Short Communications Intervirology 9: 56-59 (1978)
A Simple Method for Concentration of Enteroviruses and Rotaviruses from Cell Culture Harvests Using Membrane Filters S am uel R . F arr a h , S aga r M . G oyal , C harles P. G erba , R ic h a r d H . C o n k l in , C ra ig W allis , J oseph L. M eln ick a n d H erbert L. D u P o nt Department of Virology and Epidemiology, Baylor College of Medicine, and Department of Infectious Disease, University of Texas Health Science Center, Houston, Tex.
Key Words. Rotaviruses • Enteroviruses • Virus concentration • Cell culture harvests Summary. Organic compounds in cell culture harvests known as membrane-coating components (MCC) prevent virus adsorption to membrane filters. Blending cell culture harvests with fluorocarbon removed the MCC and permitted adsorption of virus in acidified harvests to epoxy-fiberglass filters. Subsequent elution with high pH buffer resulted in recovery of greater than 90% of the virus with concentrations of up to 100-fold. W allis and M e l n ic k [1] noted that certain metallic salts enhanced virus adsorption to membrane filters while certain organic compounds, termed membrane-coating components (MCC), interfered with virus adsorption. Removal of the MCC from cell culture harvests using a series of treatments including chloroform extraction, protamine sulfate precipitation and ionexchange chromatography permitted adsorption of virus to nitrocellulose membranes. H end erson et al. [2] used a combination of precipitation with cationic detergent and ion-exchange chromatography to remove the MCC from cell culture harvests. Virus was then adsorbed to epoxy-fiberglassasbestos membrane filters and recovered by treating the filters with a small volume of high pH buffer. In this present work, two modifications of these previously described
Received: February 23, 1977.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
Address inquiries to: Dr. Samuel R. F arrah , Department of Virology and Epidemiology, Baylor College of Medicine, Houston. TX 77030 (USA)
57
procedures were made. First, fluorocarbon was used to remove MCC. This reduced the time required for removal of MCC and simplified the procedure. Second, epoxy-fiberglass membrane filters were used as virus adsorbents. These filters are more resistant to clogging than other commercially available membrane filters but are similar to the other filters in their ability to adsorb virus [3], Plaque-purified poliovirus type 1 (strain LSc), echovirus type 1, and cox sackievirus type B3 were grown in MKHull, BGM, BSC-1 or primary baboon kidney cells. Virus was assayed as previously described [4]. Cell harvests were obtained approximately 48 h postinfection and were blended with an equal volume of trichlorotrifluoroethane (Freon 113, DuPont DeNemours Co., Wilmington, Del.) for 15 sec and centrifuged at 2,000 g for 10 min. The super natants were removed and adjusted to pH 3.5 by addition of 0.05 M glycine that had been adjusted to pH 2 by addition of 12 n HC1. The acidified entero virus harvests were then passed through a series of three epoxy-fiberglass filters (Filterite Corp., Timonium, Md.) of 3.0-, 0.45- and 0.25-pm porosities in 25-mm holders at approximately 5 ml/sec. Virus was eluted from the filters using 3 ml of 0.05 m glycine that had been adjusted to pH 10.5 by addition of 10 n NaOH. The eluates were neutralized with pH 2 glycine and assayed after dilution in Tris-buffered saline. Simian rotavirus (SA-11), which was kindly supplied by H.H. M alh erbe , was grown in primary baboon or rhesus monkey kidney cells. Virus was assayed by a quantal assay in tubes of primary primate kidney cells. The tissue-culture-infective dose 50 (TCIDso) was determined according to the formula of R eed and M u e n c h [5], Cell harvests containing SA-11 were treated with Freon and acidified as described above. The harvests were then passed through epoxy-fiberglass-asbestos filters (series AA, Cox Instrument Corp., Detroit, Mich.) or epoxy-fiberglass filters (Filterite Corp.) in 13-mm holders. The different filters tested are listed in table I. Virus adsorbed to two 13-mm, 0.25-ij.m Filterite filters was eluted using 0.35 ml of 0.05 M glycine or 50 g/1 tryptose-phosphate broth (Difco Laboratories, Detroit, Mich.) adjusted to the pH values listed in table II using 10 N NaOH. The eluates obtained were neutralized and assayed as described above. Using these procedures, enteroviruses in up to 400 ml of culture fluid could be concentrated in approximately 4 ml of neutralized filter eluate. Greater than 90% of the initial virus could be recovered (table III). If desired, greater volumes of culture harvest could be processed since neither the flow rate through the filters nor the ability of the filters to adsorb virus was diminished after processing 400 ml.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
F arrah/G oyal/G erba/C onklin /W allis/M elnick/D uP ont
58
F arrah/G oyal/G erba/C onkun /W allis/M elnick/D u Pont
Table l. Adsorption of SA-II to membrane filters1 Number and type of membrane filters
Reduction in virus titer: log
Two, Filterite 3.0-p.m Two, Filterite 0.45-|xm Two, Filterite 0.25-ixm One, Cox 2.0-¡xm One, Cox 0.45-p.m
filter influent TCIDso filter effluent TCID 50
1.0 2.6 4.2 1.5 2.3
1 Approximately 107 TCIDso of SA-11 in Freon-treated harvests at pH 3.5 was passed through the indicated filters in 13-mm holders. Virus in the influent and effluent samples was determined.
Table II. Concentration of SA-11 from cell culture harvests1 Eluent
0.05 m glycine, pH 8 0.05 m glycine, pH 9 0.05 m glycine, pH 10 0.05 m glycine, pH 11 50 g/1 tryptose-phosphate broth, pH 9
Total virus in virus harvest TCID 50
concentrate
2.3x10» 2 .3 x l0 8 2.3x10» 2.3 x 10» 4.7 x 107
6.4 x 1.2 x 2.0 x 2.0 x 5.0 x
TCID 50 10» 10» 10« 10« 107
% 3 52 1 1 106
SA-11 was similar to the enteroviruses studied in this and previous works [3, 4] in that adsorption to membrane filters occurred at low pH (table I). However, a series of elution experiments indicated that the pH 10-10.5 buffer used for elution of enteroviruses did not permit efficient elution of SA-11. Elution with pH 9 glycine buffer permitted recovery of approximately 50% of the initial virus, while elution with an organic buffer (tryptose-phosphate broth) at pH 9 resulted in recovery of essentially all of the virus present in the initial sample. Representative data from these tests are presented in table II.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
1 Virus in 25-30 ml of culture harvest was concentrated to a final volume of 0.5 ml using the procedures described in the text.
Virus Concentration from Cell Culture Harvests
59
Table III. Concentration of enteroviruses from cell culture harvests Virus
Polio 1 Polio 1 Polio 1 Polio I Polio 1 Echo 1 Coxsackie B3
Cells used to grow virus
MKHull MKHull MKHull BSC-1 BGM BGM primary baboon kidney
Virus harvest ml
Virus harvest PFU
PFU
%
24
2.9 X 10»
2.3 X 10»
20
2 .0 X 1 0 9
2 .1 X 1 0 9
79 105
160 320 400 50 50
9.0 X 109 4.4x10» 5.4 X 109 3.8 X 109 6.8 X 10»
9.0 X 109 4.7 xlO 9 4.8 X 109 3.7 X 109
Final concentrate*1
6 .2 X 1 0 9
100
107 89 97 91
Concen tration factor23456* 6
5 40 80 100 12 12
1 Concentrated to a final volume of 4 ml. 2 Volume of harvest/volume of concentrate.
1 W allis, C. and M elnick , J.L .: Concentration of enteroviruses on membrane filters. J. Virol. I: 472-477 (1967). 2 H enderson, M.; W allis, C., and M elnick, J.L .: Concentration and purification of enteroviruses by membrane chromatography. Appl. environ. Microbiol. 32: 689-693 (1976). 3 F arrah, S. R . ; G erba, C. P .; W allis, C., and M elnick, J. L .: Concentration of viruses from large volumes of tap water using pleated membrane filters. Appl. environ. Micro biol. 31 : 221-226 (1976). 4 M elnick, J.L. and W enner, H.A.: Enteroviruses; in L ennette and S chmidt Diag nostic procedures for viral and rickettsial infections; 4th ed., pp.529-602 (Am.Public Health Ass., New York 1969). 5 R eed, L. J. and M uench , H .: A simple method for estimating fifty per cent endpoints. Am. J. Hyg. 27: 493^*97 (1938). 6 W oode, G .N .; Bridger , J.C .; J ones, J.M .; F lewett, T .H .; Bryden, A.S.; D avies, H.A., and W hite, G .B .B .: Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals. Infec. Immunity 14: 804-810 (1976).
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
These procedures should prove useful for producing high-titered virus stocks and as an initial step in concentration and purification of virus stocks for biochemical and immunological studies. The ability of SA-11 to replicate in cell cultures and its antigenic similarity to the virus associated with infantile gastroenteritis [6] may make it useful as a source of antigen in studies on the distribution of the human diarrhea virus.
A Simple Method for Concentration of Enteroviruses and Rotaviruses from Cell Culture Harvests Using Membrane Filters S am uel R . F arr a h , S aga r M . G oyal , C harles P. G erba , R ic h a r d H . C o n k l in , C ra ig W allis , J oseph L. M eln ick a n d H erbert L. D u P o nt Department of Virology and Epidemiology, Baylor College of Medicine, and Department of Infectious Disease, University of Texas Health Science Center, Houston, Tex.
Key Words. Rotaviruses • Enteroviruses • Virus concentration • Cell culture harvests Summary. Organic compounds in cell culture harvests known as membrane-coating components (MCC) prevent virus adsorption to membrane filters. Blending cell culture harvests with fluorocarbon removed the MCC and permitted adsorption of virus in acidified harvests to epoxy-fiberglass filters. Subsequent elution with high pH buffer resulted in recovery of greater than 90% of the virus with concentrations of up to 100-fold. W allis and M e l n ic k [1] noted that certain metallic salts enhanced virus adsorption to membrane filters while certain organic compounds, termed membrane-coating components (MCC), interfered with virus adsorption. Removal of the MCC from cell culture harvests using a series of treatments including chloroform extraction, protamine sulfate precipitation and ionexchange chromatography permitted adsorption of virus to nitrocellulose membranes. H end erson et al. [2] used a combination of precipitation with cationic detergent and ion-exchange chromatography to remove the MCC from cell culture harvests. Virus was then adsorbed to epoxy-fiberglassasbestos membrane filters and recovered by treating the filters with a small volume of high pH buffer. In this present work, two modifications of these previously described
Received: February 23, 1977.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
Address inquiries to: Dr. Samuel R. F arrah , Department of Virology and Epidemiology, Baylor College of Medicine, Houston. TX 77030 (USA)
57
procedures were made. First, fluorocarbon was used to remove MCC. This reduced the time required for removal of MCC and simplified the procedure. Second, epoxy-fiberglass membrane filters were used as virus adsorbents. These filters are more resistant to clogging than other commercially available membrane filters but are similar to the other filters in their ability to adsorb virus [3], Plaque-purified poliovirus type 1 (strain LSc), echovirus type 1, and cox sackievirus type B3 were grown in MKHull, BGM, BSC-1 or primary baboon kidney cells. Virus was assayed as previously described [4]. Cell harvests were obtained approximately 48 h postinfection and were blended with an equal volume of trichlorotrifluoroethane (Freon 113, DuPont DeNemours Co., Wilmington, Del.) for 15 sec and centrifuged at 2,000 g for 10 min. The super natants were removed and adjusted to pH 3.5 by addition of 0.05 M glycine that had been adjusted to pH 2 by addition of 12 n HC1. The acidified entero virus harvests were then passed through a series of three epoxy-fiberglass filters (Filterite Corp., Timonium, Md.) of 3.0-, 0.45- and 0.25-pm porosities in 25-mm holders at approximately 5 ml/sec. Virus was eluted from the filters using 3 ml of 0.05 m glycine that had been adjusted to pH 10.5 by addition of 10 n NaOH. The eluates were neutralized with pH 2 glycine and assayed after dilution in Tris-buffered saline. Simian rotavirus (SA-11), which was kindly supplied by H.H. M alh erbe , was grown in primary baboon or rhesus monkey kidney cells. Virus was assayed by a quantal assay in tubes of primary primate kidney cells. The tissue-culture-infective dose 50 (TCIDso) was determined according to the formula of R eed and M u e n c h [5], Cell harvests containing SA-11 were treated with Freon and acidified as described above. The harvests were then passed through epoxy-fiberglass-asbestos filters (series AA, Cox Instrument Corp., Detroit, Mich.) or epoxy-fiberglass filters (Filterite Corp.) in 13-mm holders. The different filters tested are listed in table I. Virus adsorbed to two 13-mm, 0.25-ij.m Filterite filters was eluted using 0.35 ml of 0.05 M glycine or 50 g/1 tryptose-phosphate broth (Difco Laboratories, Detroit, Mich.) adjusted to the pH values listed in table II using 10 N NaOH. The eluates obtained were neutralized and assayed as described above. Using these procedures, enteroviruses in up to 400 ml of culture fluid could be concentrated in approximately 4 ml of neutralized filter eluate. Greater than 90% of the initial virus could be recovered (table III). If desired, greater volumes of culture harvest could be processed since neither the flow rate through the filters nor the ability of the filters to adsorb virus was diminished after processing 400 ml.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
F arrah/G oyal/G erba/C onklin /W allis/M elnick/D uP ont
58
F arrah/G oyal/G erba/C onkun /W allis/M elnick/D u Pont
Table l. Adsorption of SA-II to membrane filters1 Number and type of membrane filters
Reduction in virus titer: log
Two, Filterite 3.0-p.m Two, Filterite 0.45-|xm Two, Filterite 0.25-ixm One, Cox 2.0-¡xm One, Cox 0.45-p.m
filter influent TCIDso filter effluent TCID 50
1.0 2.6 4.2 1.5 2.3
1 Approximately 107 TCIDso of SA-11 in Freon-treated harvests at pH 3.5 was passed through the indicated filters in 13-mm holders. Virus in the influent and effluent samples was determined.
Table II. Concentration of SA-11 from cell culture harvests1 Eluent
0.05 m glycine, pH 8 0.05 m glycine, pH 9 0.05 m glycine, pH 10 0.05 m glycine, pH 11 50 g/1 tryptose-phosphate broth, pH 9
Total virus in virus harvest TCID 50
concentrate
2.3x10» 2 .3 x l0 8 2.3x10» 2.3 x 10» 4.7 x 107
6.4 x 1.2 x 2.0 x 2.0 x 5.0 x
TCID 50 10» 10» 10« 10« 107
% 3 52 1 1 106
SA-11 was similar to the enteroviruses studied in this and previous works [3, 4] in that adsorption to membrane filters occurred at low pH (table I). However, a series of elution experiments indicated that the pH 10-10.5 buffer used for elution of enteroviruses did not permit efficient elution of SA-11. Elution with pH 9 glycine buffer permitted recovery of approximately 50% of the initial virus, while elution with an organic buffer (tryptose-phosphate broth) at pH 9 resulted in recovery of essentially all of the virus present in the initial sample. Representative data from these tests are presented in table II.
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
1 Virus in 25-30 ml of culture harvest was concentrated to a final volume of 0.5 ml using the procedures described in the text.
Virus Concentration from Cell Culture Harvests
59
Table III. Concentration of enteroviruses from cell culture harvests Virus
Polio 1 Polio 1 Polio 1 Polio I Polio 1 Echo 1 Coxsackie B3
Cells used to grow virus
MKHull MKHull MKHull BSC-1 BGM BGM primary baboon kidney
Virus harvest ml
Virus harvest PFU
PFU
%
24
2.9 X 10»
2.3 X 10»
20
2 .0 X 1 0 9
2 .1 X 1 0 9
79 105
160 320 400 50 50
9.0 X 109 4.4x10» 5.4 X 109 3.8 X 109 6.8 X 10»
9.0 X 109 4.7 xlO 9 4.8 X 109 3.7 X 109
Final concentrate*1
6 .2 X 1 0 9
100
107 89 97 91
Concen tration factor23456* 6
5 40 80 100 12 12
1 Concentrated to a final volume of 4 ml. 2 Volume of harvest/volume of concentrate.
1 W allis, C. and M elnick , J.L .: Concentration of enteroviruses on membrane filters. J. Virol. I: 472-477 (1967). 2 H enderson, M.; W allis, C., and M elnick, J.L .: Concentration and purification of enteroviruses by membrane chromatography. Appl. environ. Microbiol. 32: 689-693 (1976). 3 F arrah, S. R . ; G erba, C. P .; W allis, C., and M elnick, J. L .: Concentration of viruses from large volumes of tap water using pleated membrane filters. Appl. environ. Micro biol. 31 : 221-226 (1976). 4 M elnick, J.L. and W enner, H.A.: Enteroviruses; in L ennette and S chmidt Diag nostic procedures for viral and rickettsial infections; 4th ed., pp.529-602 (Am.Public Health Ass., New York 1969). 5 R eed, L. J. and M uench , H .: A simple method for estimating fifty per cent endpoints. Am. J. Hyg. 27: 493^*97 (1938). 6 W oode, G .N .; Bridger , J.C .; J ones, J.M .; F lewett, T .H .; Bryden, A.S.; D avies, H.A., and W hite, G .B .B .: Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice and foals. Infec. Immunity 14: 804-810 (1976).
Downloaded by: Stockholm University Library 130.237.165.40 - 12/20/2018 11:05:05 PM
These procedures should prove useful for producing high-titered virus stocks and as an initial step in concentration and purification of virus stocks for biochemical and immunological studies. The ability of SA-11 to replicate in cell cultures and its antigenic similarity to the virus associated with infantile gastroenteritis [6] may make it useful as a source of antigen in studies on the distribution of the human diarrhea virus.
