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Development of a rotavirus plaque assay using Sephadex G-75
25
lour~l of Virological Methods, 28 (1990) 25-32 Elsevier VIRMET 009%
Development
of a rotavirus plaque assay using Sephadex G-75
Patricia M. Aha and Marta I. Sabara Praris Biologic-sInc., Rochester, NY 14623, U.S.A. (Accepted 29 November 1989) -.
A rapid, reproducible and easily performed plaque assay is described for use with a variety of rotavirus strains. Plaque formation was induced in MA-104 cells by the use of Sephadex G-75, instead of the traditional agar, and crystalline trypsin in the overlay. Formation of large, discrete, easily read plaques was noted in both human and non-human rotavirus strains. Rotavirus;
Plaque assay; Overlay
Introduction
In spite of the large numbers of virus particles in infective feces and the ease with which infection spreads in the field, rotaviruses have proven to be very difficult to propagate in tissue culture. In vitro propagation af rotaviruses has been desirable not only as a potential diagnostic tool but also as an aid in the molecular characterization of the virus and its replicative cycle. Several attempts were made to grow various rotaviruses in a wide variety of organ and tissue cultures. Some of these attempts were moderately successful in that the virus replicated in vitro but not to high titres. In most cases viral infectivity could not be maintained through successive passages (Femelius et al., 1972; Mebus and Mono, 197la; Mohammed et al., 1976; Welch and Twiehaus, 1973; Wyatt et al., 1974). Despite these moderate successes it became apparent that a more comprehensive understanding of the factors contributing to rotaviral infectivity was needed before rational approaches toward routine propagation in vitro could be successfully undertaken. In vivo, rotavirus replicates primarily in the small intestine villous epithelium (McNulty Correspondence to: P.M. Aha, Praxis Biologics Inc., Rochester, NY 14623, U.S.A. 0166-0934/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
26
and Pearson, 1976; Mebus and Stair, 1971; Theil et al., 1978). The restricted nature of rotavirus infections in vitro suggested that either unique mucosal factors might be involved or alternatively intraluminar factors, particularly pancreatic enzymes, could be influential determinants of rotavirus infectivity (Holmes et al., 1976; Rodger et al., 1977). The latter possibility proved to be the correct approach since pancreatic enzymes were found to be the important determinants of rotaviral infectivity in primary porcine kidney cells (Theil et al., 1977). Subsequent studies revealed that trypsin, the major enzymatic component of pancreatin, was responsible for enhancing the infectivity of several other rotaviruses (Almeida et al., 1978; Babiuk and Mohammed, 1977; McNulty et al., 1979; Sato et al., 1981; Wyatt et al., 1980). The efficient growth of rotaviruses in tissue culture prompted the development of a plaque assay for the Simian virus, SAll, in 3 continuous cell lines (MA-104, CV-1 and LLC-MK2) (Ramia et al., 1979; Smith et al., 1979) and for the neonatal calf diarrhea virus, NCDV, in one cell line (MA-104) (Matsuno et al., 1979). Historically, assays for the quantitation of human and non-human rotavirus strains have been performed using agar overlays containing DEAE-dextran and trypsin (Smith et al., 1979; Urasawa et al., 1982). These assays are technically demanding and difficult to reproduce. A sensitive and easily executed plaque assay that may be used with a variety of rotavirus serotypes would facilitate the study of rotaviruses. The purpose of this report is to describe such an assay using Sephadex G-75 as a replacement for agar in the traditional overlay. The procedure should also aid in the simplification of other bioassays performed in the study of rotavirus agents.
Materials and Methods Cells and virus
Fetal rhesus monkey MA-104 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with D-glucose at 4500 mg/l and L-glutamine (Gibco), supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine. 100 U penicillin/ml, 100 kg streptomycin/ml, and 20 mM Hepes. Stocks of C486 (bovine), SA-11 (Simian), ST-4 (human), WA (human), DS-1 (human), and RRV (Simian) rotavirus used in this study were prepared in MA104 cells. Confluent monolayers of cells in 150 cm’ flasks were washed with DMEM (no FBS) and infected with 2 ml virus. The virus was adsorbed to the cells for 1 h at 37°C with gentle rocking. At the end of this time, 20 ml of DMEM (no FBS) was added to each flask. At approximately 18-20 h, when a cytopathic effect (CPE) of >90% of the cell monolayer was noted, the cells and culture fluid were harvested. The tissue culture fluids for each strain were pooled, measured and trypsin (2.5% Hazleton) was added at 50 j&ml.
27
Trypsin
Crystalline trypsin was purchased from Sigma, with chymotrypsin, but no elastase activity (9800 BAEE units/mg of protein). The trypsin was reconstituted to a concentration of 25 mg/mf in DMEM (no supplements), aliquoted, and stored at -20°C for future use. Plaque assay
The plaque assay was performed in 24-well, tissue culture treated plates (NUNC) containing confluent monolayers of MA-104 cells. The growth medium was aspirated and each well washed with 1 ml DMEM (no FBS). Serial ten-fold dilutions of each virus were prepared in DMEM (no FBS) and each well was overlayed with 200 ~1 of diluted virus or 200 ~1 of DMEM (no FBS) as a cell control. The virus was adsorbed to the cells for 1 h at 37°C with gentle rocking. At the end of this time the inoculum was aspirated and the appropriate overlay added. The plates were then incubated for 3 days at 37”C, taking precautions to avoid agitation of the plates. Overlay
Overlay was prepared in enriched DMEM (EDMEM), supplemented with 4 mM L-glutamine, 100 U penicillin/ml, 100 p,g streptomycin/ml, 20 mM Hepes, 30 mg sodium bicarbonate/ml and crystalline trypsin at concentrations ranging from 100 kg/ml to 0.25 pg/ml. Sephadex G-75 beads (Pharmacia), rehydrated in 1 M phosphate buffered saline (PBS) to a concentration of 4% (w/v), were measured out, spun gently for 3 min and resuspended in EDMEM to the desired percent concentration, ranging from 1 to 4%. Plate staining
On day three post-infection, the plates were gently swirled to resuspend the settled beads and the overlay was aspirated from each well. The wells were incubated with 0.75 ml 0.5% crystal violet&O% methanol/PBS for 20 mitt at room temperature. The plates were then washed under a gentle stream of tap water and inverted to dry.
Ened
of trypsin on virus plaque formation
Trypsin concentrations in the various Sephadex overlays ranged from 100 &ml to 0.25 Fg/rnl or 980 BAEE units to 2.45 BAEE units. As illustrated in Fig. 1,
28
Fig. 1. The effect of trypsin concentration on plaque formation under 2% Sephadex using lo-fold serial dilutions of the C486 rotavirus strain, with the lowest dilution at the top and the highest dilution at the bottom. The bottom row was left uninfected as a cell control. (A), 0.50 &ml; (B), 0.75 kg/ml; (C), 1.0 &ml; (D), 5.0 pgiml.
A
B
C
D
Fig. 2. The effect of Sephadex G-75 concentration on plaque formation using lo-fold serial dilutions of the WA rotavirus strain, with the lowest dilution at the left and the highest dilution at the right. The far rightcolumn represents the uninfected cell control. (A), 4.0%; (B), 3.0%; (C), 2.0%; (D), 1.0%.
29
Fig. 3. Examples of plaque formation with a variety of human and adex with 0.75 pg/ml crystalline trypsin. The viruses were applied lowest dilution at the left and the highest dilution at the right and uninfected cell control. (A), RRV; (B), WA; (C), ST-4; (D),
non-human strains under 2% Sephin lo-fold serial dilutions with the the far right column was left as an DS-1; (E), SA-11; (F), C486.
MA-104 ceils were not able to survive concentrations greater than 1 &ml. However, at lower concentrations, the cell monolayers remained intact. Using C486 rotavirus as the prototype virus, plaque formation was observed in even the lowest dose of trypsin. Plaque diameter was found to be directly proportional to the amount of trypsin in the overlay. Effect of Sephadex on virus plaque formation
Sephadex G-75 was used in place of the traditional agar at concentrations of 1 to 4% (w/v). As shown in Fig. 2, MA-104 cells were relatively intolerant of the highest bead concentration. Specifically, cell monolayers were still visible upon staining however the dye uptake was less than in the lower concentrations of Sephadex denoting a higher incidence of generalized cell death. Plaque formation was observed with all the bead concentrations, plaque diameter being directly proportional to the amount of Sephadex in the overlay. All the strains tested showed comparable plaquing patterns with the varying bead concentrations, the most discrete and easily read being found in the range of 2-3% Sephadex (Fig. 3).
30
Discussion
The use of a semi-solid overlay such as that provided by Sephadex G-75 beads has been documented for other viruses, most notably for rabies virus (Smith et al., 1977) as well as for respiratory syncytial virus (personal communication, S. Hildreth, Praxis Biologics, Rochester, New York). In both cases the plaque assay boasts consistent success using this type of overlay. Several human and non-human rotavirus strains displayed plaque formation under a semi-solid overlay containing Sephadex G-75 and crystalline trypsin. The agar overlays used in the past have been both technically demanding and difficult to reproduce. The method described here circumvents both of these problems by providing reproducibility, ease of application and speedy acquisition of results, in some cases decreasing the time needed for plaque formation in half (Urasawa et al., 1982). MA-104 cells exhibit plaque formation, with large, discrete, easily read plaques under a 2-3% Sephadex overlay with 0.5-0.75 Fg/ml crystalline trypsin (4.9-7.4 units BAEE activity). These were determined to be the optimum conditions for plaque formation in all the rotavirus strains tested. The discrete nature of the plaques supports the theory that one infectious particle can initiate infection and that this overlay is capable of quantitating rotavirus plaque forming units. The described plaque assay has many potential uses. Along with being a reliable quantitator of infectivity, it may also be used in plaque reduction assays to determine levels of neutralizing antibody. Also, we have found that by rinsing off the bead overlay on day two post-infection and adding a traditional agar overlay (Smith et al., 1979), plaques may be isolated and picked to produce pure, high titered virus stocks. This procedure has been used in this lab with many human and nonhuman rotavirus strains with great success. References Almeida, J.D., Hall, T., Banatuala, J.E., Totterdell, B.M. and Chrystie, I.L. (1978) The effect of trypsin on the growth of rotavirus. J. Gen. Viral. 40, 213-218. Babiuk, L.A., Mohammed, K., Spence, L., Fauvel, M. and Petro, R. (1977) Rotavirus isolation and cultivation in the presence of trypsin. J. Clin. Microbial. 6, 610-617. Fernelius, A.L., Ritchie, A.E., Classick, L.G., Norman, J.O. and Mebus, C.A. (1972) Cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in Nebraska. Arch. Ges. Virusforsch. 37, 114-130. Holmes, I.H., Rodger, SM., Schnagl, R.D., Ruck, B.T., Gust, I.D., Bishop, R.F. and Barres, G.L. (1976) Is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses? Lancet i, 1387-1388. Matsuno, S., Inouye, S. and Kono, R. (1977) Plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human serum. J. Clin. Microbial. 5, l-4. McNulty, MS., Pearson, G.R., McFerran, J.B., Collins, D.S. and Allan, G.M. (1976) A reovirus-like agent (rotavirus) associated with diarrhoea in neonatal pigs. Vet. Microbial. 1, 55-63. McNulty, M.S., Allan, G.M., Todd, D. and McFerran, J.B. (1979) Isolation and cell culture propagation of rotaviruses from turkeys and chickens. Arch. Viral. 61, 13-21. Mebus, C.A., Kono, M., Underdahl, N.R. and Twiehaus, M.J. (1971a) Cell culture propagation of neonatal calf diarrhea (scours) virus. Can. Vet. J. 12, 69-72.
31 Mebus, C.A., Stair, E.L. and Underdahl, N.R. (1971) Pathology of neonatal calf diarrhea induced by a reo-like virus. Vet. Pathol. 8490-505. Mohammed, K.A. and Saunders, J.R. (1976) Propagation of the rotavirus of neonatal calf diarrhea in fetal intestinal cell culture. Can. J. Comp. Med. 41, 226-229. Ramia, S. and Saltar, S.A. (1979) Simian rotavirus SAll plaque formation in the presence of trypsin. J. Clin. Microbial. 5, l-4. Rodger, S.M., Schnagl, R.D. and Holmes, I.H. (1977) Further biochemical characterization including the detection of surface glycoproteins of human, calf and simian rotaviruses. J. Virol. 24, 91-98. Sato, K., Inaba, Y., Shinoznki, T., Fujii, R. and Matumoto, M. (1981) Isolation of human rotavirus in cell cultures. Arch. Virol. 69, 155-160. Smith, A.L., Tignor, G.H., Mifune, K. and Motokoski, T. (1977) Isolation and assay of rabies serogroup viruses in CER cells. Intervirology 8, 92-99. Smith, E.M., Estes, M.K., Graham, D.Y. and Gerba, C.P. (1979) A plaque assay for the simian rotavirus SAll. J. Gen. Virol. 43, 513-519. Thiel, K.W., Bohl, E.H. and Arden, A.G. (1977) Cell culture propagation of porcine rotavirus (reovirus-like agent). Am. J. Vet. Res. 38, 1765-1768. Thiel, K.W., Bohl, E.H. and Cross, R.F. (1978) Pathogenesis of porcine rotaviral infection on experimentally inoculated gnotobiotic pigs. Am. J. Vet. Res. 39, 213-220. Urasawa, S., Urasawa, T. and Taniguchi, K. (1982) Three human rotavirus serotypes demonstrated by plaque neutralization of isolated strains. Infect. Immun. 38, 781-784. Welch, A.B. and Twiehaus, M.J. (1973) Cell culture studies of a neonatal calf diarrhea virus. Can. J. Comp. Med. 37,287-294. Wyatt, R.G., Kapikian, A.Z., Thomhill, T.S., Sereno, M.M., Kim, H. W. and Chanock, R.M. (1974) In vitro cultivation in human fetal intestinal organ culture of a reovirus-like agent associated with non-bacterial gastroenteritis in infants and children. J. Infect. Dis. 130, 523-528. Wyatt, R.G., James, W.D., Bohl, E.H., Theil, K.W., Saif, L.J., Kalica, A.R., Greenberg, H.B., Kapikian, A.Z. and Chanock, R.M. (1980) Human rotavirus type 2: cultivation in vitro. Science 207, 189-191.
lour~l of Virological Methods, 28 (1990) 25-32 Elsevier VIRMET 009%
Development
of a rotavirus plaque assay using Sephadex G-75
Patricia M. Aha and Marta I. Sabara Praris Biologic-sInc., Rochester, NY 14623, U.S.A. (Accepted 29 November 1989) -.
A rapid, reproducible and easily performed plaque assay is described for use with a variety of rotavirus strains. Plaque formation was induced in MA-104 cells by the use of Sephadex G-75, instead of the traditional agar, and crystalline trypsin in the overlay. Formation of large, discrete, easily read plaques was noted in both human and non-human rotavirus strains. Rotavirus;
Plaque assay; Overlay
Introduction
In spite of the large numbers of virus particles in infective feces and the ease with which infection spreads in the field, rotaviruses have proven to be very difficult to propagate in tissue culture. In vitro propagation af rotaviruses has been desirable not only as a potential diagnostic tool but also as an aid in the molecular characterization of the virus and its replicative cycle. Several attempts were made to grow various rotaviruses in a wide variety of organ and tissue cultures. Some of these attempts were moderately successful in that the virus replicated in vitro but not to high titres. In most cases viral infectivity could not be maintained through successive passages (Femelius et al., 1972; Mebus and Mono, 197la; Mohammed et al., 1976; Welch and Twiehaus, 1973; Wyatt et al., 1974). Despite these moderate successes it became apparent that a more comprehensive understanding of the factors contributing to rotaviral infectivity was needed before rational approaches toward routine propagation in vitro could be successfully undertaken. In vivo, rotavirus replicates primarily in the small intestine villous epithelium (McNulty Correspondence to: P.M. Aha, Praxis Biologics Inc., Rochester, NY 14623, U.S.A. 0166-0934/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
26
and Pearson, 1976; Mebus and Stair, 1971; Theil et al., 1978). The restricted nature of rotavirus infections in vitro suggested that either unique mucosal factors might be involved or alternatively intraluminar factors, particularly pancreatic enzymes, could be influential determinants of rotavirus infectivity (Holmes et al., 1976; Rodger et al., 1977). The latter possibility proved to be the correct approach since pancreatic enzymes were found to be the important determinants of rotaviral infectivity in primary porcine kidney cells (Theil et al., 1977). Subsequent studies revealed that trypsin, the major enzymatic component of pancreatin, was responsible for enhancing the infectivity of several other rotaviruses (Almeida et al., 1978; Babiuk and Mohammed, 1977; McNulty et al., 1979; Sato et al., 1981; Wyatt et al., 1980). The efficient growth of rotaviruses in tissue culture prompted the development of a plaque assay for the Simian virus, SAll, in 3 continuous cell lines (MA-104, CV-1 and LLC-MK2) (Ramia et al., 1979; Smith et al., 1979) and for the neonatal calf diarrhea virus, NCDV, in one cell line (MA-104) (Matsuno et al., 1979). Historically, assays for the quantitation of human and non-human rotavirus strains have been performed using agar overlays containing DEAE-dextran and trypsin (Smith et al., 1979; Urasawa et al., 1982). These assays are technically demanding and difficult to reproduce. A sensitive and easily executed plaque assay that may be used with a variety of rotavirus serotypes would facilitate the study of rotaviruses. The purpose of this report is to describe such an assay using Sephadex G-75 as a replacement for agar in the traditional overlay. The procedure should also aid in the simplification of other bioassays performed in the study of rotavirus agents.
Materials and Methods Cells and virus
Fetal rhesus monkey MA-104 cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) with D-glucose at 4500 mg/l and L-glutamine (Gibco), supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine. 100 U penicillin/ml, 100 kg streptomycin/ml, and 20 mM Hepes. Stocks of C486 (bovine), SA-11 (Simian), ST-4 (human), WA (human), DS-1 (human), and RRV (Simian) rotavirus used in this study were prepared in MA104 cells. Confluent monolayers of cells in 150 cm’ flasks were washed with DMEM (no FBS) and infected with 2 ml virus. The virus was adsorbed to the cells for 1 h at 37°C with gentle rocking. At the end of this time, 20 ml of DMEM (no FBS) was added to each flask. At approximately 18-20 h, when a cytopathic effect (CPE) of >90% of the cell monolayer was noted, the cells and culture fluid were harvested. The tissue culture fluids for each strain were pooled, measured and trypsin (2.5% Hazleton) was added at 50 j&ml.
27
Trypsin
Crystalline trypsin was purchased from Sigma, with chymotrypsin, but no elastase activity (9800 BAEE units/mg of protein). The trypsin was reconstituted to a concentration of 25 mg/mf in DMEM (no supplements), aliquoted, and stored at -20°C for future use. Plaque assay
The plaque assay was performed in 24-well, tissue culture treated plates (NUNC) containing confluent monolayers of MA-104 cells. The growth medium was aspirated and each well washed with 1 ml DMEM (no FBS). Serial ten-fold dilutions of each virus were prepared in DMEM (no FBS) and each well was overlayed with 200 ~1 of diluted virus or 200 ~1 of DMEM (no FBS) as a cell control. The virus was adsorbed to the cells for 1 h at 37°C with gentle rocking. At the end of this time the inoculum was aspirated and the appropriate overlay added. The plates were then incubated for 3 days at 37”C, taking precautions to avoid agitation of the plates. Overlay
Overlay was prepared in enriched DMEM (EDMEM), supplemented with 4 mM L-glutamine, 100 U penicillin/ml, 100 p,g streptomycin/ml, 20 mM Hepes, 30 mg sodium bicarbonate/ml and crystalline trypsin at concentrations ranging from 100 kg/ml to 0.25 pg/ml. Sephadex G-75 beads (Pharmacia), rehydrated in 1 M phosphate buffered saline (PBS) to a concentration of 4% (w/v), were measured out, spun gently for 3 min and resuspended in EDMEM to the desired percent concentration, ranging from 1 to 4%. Plate staining
On day three post-infection, the plates were gently swirled to resuspend the settled beads and the overlay was aspirated from each well. The wells were incubated with 0.75 ml 0.5% crystal violet&O% methanol/PBS for 20 mitt at room temperature. The plates were then washed under a gentle stream of tap water and inverted to dry.
Ened
of trypsin on virus plaque formation
Trypsin concentrations in the various Sephadex overlays ranged from 100 &ml to 0.25 Fg/rnl or 980 BAEE units to 2.45 BAEE units. As illustrated in Fig. 1,
28
Fig. 1. The effect of trypsin concentration on plaque formation under 2% Sephadex using lo-fold serial dilutions of the C486 rotavirus strain, with the lowest dilution at the top and the highest dilution at the bottom. The bottom row was left uninfected as a cell control. (A), 0.50 &ml; (B), 0.75 kg/ml; (C), 1.0 &ml; (D), 5.0 pgiml.
A
B
C
D
Fig. 2. The effect of Sephadex G-75 concentration on plaque formation using lo-fold serial dilutions of the WA rotavirus strain, with the lowest dilution at the left and the highest dilution at the right. The far rightcolumn represents the uninfected cell control. (A), 4.0%; (B), 3.0%; (C), 2.0%; (D), 1.0%.
29
Fig. 3. Examples of plaque formation with a variety of human and adex with 0.75 pg/ml crystalline trypsin. The viruses were applied lowest dilution at the left and the highest dilution at the right and uninfected cell control. (A), RRV; (B), WA; (C), ST-4; (D),
non-human strains under 2% Sephin lo-fold serial dilutions with the the far right column was left as an DS-1; (E), SA-11; (F), C486.
MA-104 ceils were not able to survive concentrations greater than 1 &ml. However, at lower concentrations, the cell monolayers remained intact. Using C486 rotavirus as the prototype virus, plaque formation was observed in even the lowest dose of trypsin. Plaque diameter was found to be directly proportional to the amount of trypsin in the overlay. Effect of Sephadex on virus plaque formation
Sephadex G-75 was used in place of the traditional agar at concentrations of 1 to 4% (w/v). As shown in Fig. 2, MA-104 cells were relatively intolerant of the highest bead concentration. Specifically, cell monolayers were still visible upon staining however the dye uptake was less than in the lower concentrations of Sephadex denoting a higher incidence of generalized cell death. Plaque formation was observed with all the bead concentrations, plaque diameter being directly proportional to the amount of Sephadex in the overlay. All the strains tested showed comparable plaquing patterns with the varying bead concentrations, the most discrete and easily read being found in the range of 2-3% Sephadex (Fig. 3).
30
Discussion
The use of a semi-solid overlay such as that provided by Sephadex G-75 beads has been documented for other viruses, most notably for rabies virus (Smith et al., 1977) as well as for respiratory syncytial virus (personal communication, S. Hildreth, Praxis Biologics, Rochester, New York). In both cases the plaque assay boasts consistent success using this type of overlay. Several human and non-human rotavirus strains displayed plaque formation under a semi-solid overlay containing Sephadex G-75 and crystalline trypsin. The agar overlays used in the past have been both technically demanding and difficult to reproduce. The method described here circumvents both of these problems by providing reproducibility, ease of application and speedy acquisition of results, in some cases decreasing the time needed for plaque formation in half (Urasawa et al., 1982). MA-104 cells exhibit plaque formation, with large, discrete, easily read plaques under a 2-3% Sephadex overlay with 0.5-0.75 Fg/ml crystalline trypsin (4.9-7.4 units BAEE activity). These were determined to be the optimum conditions for plaque formation in all the rotavirus strains tested. The discrete nature of the plaques supports the theory that one infectious particle can initiate infection and that this overlay is capable of quantitating rotavirus plaque forming units. The described plaque assay has many potential uses. Along with being a reliable quantitator of infectivity, it may also be used in plaque reduction assays to determine levels of neutralizing antibody. Also, we have found that by rinsing off the bead overlay on day two post-infection and adding a traditional agar overlay (Smith et al., 1979), plaques may be isolated and picked to produce pure, high titered virus stocks. This procedure has been used in this lab with many human and nonhuman rotavirus strains with great success. References Almeida, J.D., Hall, T., Banatuala, J.E., Totterdell, B.M. and Chrystie, I.L. (1978) The effect of trypsin on the growth of rotavirus. J. Gen. Viral. 40, 213-218. Babiuk, L.A., Mohammed, K., Spence, L., Fauvel, M. and Petro, R. (1977) Rotavirus isolation and cultivation in the presence of trypsin. J. Clin. Microbial. 6, 610-617. Fernelius, A.L., Ritchie, A.E., Classick, L.G., Norman, J.O. and Mebus, C.A. (1972) Cell culture adaptation and propagation of a reovirus-like agent of calf diarrhea from a field outbreak in Nebraska. Arch. Ges. Virusforsch. 37, 114-130. Holmes, I.H., Rodger, SM., Schnagl, R.D., Ruck, B.T., Gust, I.D., Bishop, R.F. and Barres, G.L. (1976) Is lactase the receptor and uncoating enzyme for infantile enteritis (rota) viruses? Lancet i, 1387-1388. Matsuno, S., Inouye, S. and Kono, R. (1977) Plaque assay of neonatal calf diarrhea virus and the neutralizing antibody in human serum. J. Clin. Microbial. 5, l-4. McNulty, MS., Pearson, G.R., McFerran, J.B., Collins, D.S. and Allan, G.M. (1976) A reovirus-like agent (rotavirus) associated with diarrhoea in neonatal pigs. Vet. Microbial. 1, 55-63. McNulty, M.S., Allan, G.M., Todd, D. and McFerran, J.B. (1979) Isolation and cell culture propagation of rotaviruses from turkeys and chickens. Arch. Viral. 61, 13-21. Mebus, C.A., Kono, M., Underdahl, N.R. and Twiehaus, M.J. (1971a) Cell culture propagation of neonatal calf diarrhea (scours) virus. Can. Vet. J. 12, 69-72.
31 Mebus, C.A., Stair, E.L. and Underdahl, N.R. (1971) Pathology of neonatal calf diarrhea induced by a reo-like virus. Vet. Pathol. 8490-505. Mohammed, K.A. and Saunders, J.R. (1976) Propagation of the rotavirus of neonatal calf diarrhea in fetal intestinal cell culture. Can. J. Comp. Med. 41, 226-229. Ramia, S. and Saltar, S.A. (1979) Simian rotavirus SAll plaque formation in the presence of trypsin. J. Clin. Microbial. 5, l-4. Rodger, S.M., Schnagl, R.D. and Holmes, I.H. (1977) Further biochemical characterization including the detection of surface glycoproteins of human, calf and simian rotaviruses. J. Virol. 24, 91-98. Sato, K., Inaba, Y., Shinoznki, T., Fujii, R. and Matumoto, M. (1981) Isolation of human rotavirus in cell cultures. Arch. Virol. 69, 155-160. Smith, A.L., Tignor, G.H., Mifune, K. and Motokoski, T. (1977) Isolation and assay of rabies serogroup viruses in CER cells. Intervirology 8, 92-99. Smith, E.M., Estes, M.K., Graham, D.Y. and Gerba, C.P. (1979) A plaque assay for the simian rotavirus SAll. J. Gen. Virol. 43, 513-519. Thiel, K.W., Bohl, E.H. and Arden, A.G. (1977) Cell culture propagation of porcine rotavirus (reovirus-like agent). Am. J. Vet. Res. 38, 1765-1768. Thiel, K.W., Bohl, E.H. and Cross, R.F. (1978) Pathogenesis of porcine rotaviral infection on experimentally inoculated gnotobiotic pigs. Am. J. Vet. Res. 39, 213-220. Urasawa, S., Urasawa, T. and Taniguchi, K. (1982) Three human rotavirus serotypes demonstrated by plaque neutralization of isolated strains. Infect. Immun. 38, 781-784. Welch, A.B. and Twiehaus, M.J. (1973) Cell culture studies of a neonatal calf diarrhea virus. Can. J. Comp. Med. 37,287-294. Wyatt, R.G., Kapikian, A.Z., Thomhill, T.S., Sereno, M.M., Kim, H. W. and Chanock, R.M. (1974) In vitro cultivation in human fetal intestinal organ culture of a reovirus-like agent associated with non-bacterial gastroenteritis in infants and children. J. Infect. Dis. 130, 523-528. Wyatt, R.G., James, W.D., Bohl, E.H., Theil, K.W., Saif, L.J., Kalica, A.R., Greenberg, H.B., Kapikian, A.Z. and Chanock, R.M. (1980) Human rotavirus type 2: cultivation in vitro. Science 207, 189-191.