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Evaluation of procedures for recovery of viruses from water—II detection systems
Water Research VoL 14. pp. 795 1o 798 Pergamon Press Lid 1980. Printed in Great Britain
0043-1354/80/07014~795$02.00/0
EVALUATION OF PROCEDURES FOR. RECOVERY OF VIRUSES FROM WATER--II DETECTION SYSTEMS R, MOI~RIS* and W. M. WAITE Regional Laboratory, Severn Trent Water Authority. St Martins Road, Coventry, England Abstract--Three cell lines used in three assay procedures have been evaluated for their suitability in detecting viruses in concentrates of river water samples. Vero cells proved to be relatively inefficient, BGM cells gave the best recoveries in general, whilst RD cells were variable. The mode of presentation of the cell system was of importance, monolayers in flasks giving low levels of virus, tube cultures being intermediate, whilst the agar suspended cell mode giving best recoveries. Using the BGM-agar suspended cell system the River Sowe gave virus levels varying from 145-620pfu 1-1 whilst the River Avon generally gave much lower levels with evidence of occasional high levels of contamination (540 pfu 1- l). It is suggested that BGM and RD cells used as agar suspended cell cultures are suitable for routine surveillance of the aquatic environment.
INTRODUCTION Infectivity of viruses, and hence numbers, can be measured using a variety of biological methods. Tissue culture systems afford simple, cheap and reproducible procedures for assaying viruses. The presentation of cells for virus infectivity has involved the use of monolayers in tubes (Chang et al., 1958), flasks (Hsuing & Me[nick, 1955) and petri dishes (Kjellen 1961). A method using cells suspended in agar has been described by Cooper (1967). The cell system used for virus isolation has depended on the range of viruses to be detected, the numbers of cells produced and the ease of handling. As most work on viruses has been on human types, the emphasis has been on the use of human and monkey cells as substrates. Primary cultures tend to be very sensitive but are increasingly more expensive. However, the introduction of continuous cell lines has proved useful for the isolation of viruses from clinical specimens because of the greater control over assay systems. Clinical virology concentrates on isolating and identifying dominant viruses in cases of illness, whereas virus detection in water samples aims at recovering the maximum number of viruses and virus types. The present study examined the suitability of three cell systems (one human, two monkey), presented in three ways (monolayers in tubes and flasks, agar suspended cells), for detecting enteroviruses in river water.
MATERIALS AND METHODS Cell cultures
The Veto cells used in this study have been described previously by Morris & Waite (1980). The monkey kidney cell line designated BGM and established by Barton et al. (1970) was supplied b~, Flow Laboratories, Scotland. A cell * To whom all correspondence should be addressed. 795
line derived from a human rhabdomyosarcorna by MeAllister et al. (1969) and designated RD, was a gift from Dr E. Bell, Glasgow, Scotland. BGM cells were grown in roller bottle culture using a mixture of Eagle's MEM and Liebowitz (LI5) media whilst RD cells were propagated on Eagle's MEM in plastic flasks (175era 2, Nunc). Both growth media contained foetal calf serum (10%), penicillin ( 100 units ml - i ) and streptomycin (100/zg ml - i), (GibcoBiocuit). Assay systems (a) Monolayer technique (Hsuiag & Melnick 1955). Plastic flasks (75 crn2, Nunc) were seeded with 1.5-2.0 x 106 cells in 25 ml of appropriate growth medium. Confluence was reached in 3-5 days at which time the cultures were drained and washed once with either medium 199 (BGM and Veto) or Eagle's MEM (RD cells). Sample concentrate (mixed with an equal volume of double strength wash medium) was allowed to adsorb for 1½h at room temperature, then washed off with a further aliquot of wash medium. BGM and Veto cultures were overlaid with the agar medium described by Morris and Waite (1980) whilst RD cells were overlaid with an Eagle's MEM based agar medium. Plaques were counted daily over nine days incubation at 37°C. Three replicates per sample were assayed. Tube technique. Soda glass tissue culture tubes (Flow) were seeded with approximately 3 x 10~ cells in 2 ml of appropriate growth medium and after 24 hr cultures were changed to maintenance medium. BGM cultures were maintained on iactalbumin hydrolysate, Veto cultures on medium 199 and RD cultures on Eagles MEM, all containing foetal calf serum (5%) and the full range of antibiotics previously described (Morris & Waite 1980). Sample concentrates were diluted in appropriate maintenance medium and 0.1 ml volumes added to each of a set of five tubes. Dilutions were 4-fold ranging from neat to 1:64. Tubes were examined daily for evidence of cytopathic effect (cpe) over a period of seven days incubation at 37°C. Most probable numbers (MPN) were determined using the tables of Chang et al. (1958). (c) Agar suspended cell technique. This technique, described by Cooper (1967), was adopted for use with water samples by Slade (1978). A cell suspension of l0 T cells/ml was prepared from stock cultures by centrifugation and resuspension. Sample concentrate (1 ml) was mixed with 2ml of cell suspension and 10ml of overlay medium described previously, before pouring into a plastic, 90 mm diameter, vented bacteriological grade petri dish (Sterilin).
796
R MORRISand W. M ~'AIr~ Table 1. Bacteriological levels of river Sowe and river Avon River Sowe Total coliforms/100ml
Mean Range Mean Range Mean Range
E. coli/lOOml Faecal streptococci/100ml
1.8 0.5 9.8 0.2 1.3 0.3
x
l0 6
x x x x x
10~-5.3 x 106 I0"~ 105-2.7 x 105 104 10~-2.7 x 10't
When set, plates were incubated inverted at 37°C in an atmo~hcre of 5% carbon dioxide in air, plaques being counted daily over seven days. Three replicates per sample were examined. Waters Two river waters were examined for virus levels using the methods de~ribed above. The River Avon was sampied approx 25 km downstream of Rugby Water Reclamation Works; the River Sowe was sampled approximately 2 km downstream of Finham Water Reclamation Works (Severn Trent Water Authority, 1978). Bacterial data for both rivers over the period of the present evaluation are shown in Table I. Sample processing Samples of both river waters (51) were conditioned and concentrated as described previously (Morris & Waite 1980) using the Balston filter system in the presence of aluminium cations. The final concentrate (25 ml) was held at -20°C prior to assay.
RESULTS AND DISCUSSION levels of virus found in samples from two rivers concentrated by a single procedure and assayed by three techniques using three cell lines are given in Table 2. Virus levels obtained with Vero cells as the substrate were invariably low regardless of the assay system used. On no occasion were viruses demonstrated using the tube technique with Vero cells and when using the cell line as monolayers in flasks, viruses
7.1 1.0 2.3 0.1 2.5 0.1
River Avon × 104 x 10"t--2.0 × 105 × 10'~ x 10a-.7.5 x 10't x l0 g × 103--5 X 1 0 3
were demonstrated in only one of six samples of the river Avon and three of four samples of the river Sowe, counts in all cases being very low. The agar suspended cell technique with Vero performed slightly better, yielding viruses from all samples found positive by the monolayer method and also from two samples which yielded no viruses with the other techniques. The highest recovery from any sample using Vero cells was 20pfu, this being obtained with a sample of river Sowe using the agar suspended cell procedure. Recoveries of viruses using BGM cells were very much greater, the tube technique giving viruses from all but two samples, these two, however, giving low counts by both flask monotayer and agar suspended cell methods. The highest count obtained with BGM tubes was an MPN of 5851-1. Flask monolayers recovered viruses from all but one of the samples but recoveries were low, a maximum of 25 pfu 1- ~ being achieved. The numbers of viruses recovered b y t h i s method did not differ appreciably from those obtained by the Vero monolayer technique. The use of BGM in agar suspended cell cultures proved to be the most sensitive of all the methods evaluated, viruses being detected in all samples with a maximum recovery of 620 pfu 1- z. Results obtained with RD cells were variable when compared with those obtained with the other cell lines. Recoveries with flask monolayers~ however, remained low. On two occasions the findings with the
Table 2. Enterovirus levels in two rivers by nine assay methods Cell system Assay system Date 17.11.78 23.11.78 7.12.78 18.12.78 20.12.78 27.12.78
Riper Avon Avon Sowe Avon Sowe Avon Avon Sowe Avon Sowe
RD T*
BGM
Vero
MLt
SC~
T
ML
SC
T
ML
SC
ND~ ND ND 8 8 27 ND 5 8 ND ND 3 8 ND 94 5 8 8 200 7
2 ND 185 310 170 ND ND ND ND 3
ND ND 72 151 585 8 18 94 8 161
2 2 7 8 13 5 2 2 ND 25
2 5 145 540 620 8 15 310 34 562
ND ND ND ND ND ND ND ND ND ND
ND ND 5 8 3 ND ND ND ND 5
ND ND 8 7 20 ND ND 5 11 7
* Tube MPN I- z. t Monolayer pfu I- z. :~Agar suspended cell pfu 1- l None detected.
Evaluation of procedures for recovery of viruses from water--lI tube procedures were comparable with those obtained with agar suspended cell cultures but remits on all other samples were low or virus was not detected. Similarly the agar suspended cell method gave very high counts on three samples but failed to detect viruses in a further five samples. The variable recovery with RD cells may possibly reflect the different spectrum of virus susceptibility reported by some workers (Schmidt et al. 1978; Wecker & ter Muelen 1977). The high plating efficiency associated with BGM cells used in agar suspension may be attributed to the availability of a large number of virus adsorbing sites on the cell surfaces because of the high numbers of cells used in the assay system and the availability of the whole cell surface for adsorption. The low efficiency of plaquing associated with monolayer cultures of all three ceils possibly reflects the limited availability of adsorption sites, firstly due to the substantially fewer number of ceils used in the system, and secondly because only part of the cell surface was available for virus adsorption. Cooper (1967) compared the merits of monolayer and agar suspended cell techniques and found that the latter had a higher efficiency of plating for free virus of the small and medium sized groups and that the system was much easier to handle. Additionally the agar suspended cell procedure did not require high grade tissue culture ware, no virus adsorption time was required, media requirements were less, and as ceils were harvested as a suspension it was convenient to use them in this form. Monolayer cultures were much better for large virus types and were generally more economical on cell usage. Probably the main advantage in using an agar suspended cell system was in the shortening of the incubation period necessary before results could be read. Chang et al. (1958) described a "most probable number" technique using tube cultures for the enumeration of coxsackie B1 virus. The advantage of the system is that it detects those viruses which produce microscopically visible cytopathic effect but fail to produce plaques under overlay. They point out that while it is desirable that results from a particular dilution series bracket the 50% response dilution, reasonable estimates of virus concentration can be made when this is not the case; an estimate can be obtained from response data of a single dilution, provided the data includes both positive and negative tubes. When considering the data presented in Table 2, it is obvious that whilst the tube methods often do detect significant numbers of viruses, there is variation of results between the different cell lines used and discrepancies exist between levels of virus obtained by the agar suspended cell method and the MPN method. No relationship between MPN and pfu (suspended cell) is evident. The susceptibility of Vero cells to enteroviruses was examined by Davis & Phillpotts (1974) who found that Vero was capable of supporting as wide a spectrum of viruses as primary rhesus monkey kidney
797
ceils. However, they noted that when isolating viruses from clinical specimens containing multiple types of virus there was a tendency to fail in isolating polioviruses types 1 and 3 in the presence of poliovirus type 2. They also demonstrated a variation in susceptibility of the cell line to variants of the same virus serotype and came to the conclusion that whilst Vero was suitable for propagating isolates, the line was not suitable for use as the initial isolation procedure with clinical samples. Hasler & Wigand (1978) examined the usefulness of Vero in supporting growth of adenoviruses but concluded that the sensitivity of Vero to small amounts of virus was lower than when using Hela cells, and that Vero was thus not suitable for the isolation of adenoviruses from clinical specimens. The poor performance of Vero cells in recovering naturally occurring viruses has been demonstrated in Table 2 whilst the ability of the cell line to support virus growth is shown by the relative titres obtained by assay of stock virus preparations in Veto monolayers and BGM agar suspended cell cultures (Table 3). Schmidt et al. (1975) used RD cells for the isolation of type A coxsackie viruses from clinical samples. They found that the line supported growth of many of the serotypes which previously had only been propagated in suckling mice. Whilst the cell line was slightly less sensitive than mice for isolation work, the adoption of the cell line as an alternative for the screening for coxsackie A viruses was feasible. Wecker & ter Muelen (1977) examined the growth of other enteroviruses in RD cells and found that many echoviruses gave higher titres than when grown in other human cell lines. In a comparison of RD and Vero ceils they found that RD gave higher titres and showed cpe much earlier than in Vero. Dahling et al. (1974) described the use of BGM cells (as monolayers) for the detection of viruses in water samples. They found that BGM ceils were more sensitive than either primary rhesus or primary african green monkey kidney ceils for detecting enteroviruses and that plaques tended to develop earlier, gave higher titres and were much more well defined. Using sewage and effluent samples the number of viruses recovered using the BGM system were 2-3 times higher than rhesus kidney, sometimes as much as
Table 3. Titres of monodispersed stock viruses by two assay systems (pfu ml- ~) Virus type Polio 1 Polio 2 Coxsackie A9 Coxsackie B2 Coxsackie 134 Coxsackie B5 Echo 1
Vero monolayers 5.5 x 4.0 x 1.1 x 1.5 x 1.6 x 1.0 x 1.5 x
107 107 107 107 106 106 107
BGM agar suspended cell 1.6 7.3 2.1 3.5 1.8 1.l 4.9
x x x x x x x
10° 108 l0 s 106 106 107 107
798
R, Morris and W, M. WAIt;
20 × higher. The incidence of positive growth of isolates on passage was also increased. They concluded that BGM was 3-10 × more sensitive than rhesus kidney and 2-5 × more sensitive than african green monkey kidney cells for recovering viruses from wastewater. Schmidt et aL (1978) examined the suitability of five cell systems (including BGM and RD) for detection of viruses in water samples. They found that BGM was not satisfactory for the isolation of naturally occurring echoviruses and group A coxsackie viruses and was not as sensitive as rhesus monkey kidney cells for reovirus. However, susceptibility to polioviruses and coxsackie B viruses was acceptable. RD cells were useful in recovering coxsackie type A viruses and echoviruses. They also reported on the tendency of BGM to give false plaques because of localized toxicity caused by small particulate matter. This emphasised the need to confirm plaques by serial passage. Monolayer cultures carried out in petri dishes in a carbon dioxide atmosphere were not suitable for isolation of echoviruses whereas closed cultures were suitable. The difference in numbers between cell types presented in Table 2 may be explained by (a) high sensitivity of BGM to some viruses, (b) high sensitivity of RD to viruses other than those covered by BGM, and (c) the low sensitivity of Vero cells regardless of virus types. In this study over 90°,,, of plaques picked off from BGM and RD agar suspended celt cultures were successfully passaged. The use of the agar suspended cell technique using BGM cells has recently been reported by Slade (1978). In a study of slow sand filtered water, he found low numbers of viruses in seven of nineteen samples, with one sample yielding viruses in the absence of E. coll.
CONCLUSIONS The data presented above show that the BGM cell line is the most sensitive cell system, of those tested, for the routine detection of viruses in water, with the overall efficiency being increased substantially by the use of the agar suspended cell technique. While this cell line is suitable for routine screening it is desirable in any definitive study, especially when only low numbers of viruses are expected, to use additional cell lines, such as RD cells, to increase the spectrum of virus types which might be recovered. The use of the agar suspended cell technique is relatively expensive in terms of cell numbers used but this is more than
offset by the increased sensitivity and the ease of handling of the system. Acknowledgements--We thank D. Sharp for technical as-
sistance, K. Bamford and his staff for chemical analyses. and Mrs S Barr for the typescript. G. J. Holland. J. S. Leahy and W. J.. Bell were kind enough to review the manuscripts and we thank W. F. Lester. Director of Scientific Services, Severn Trent Water Authority for permission to publish. REFERENCES
Barron A. L. OlsheveskyC. and Cohen M. M. (1970)Characteristics of the BGM line of cells from Africa green monkey kidney. Arch. ges. Virusforsch. 32, 389--392. Chang S. K. Berg G., Busch K, A. Stevenson R. F. Clarke N. A. and Kabler P. W. (1958) Application of the "most probable number" method for estimating concentrations of animal viruses by the tissue culture technique. ViroIocdy 6, 27-42. Cooper P. D. (1967) The plaque assay of animal viruses. Adv. Virus Res. $, 319-378. Dahling D. R., Berg G. and Berman D. (1974) BGM, a continuous cell line more sensitive than primary rhesus and African green monkey kidney cells for the recovery of viruses from water. Health Lab. Sci. II, 275-282. Davis P. M. and PhiUpotts R. J. (1974) Susceptibility of the Vero line of African green monkey kidney cells to human enteroviruses. J. Hyg., Camb. 72, 23-30. Hasler P. and Wigand R. (1978) The susceptibility of Vero cell cultures for human adenoviruses. Mpd. Microbiol. Immunol. 164~267-275. Hsuing G. D. and Meinick J. L. (1955) Plaque formation with poliomyelitis, coxsackie and orphan (echo) viruses in bottle cultures of monkey epithelial cells. Virology I, 533-535. Kjelkm L. (1961) A study of adenovirus-host cell system by the plaque technique. Virolo~jy 14, 234-239. McAllister R. M., Melnyk J., Finklestein J. Z., Adams E. C., Jr & Gardner M. B. (1969) Cultivation in ritro of cell derived from a human rhabdomyosarcoma. Cancer 24, 520-526. Morris R. and Walte W. M. (1980) Evaluation of procedures for recovery of viruses from water--I. Concentration systems. Water Res. 14, 795-798. Schmidt N. J., Ho H. H. and Lennette E. H. (1975) Propagation and isolation of group A coxsackie viruses in RD cells J. clin. MicrobioL 2, 183-185. Schmidt N. J., Ho H. H., Riggs J. L. and Lennette E. H. (1978) Comparative sensitivity of various cell culture systems for isolation of viruses from wastewater and faecal samples. Appl. & Environ. Microbiol. 36, 480--486. Severn Trent Water Authority (1978) Water Quality 1977/78. Appendix 3--Avon Division. Slade J. S. (1978) Enteroviruses in slow sand filtered water. J. Inst. Water Engrs ScL 32, 530-536. Wecker J. and ter Muelen V. (1977) RD cells in the laboratory diagnosis of enteroviruses. Med. MicrobioL Immunol. 163. 233-240.
0043-1354/80/07014~795$02.00/0
EVALUATION OF PROCEDURES FOR. RECOVERY OF VIRUSES FROM WATER--II DETECTION SYSTEMS R, MOI~RIS* and W. M. WAITE Regional Laboratory, Severn Trent Water Authority. St Martins Road, Coventry, England Abstract--Three cell lines used in three assay procedures have been evaluated for their suitability in detecting viruses in concentrates of river water samples. Vero cells proved to be relatively inefficient, BGM cells gave the best recoveries in general, whilst RD cells were variable. The mode of presentation of the cell system was of importance, monolayers in flasks giving low levels of virus, tube cultures being intermediate, whilst the agar suspended cell mode giving best recoveries. Using the BGM-agar suspended cell system the River Sowe gave virus levels varying from 145-620pfu 1-1 whilst the River Avon generally gave much lower levels with evidence of occasional high levels of contamination (540 pfu 1- l). It is suggested that BGM and RD cells used as agar suspended cell cultures are suitable for routine surveillance of the aquatic environment.
INTRODUCTION Infectivity of viruses, and hence numbers, can be measured using a variety of biological methods. Tissue culture systems afford simple, cheap and reproducible procedures for assaying viruses. The presentation of cells for virus infectivity has involved the use of monolayers in tubes (Chang et al., 1958), flasks (Hsuing & Me[nick, 1955) and petri dishes (Kjellen 1961). A method using cells suspended in agar has been described by Cooper (1967). The cell system used for virus isolation has depended on the range of viruses to be detected, the numbers of cells produced and the ease of handling. As most work on viruses has been on human types, the emphasis has been on the use of human and monkey cells as substrates. Primary cultures tend to be very sensitive but are increasingly more expensive. However, the introduction of continuous cell lines has proved useful for the isolation of viruses from clinical specimens because of the greater control over assay systems. Clinical virology concentrates on isolating and identifying dominant viruses in cases of illness, whereas virus detection in water samples aims at recovering the maximum number of viruses and virus types. The present study examined the suitability of three cell systems (one human, two monkey), presented in three ways (monolayers in tubes and flasks, agar suspended cells), for detecting enteroviruses in river water.
MATERIALS AND METHODS Cell cultures
The Veto cells used in this study have been described previously by Morris & Waite (1980). The monkey kidney cell line designated BGM and established by Barton et al. (1970) was supplied b~, Flow Laboratories, Scotland. A cell * To whom all correspondence should be addressed. 795
line derived from a human rhabdomyosarcorna by MeAllister et al. (1969) and designated RD, was a gift from Dr E. Bell, Glasgow, Scotland. BGM cells were grown in roller bottle culture using a mixture of Eagle's MEM and Liebowitz (LI5) media whilst RD cells were propagated on Eagle's MEM in plastic flasks (175era 2, Nunc). Both growth media contained foetal calf serum (10%), penicillin ( 100 units ml - i ) and streptomycin (100/zg ml - i), (GibcoBiocuit). Assay systems (a) Monolayer technique (Hsuiag & Melnick 1955). Plastic flasks (75 crn2, Nunc) were seeded with 1.5-2.0 x 106 cells in 25 ml of appropriate growth medium. Confluence was reached in 3-5 days at which time the cultures were drained and washed once with either medium 199 (BGM and Veto) or Eagle's MEM (RD cells). Sample concentrate (mixed with an equal volume of double strength wash medium) was allowed to adsorb for 1½h at room temperature, then washed off with a further aliquot of wash medium. BGM and Veto cultures were overlaid with the agar medium described by Morris and Waite (1980) whilst RD cells were overlaid with an Eagle's MEM based agar medium. Plaques were counted daily over nine days incubation at 37°C. Three replicates per sample were assayed. Tube technique. Soda glass tissue culture tubes (Flow) were seeded with approximately 3 x 10~ cells in 2 ml of appropriate growth medium and after 24 hr cultures were changed to maintenance medium. BGM cultures were maintained on iactalbumin hydrolysate, Veto cultures on medium 199 and RD cultures on Eagles MEM, all containing foetal calf serum (5%) and the full range of antibiotics previously described (Morris & Waite 1980). Sample concentrates were diluted in appropriate maintenance medium and 0.1 ml volumes added to each of a set of five tubes. Dilutions were 4-fold ranging from neat to 1:64. Tubes were examined daily for evidence of cytopathic effect (cpe) over a period of seven days incubation at 37°C. Most probable numbers (MPN) were determined using the tables of Chang et al. (1958). (c) Agar suspended cell technique. This technique, described by Cooper (1967), was adopted for use with water samples by Slade (1978). A cell suspension of l0 T cells/ml was prepared from stock cultures by centrifugation and resuspension. Sample concentrate (1 ml) was mixed with 2ml of cell suspension and 10ml of overlay medium described previously, before pouring into a plastic, 90 mm diameter, vented bacteriological grade petri dish (Sterilin).
796
R MORRISand W. M ~'AIr~ Table 1. Bacteriological levels of river Sowe and river Avon River Sowe Total coliforms/100ml
Mean Range Mean Range Mean Range
E. coli/lOOml Faecal streptococci/100ml
1.8 0.5 9.8 0.2 1.3 0.3
x
l0 6
x x x x x
10~-5.3 x 106 I0"~ 105-2.7 x 105 104 10~-2.7 x 10't
When set, plates were incubated inverted at 37°C in an atmo~hcre of 5% carbon dioxide in air, plaques being counted daily over seven days. Three replicates per sample were examined. Waters Two river waters were examined for virus levels using the methods de~ribed above. The River Avon was sampied approx 25 km downstream of Rugby Water Reclamation Works; the River Sowe was sampled approximately 2 km downstream of Finham Water Reclamation Works (Severn Trent Water Authority, 1978). Bacterial data for both rivers over the period of the present evaluation are shown in Table I. Sample processing Samples of both river waters (51) were conditioned and concentrated as described previously (Morris & Waite 1980) using the Balston filter system in the presence of aluminium cations. The final concentrate (25 ml) was held at -20°C prior to assay.
RESULTS AND DISCUSSION levels of virus found in samples from two rivers concentrated by a single procedure and assayed by three techniques using three cell lines are given in Table 2. Virus levels obtained with Vero cells as the substrate were invariably low regardless of the assay system used. On no occasion were viruses demonstrated using the tube technique with Vero cells and when using the cell line as monolayers in flasks, viruses
7.1 1.0 2.3 0.1 2.5 0.1
River Avon × 104 x 10"t--2.0 × 105 × 10'~ x 10a-.7.5 x 10't x l0 g × 103--5 X 1 0 3
were demonstrated in only one of six samples of the river Avon and three of four samples of the river Sowe, counts in all cases being very low. The agar suspended cell technique with Vero performed slightly better, yielding viruses from all samples found positive by the monolayer method and also from two samples which yielded no viruses with the other techniques. The highest recovery from any sample using Vero cells was 20pfu, this being obtained with a sample of river Sowe using the agar suspended cell procedure. Recoveries of viruses using BGM cells were very much greater, the tube technique giving viruses from all but two samples, these two, however, giving low counts by both flask monotayer and agar suspended cell methods. The highest count obtained with BGM tubes was an MPN of 5851-1. Flask monolayers recovered viruses from all but one of the samples but recoveries were low, a maximum of 25 pfu 1- ~ being achieved. The numbers of viruses recovered b y t h i s method did not differ appreciably from those obtained by the Vero monolayer technique. The use of BGM in agar suspended cell cultures proved to be the most sensitive of all the methods evaluated, viruses being detected in all samples with a maximum recovery of 620 pfu 1- z. Results obtained with RD cells were variable when compared with those obtained with the other cell lines. Recoveries with flask monolayers~ however, remained low. On two occasions the findings with the
Table 2. Enterovirus levels in two rivers by nine assay methods Cell system Assay system Date 17.11.78 23.11.78 7.12.78 18.12.78 20.12.78 27.12.78
Riper Avon Avon Sowe Avon Sowe Avon Avon Sowe Avon Sowe
RD T*
BGM
Vero
MLt
SC~
T
ML
SC
T
ML
SC
ND~ ND ND 8 8 27 ND 5 8 ND ND 3 8 ND 94 5 8 8 200 7
2 ND 185 310 170 ND ND ND ND 3
ND ND 72 151 585 8 18 94 8 161
2 2 7 8 13 5 2 2 ND 25
2 5 145 540 620 8 15 310 34 562
ND ND ND ND ND ND ND ND ND ND
ND ND 5 8 3 ND ND ND ND 5
ND ND 8 7 20 ND ND 5 11 7
* Tube MPN I- z. t Monolayer pfu I- z. :~Agar suspended cell pfu 1- l None detected.
Evaluation of procedures for recovery of viruses from water--lI tube procedures were comparable with those obtained with agar suspended cell cultures but remits on all other samples were low or virus was not detected. Similarly the agar suspended cell method gave very high counts on three samples but failed to detect viruses in a further five samples. The variable recovery with RD cells may possibly reflect the different spectrum of virus susceptibility reported by some workers (Schmidt et al. 1978; Wecker & ter Muelen 1977). The high plating efficiency associated with BGM cells used in agar suspension may be attributed to the availability of a large number of virus adsorbing sites on the cell surfaces because of the high numbers of cells used in the assay system and the availability of the whole cell surface for adsorption. The low efficiency of plaquing associated with monolayer cultures of all three ceils possibly reflects the limited availability of adsorption sites, firstly due to the substantially fewer number of ceils used in the system, and secondly because only part of the cell surface was available for virus adsorption. Cooper (1967) compared the merits of monolayer and agar suspended cell techniques and found that the latter had a higher efficiency of plating for free virus of the small and medium sized groups and that the system was much easier to handle. Additionally the agar suspended cell procedure did not require high grade tissue culture ware, no virus adsorption time was required, media requirements were less, and as ceils were harvested as a suspension it was convenient to use them in this form. Monolayer cultures were much better for large virus types and were generally more economical on cell usage. Probably the main advantage in using an agar suspended cell system was in the shortening of the incubation period necessary before results could be read. Chang et al. (1958) described a "most probable number" technique using tube cultures for the enumeration of coxsackie B1 virus. The advantage of the system is that it detects those viruses which produce microscopically visible cytopathic effect but fail to produce plaques under overlay. They point out that while it is desirable that results from a particular dilution series bracket the 50% response dilution, reasonable estimates of virus concentration can be made when this is not the case; an estimate can be obtained from response data of a single dilution, provided the data includes both positive and negative tubes. When considering the data presented in Table 2, it is obvious that whilst the tube methods often do detect significant numbers of viruses, there is variation of results between the different cell lines used and discrepancies exist between levels of virus obtained by the agar suspended cell method and the MPN method. No relationship between MPN and pfu (suspended cell) is evident. The susceptibility of Vero cells to enteroviruses was examined by Davis & Phillpotts (1974) who found that Vero was capable of supporting as wide a spectrum of viruses as primary rhesus monkey kidney
797
ceils. However, they noted that when isolating viruses from clinical specimens containing multiple types of virus there was a tendency to fail in isolating polioviruses types 1 and 3 in the presence of poliovirus type 2. They also demonstrated a variation in susceptibility of the cell line to variants of the same virus serotype and came to the conclusion that whilst Vero was suitable for propagating isolates, the line was not suitable for use as the initial isolation procedure with clinical samples. Hasler & Wigand (1978) examined the usefulness of Vero in supporting growth of adenoviruses but concluded that the sensitivity of Vero to small amounts of virus was lower than when using Hela cells, and that Vero was thus not suitable for the isolation of adenoviruses from clinical specimens. The poor performance of Vero cells in recovering naturally occurring viruses has been demonstrated in Table 2 whilst the ability of the cell line to support virus growth is shown by the relative titres obtained by assay of stock virus preparations in Veto monolayers and BGM agar suspended cell cultures (Table 3). Schmidt et al. (1975) used RD cells for the isolation of type A coxsackie viruses from clinical samples. They found that the line supported growth of many of the serotypes which previously had only been propagated in suckling mice. Whilst the cell line was slightly less sensitive than mice for isolation work, the adoption of the cell line as an alternative for the screening for coxsackie A viruses was feasible. Wecker & ter Muelen (1977) examined the growth of other enteroviruses in RD cells and found that many echoviruses gave higher titres than when grown in other human cell lines. In a comparison of RD and Vero ceils they found that RD gave higher titres and showed cpe much earlier than in Vero. Dahling et al. (1974) described the use of BGM cells (as monolayers) for the detection of viruses in water samples. They found that BGM ceils were more sensitive than either primary rhesus or primary african green monkey kidney ceils for detecting enteroviruses and that plaques tended to develop earlier, gave higher titres and were much more well defined. Using sewage and effluent samples the number of viruses recovered using the BGM system were 2-3 times higher than rhesus kidney, sometimes as much as
Table 3. Titres of monodispersed stock viruses by two assay systems (pfu ml- ~) Virus type Polio 1 Polio 2 Coxsackie A9 Coxsackie B2 Coxsackie 134 Coxsackie B5 Echo 1
Vero monolayers 5.5 x 4.0 x 1.1 x 1.5 x 1.6 x 1.0 x 1.5 x
107 107 107 107 106 106 107
BGM agar suspended cell 1.6 7.3 2.1 3.5 1.8 1.l 4.9
x x x x x x x
10° 108 l0 s 106 106 107 107
798
R, Morris and W, M. WAIt;
20 × higher. The incidence of positive growth of isolates on passage was also increased. They concluded that BGM was 3-10 × more sensitive than rhesus kidney and 2-5 × more sensitive than african green monkey kidney cells for recovering viruses from wastewater. Schmidt et aL (1978) examined the suitability of five cell systems (including BGM and RD) for detection of viruses in water samples. They found that BGM was not satisfactory for the isolation of naturally occurring echoviruses and group A coxsackie viruses and was not as sensitive as rhesus monkey kidney cells for reovirus. However, susceptibility to polioviruses and coxsackie B viruses was acceptable. RD cells were useful in recovering coxsackie type A viruses and echoviruses. They also reported on the tendency of BGM to give false plaques because of localized toxicity caused by small particulate matter. This emphasised the need to confirm plaques by serial passage. Monolayer cultures carried out in petri dishes in a carbon dioxide atmosphere were not suitable for isolation of echoviruses whereas closed cultures were suitable. The difference in numbers between cell types presented in Table 2 may be explained by (a) high sensitivity of BGM to some viruses, (b) high sensitivity of RD to viruses other than those covered by BGM, and (c) the low sensitivity of Vero cells regardless of virus types. In this study over 90°,,, of plaques picked off from BGM and RD agar suspended celt cultures were successfully passaged. The use of the agar suspended cell technique using BGM cells has recently been reported by Slade (1978). In a study of slow sand filtered water, he found low numbers of viruses in seven of nineteen samples, with one sample yielding viruses in the absence of E. coll.
CONCLUSIONS The data presented above show that the BGM cell line is the most sensitive cell system, of those tested, for the routine detection of viruses in water, with the overall efficiency being increased substantially by the use of the agar suspended cell technique. While this cell line is suitable for routine screening it is desirable in any definitive study, especially when only low numbers of viruses are expected, to use additional cell lines, such as RD cells, to increase the spectrum of virus types which might be recovered. The use of the agar suspended cell technique is relatively expensive in terms of cell numbers used but this is more than
offset by the increased sensitivity and the ease of handling of the system. Acknowledgements--We thank D. Sharp for technical as-
sistance, K. Bamford and his staff for chemical analyses. and Mrs S Barr for the typescript. G. J. Holland. J. S. Leahy and W. J.. Bell were kind enough to review the manuscripts and we thank W. F. Lester. Director of Scientific Services, Severn Trent Water Authority for permission to publish. REFERENCES
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