 1 (Brunhilde strain) was grown and assayed by plaque assay in BGM (African green monkey kidney) cells as previously described (Kedmi and Fattal, 1981). 85

\ a , ( : d l (JLTT',IA\-BANS C: ~ :

[able i Po::o~was concen'rat:on % etectronega::'.e filters"

Filter t?pe

No. of exp.

Cox

3 3 6 4 2

Balston

[ 1 3

~a~er ~,3

[.qpU!

t[.J

Ipfu)

2 2 1¢)0 0.5 5 5 5 5

3.0 ,< t0 s +23 11-42 1.0 x t0 ~ 1.8 x 10 ~ 1.8 × I 0 ~

,,I r us

Eluent 1",, I", I',, _.7""". l°,, 1~o

Fdtrate

Elaate

0 -

110 ___5

BE BE

. . . . .

TPB

0 87 _- 5 94 __- 0 . . . . ---

BE

BE BE

l"., BE 1°,, BE

2,0 × tO "~ 1.8 x 10 ~

-

Organ;c fl,)c [ I l - '~ -I 221 % : b3 -~7 : 4 04 b) 92 : 18

*Poliovirus was seeded into tap water and concentrated by the indicated filters. The p H o f the input water was 3.5 and the eluent p H was 9.0. BE = beef extract and T P B = tryptose phosphate broth. The size o f the C o x filters was 142 m m for the small volume and 267 m m for the 100 I. experiments. Results are expressed as average per cent o f the input virus in the indicated fraction -- SD.

1975) and low ( < 3 0 % ; Sobsey et al., 1980) efficiencies, were tested for their ability to concentrate poliovirus from large and small volumes of tap water (Table I). Virus recovery after reconcentration by organic flocculation was also determined. In general, virus recovery was good, both in the eluate and in the organic floc formed from the etuate. In these experiments the concentration of beef extract was reduced to 1% in place of the previously used 3":; (Katzenetson et aL, 1976), with no reduction in virus recovery. Essentially all of the input virus was recovered by the two-step technique, with an overall average recovery of 91%. A slightly lower recovery was observed when small virus numbers were seeded, but this may have been due to the imprecise measurement of input titer when low virus concentrations are used. Using large volumes of water, virus recovery in individual experiments ranged from 29 to 200% showing a wide range of recoveries, Another protein-rich eluent, tryptose phosphate broth, was used to etute the virus, and was found to be efficient (Table I). However, this broth did not form floes at pH 3.5, and thus could not be used to further concentrate the virus by organic flocculation, but it might be used as a substitute eluent if organic flocculation is not required. A second type of filter, the Balston cartridge filter, was assayed for its ability to concentrate poliovirus. As with the Cox filters, this type of filter has been reported to be relatively efficient ( > 40%; Jakubowski al.,

et al., 1974, 1975; Guttman-Bass et al., 1981: Kedmi

and Fatta[, 1981) and inefficient ( < 20%; Morris and Waite, 1980: Sobsey et al., 1980) for virus concentration from tap water. The efficiency of virus recovery by the Balston filters followed by organic flocculation was investigated here using small and large volumes of input water. In the experiments using small volumes and various concentrations of virus, the filters were found to be very efficient virus adsorbers, and an average of 890.0 of the virus was found in the concentrate after the two-step concentration procedure (Table I). It should be noted that adsorbent aids such as muhivalent cations were not used in these experiments and were not found to be necessary. In these experiments, 1% beef extract was used to elute the virus and proved to be as efficient as in previously reported work using filters with a smaller pore size (Kedmi and FattaI, 1981). In addition, the results indicated a high degree of reproducibility in the experiments, with an average organic flocculation efficiency of 92%. The range of recoveries was 78-1 t2% for the combined two-step technique. The Balston filters have been recommended for use in recovering virus from large volumes of water in conjunction with a proportioner pump which conditions the water on-line. This portable virus concentration apparatus (Jakubowski et al., 1978) can be used to process up to 1900 1. of water in the field using three Balston filters in parallel (Hill et aL, 1976). In

T a b l e 2. Poliovirus concentration

by a portable virus concentrator*

pH range

No.

o f mixing

chambers I

2

........................................... Before filter After 2.9-.~..1

3.3-3.7

filter

3,3-3.7

3.5

[flput water vol.

Input virus

"~ Virus

(I.)

(pfu)

recovery

1.6 × 10z x I0"

24 26

200

200 I00 100 I00 100 400

1.6 8.0 8.0 2.0 2.0 7.2

x 105 x 10-~ x I0" x I0 v x 10 z

96 79 63 81 99

*Poliovirus was seeded into dechlorinated tap water and the water was passed through a proportioner pump which adjusted the p H to 3.5. T h e p H was m o n i t o r e d before and after the water passed through the B a l g o n grade C adsorbing filter. Elation o f the virus with I% beef extract and organic flocculation were as described above. % Virus recovery is the per cent o f the input virus recovered in the organic floe.

Current methods of poliovirus concentration from tap water the first set of experiments performed to test the efficiency of the method, the ori~nal model of the apparatus was used, albeit with a single filter, with low virus recover)' (Table 2). The pH of the water was monitored continuously both before and after the filter, and found to vary widely. This may account for the low virus recovery, due possibly to tow pH inactivation of the virus or the passage of water at a pH not conducive to virus adsorption. In order to smooth out the pH fluctuations, an additional mixing chamber was added in series with the first one (situated between the proportioner pump and the filter). This damped out the pH swings, and ted to an increase in virus recovery, with an average of 84 +_ [ 500. Good virus recovery was obtained from up to 400 1. of water containing a low concentration of virus. It should also be noted that the water was passed through a single filter, without a prefilter and not using three parallel filters, both of which adjustments would increase the volume of water which could be processed and the volume of eluent necessary to elute the virus. Finally, the addition of a second mixing chamber to the virus concentration apparatus is a relatively simple alteration, in comparison with the use of a quadriplex proportioner pump, as has recently been recommended APHA, 1981).

Poliovirus concentration from electroposittve filters Recently, electropositive filters have been suggested for use as virus adsorbents and might be practical alternatives to the more electronegative types of filters (Sobsey et al., 1981). One advantage of these filters is that they do not require drastic lowering of the pH of the input water in order to adsorb viruses. At ambient pH levels, the virus, with its negative charge, should adsorb more easily to more positively charged filters. Such relatively electropositive filters have been reported to successfully concentrate viruses from tap and waste waters (Guttman-Bass, 1983). The usefulness of the filters for the concentration of poliovirus, using beef extract as an e[uent and organic flocculation as a second concentration step was investigated and the results are shown in Table 3. Poliovirus adsorbed to and eluted from the filters efficiently, with both beef

87

extract and glycine-NaOH as eluents, although beef extract was more efficient. Adsorption was efficient at a range of pH from 6.0 to 7.4, but recovery in the eluent was best at pH 6.4 or below. Sobsey and Jones (1979) similarly reported lower recoveries above pH 6.0. apparently due to lower adsorption efficiency, while in this case the elution efficiency was affected. Since maximal recovery requires pH control, as has been found for electronegative filters, equipment to condition the water should probably be used with these filters as well. The use of less extreme pH levels with these filters may not be a significant advantage for po[iovirus recovery, but may enable the more efficient recovery of more acid-sensitive viruses. Initially, 1~ rather than 3?° beef extract was used to elute the virus to minimize the concentration used. As described above, this lower concentration was found to be sufficient for elution from Cox and Balston filters, followed by organic flocculation of the eluate, with recovery of most of the virus in the concentrate. However, with Zeta Plus filters, reduction of the pH of the eluate to 3.5 did not always result in floc formation; in some experiments no visible floc was formed. In order to aid the organic flocculation, the concentration of beef extract in eluate was increased to 3~o. At this concentration, floc formation was observed and a visible pellet was formed after centrifugation. Even under these conditions there was inconsistent virus recovery. On the average approximately half of the virus in the eluate was recovered in the resuspended floc, and in individual experiments the recovery efficiency of the organic flocculation step ranged from 22 to 76~o. The recovery of poliovirus by the two-step concentration technique ranged from 19 to 64~o of the input. In an attempt to locate the missing virus, the supernatant of the organic floc was assayed and little or no virus was found (data not shown). In addition, the disaggregation of virus clumps which might have formed during the organic flocculation step was attempted by subjecting the resuspended floc to mild sonication for 10 rain in a water bath sonicator but this treatment did not increase the virus titer (data not shown). That the efficiency of organic flocculation from an eluate could be influenced by the type of filter eluted has also been reported by Sobsey et al. (1981). In

Table 3. Poliovirus concentration by Zeta Plus filters* Filter dia. (ram) 47

142

No. o f exp.

Input water vol. (I.)

% Virus recovered in

1

2

2.5

x

I 1 1 4 4 2 2 2

2 2 2 I 2 2 2 2

5.0 2.5 5.0 t.2 4.0 6.7 6.7 6.7

x × x x x x × x

Input virus (pfu) 102 103 102 103 107 104 103 IO" l0 t

Input pH

Eluent

Filtrate

Eluate

6.0 6.0 6.0 6.0 7.4 6.6 6.4 6.4 6.4

I ~ BE lYo BE GIy-NaOH Gly-NaOH 3 ~ BE 37/~ BE 3% BE 3 ~ BE 3 ~ BE

2 0 1 0 0.25 4- 0.05 -----

166 104 80 72 69 4- 7 73 _ 5 81 4- 4 I00 4- I l --

Organic floc --

----52 _ 3 22 + 3 61 4- 3 55 4- 7

*Concentration o f poliovirus was as described in the legend to Table I, except that the p H of the input was as indicated, and the eluent G l y - N a O H was 0.05 M g l y c i n e - N a O H , p H 9.9

~>;

NAOMI GUTTMAN-BA&~,e: a:.

their case, even the addition of flesh beef extract prior to floc formation did not alleviate this effect. Using different filters and viruses than those tested here, they found that organic flocculation from a positively charged filter eluate was more efficient than from a negatively charged filter eluate. In contrast, we have found that the negatively charged filters used here did not interfere with organic flocculation from the eluate. but that the relatively positively charged Zeta Plus filters resulted in less efficient and less consistent recovery by organic flocculation. In both reports, virus was not detected in the supernatant but only in the floc. Therefore, it may be that the composition of the eluent is altered upon passage through particular filters, with certain components retained. That some components are adsorbed and not eluted after filtration through Zeta Plus filters is evidenced by the reduced ability of the eluate to form flocs at lower beef extract concentrations. The changed balance of the eluate may then provide less stable conditions for the virus, especially at the low pH used in organic flocculation. Thus, modification of the procedure, such as the use of a different proteinaceous eluent, followed by the addition of fresh beef extract for organic floculation might lead to improved recovery in the two-step concentration technique using the problematic filters. CONCLUSIONS The aim of this paper was to assess a number o f virus concentration techniques which are potentially standard methods for the concentration of viruses from large volumes of tap water. Both Balston and Cox filters recovered poliovirus with good efficiency, and worked well in combination with organic flocculation as two-step concentration methods. Cox filters, however, had variable virus recoveries from large volumes of water. Balston filters in particular are easy to handle with large volumes of water in a slightly modified portable apparatus. Zeta Plus filters, were found to recover poliovirus with good efficiency at more moderate p H levels than the more electronegative filters. However, organic flocculation of the etuates of these filters did not result in consistent virus recovery and requires further investigation. In summary, all of the methods tested were found to be comparable for the concentration of poliovirus from tap water although results for the two-step method using Balston filters were the most consistent. The ultimate choice of a method may thus have to be made on the basis of performance using a wider range of virus types, since different types and strains may vary in their adsorptive characteristics (Landry et al., 1979), and more highly polluted water. Acknowledgements--Although the research described in this article has been funded wholly or in part by the United

States Envtronmental Protection Agenc.,, throtagn Grant> No. CR-806588-03-2 to H. I. Shu',al and N. Guttman-Bas_, and No. CR 806416-01 to H. I. Shu',al. M. Davies ard B. Fattal. it has not been subjected to Agency rc'~iew and therefore does not necessarily reflect the views of the Agent.', and no official endorsement should be inferred.

REFERENCES

APHA (1976) Standard Methods Jbr the Examination of Water and Wastewater, 14th Edition. pp. 971-975. American Public Health Association, Washington, DC. APHA (t981) Standard Methods for the Examination of Water and Wastewater, 15th Edition. pp. 851-859. American Public Health Association. Washington, DC. Belfort G, and Dziewulski D. M. (1982)Concentration of viruses from aqueous environments: a review of the methodology. In Water Reuse (Edited by Middlebrooks E. J.), pp. 679-750. Ann Arbor Science, Ann Arbor, MI. Guttman-Bass N. (1983) Developments in methods for the detection of viruses in large volumes of water. War. Sci. Technol. 15, 59--67. Guttman-Bass N., Tchorsh Y., Nasser A. and Katzenetson E. (1981) Rapid detection of enteroviruses in water by a quantitative fluorescent antibody technique, tn Viruses and Wastewater Treatment (Edited by Goddard M. and Butler M.), pp. 247-251. Pergamon Press, Oxford. Hill W. F. Jr, Jakubowski W., Akin E. W. and Clarke N. A. (1976) Detection of virus in water: sensitiyity of the tentative standard method for drinking water. Appl. eniqr. Microbiol. 31, 254-261. Jakubowski W., Hill W. F. Jr and Clarke N. A (1975) Comparative study of four microporous filters for concentrating viruses from drinking water. Appl. Microbiol. 30, 58-65. Jakubowski W., Hoff J. C., Anthony N. C. and Hill W. F. Jr (1974) Epoxy-fiberglass adsorbent for concentrating viruses from large volumes of potable water. Appl. MicrobioL 28, 501-502. Jakubowski W., Chang S.-L., Ericksen T. H., Lippy E, C. and Akin E. W. (1978) Large-volume sampling of water supplies for microorganisms. J. Am. Wat. Wks Ass. 70, 702-706. Katzenelson E., Fattal B. and Hostovesky T. (1976) Organic flocculation: an efficient second step concentration method for the detection of viruses in tap water. AppL envir. Microbiol. 32, 638-639. Kedmi S. and Fattal B. (1981) Effectiveness of the organic flocculation method in concentrating echovirus 7 and coxsackievirus A9 from water. Water Res. 15, 13-15. Landry E. F., Vaughn J. M., Thomas M. Z. and Beckwith C. A. (1979) Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater. Appl. envir. Microbiol. 38, 680--687. Morris R. and Waite W. M. (1980) Evaluation of proecedures for recovery of viruses from water--I. Concentration systems. Water Res. 14, 791-793. Sobsey M. D. and Jones B. L. (1979) Concentration of poliovirus from tap water using positively charged microporous filters. AppL envir. Microbiol. 37, 588-595. Sobsey M. D., Moore R. S. and Glass J. S. (1981) Evaluating adsorbent filter performance for enteric virus concentrations in tap water. J. Am. Wat. Wks Ass. 73, 542-548. Sobsey M. D., Wallis C., Henderson M. and Melnick J. L. (1973) Concentration of enteroviruses from large volumes of water. AppL MicrobioL 26, 529-534. Sobsey M. D., Glass J. S., Carrick R. J., Jacobs R. R. and Rutala W. A. (1980) Evaluation of the tentative standard method for enteric virus concentration from large volumes of tap water. J. Am. Wat. Wks Ass. 72, 292-299.