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Improved conditions for the production of arthropod-borne viral hemagglutinins in infected HeLa cell cultures
DISCUSSION
AND
PRELIMINARY
REFERENCES 1. FUJII\IOTO, M., and MURASE, Resins” (M. Honda, H. Yoshino, eds.), pp. 25-28. kawa Publishing Co., Tokyo, 2. MEITFS, L., and Thomas, Analytical Chemistry,” pp. Hill, New York, 1958.
T., “Ion Exchange Kakihana, and Y. In Japanese. Hiro1957. H. C., “Advanced 375-378. McGraw-
TAKESHI
TA;\TIGUCHI
Plant Pathology Laboratory Faculty of Agriculture, Nagoya University Anjyo, Japan Received July 23, 1962
Comment
on “Aphid Vector Electrostatics”
Affected
by
Bradley (1) has recently described experiments in which he exposed the stylets of Myzus persicae to “static” electricity from a charged electroscope, and observed that the insects lost their ability to transmit virus and showed a disinclination to feed. It would seemthat a simple reason for this disinclination to feed exists, which hinges around the necessity of taking dimensions into account when applying physical forces to small objects. It does not seem to be appreciated that electrostatic charges on glass rods, etc., differ only in degree from charges on capacitors which may be extremely dangerous to man. An idea of the magnitude of the charge used by Bradley may be gained from the following considerations. First, the voltage on the electroscope would be about 300-500 volts, values given for a charged gold-leaf electroscope (2). Secondly, the capacity of the electroscope was estimated experimentally on a dummy electroscope having leaves of the same size (10 X 45 mm) set permanently apart at about 60 degrees. It was found to be lo-l1 farad (10 pF) by resonating it with a small inductance and then substituting the capacity of the electroscope by a known capacity.l The resonant frequency was 33 megacycles, both with the electroscope and a capacity of the above value. The capacity of an aphid in ‘This value is about three to four times quoted by Neher (2) for a typical gold-leaf troscope.
that elec-
REPORTS
647
free space was taken as being about that of a sphere 2 mm in diameter, which is about 0.11 pF (5), an estimate which cannot be seriously in error. Consequently, if an aphid were to be charged to the potential of the electroscope, it could only abstract about 1% of the t,otal charge when removed. As the electroscope leaves collapsed “instantly” it follows that a current was flowing through the aphis, the damp brush, and the operator. The approximate magnitude of this current can be estimated if one interprets “instantly” as meaning one-tenth second or less; it is of the order of 5 x lo-* amperes. This may seem to be quite small, but it would probably result in the flow of several amperes per square centimeter at the stylet t,ip. This would undoubtedly cause severe local damage. In addition, the whole current flow would pass along the entire central nervous system of the aphis and might well cause damage there too. It is not altogether surprising that the aphids failed to act as virus vectors after such treatment. REFERENCES R. H. E., ViroEogy 17,95 (1962). H. V., in STRONG, J. “Modern Physical Laboratory Practice,” p. 225. Blackie, London and Glasgow (1949). 3. TERSIAN, F. E. Radio Engineers’ Handbook, p. 113. McGraw-Hill, New York (1950). ROI- MARKHAM Agricultural Research Council Viws Research Unit Huntingdon Rond Cambridge, England
1. BRADLEY, 2. NEHER,
“The “control” experiment used by Bradley in which a hair of a dry brush was applied to the electroscope merely shows that a dry hair is a better insulator than is a damp brush.
Improved Conditions for the Production Arthropod-Borne Viral Hemagglutinins in Infected HeLa Cell Cultures
of
Production of hemagglutinins in HeLa cells infected with arthropod-borne viruses has been reported previously from this laboratory (1-s). In t’he course of further studies, it was found that titers of hemagglu-
648
DISCUSSION
AND
PRELIMINARY
tinins produced in cell cultures could be increased consistently by increasing the number of infected cells, by incubating infected cells at well-defined optimal temperatures, by omitting phenol red from Hanks’ balanced salt solution, a constituent of the maintenance medium, and by concentrating the hemagglutinins in cell culture fluids by ultrafiltration. Large amounts of infected fluids were obtained by propagating various arthropodborne viruses in HeLa cells grown in 16 x 150-mm screw-cap tubes or in 160-ml French square bottles. Following viral inoculat,ion, HeLa cells were maintained in 97% Eagle’s basal medium and 3% fetal bovine serum. Fluids were collected at S-day intervals following inoculation; the infected HeLa cells were refed at the same time and kept for periods of up to 6 weeks after inoculation. Different lots of infected fluids were stored at -70”. They were then thawed and TABLE
1
COMPARISON OF HI TITERS OF CENTRAL EUROPEAN TICK-BORNE ENCEPHALITIS VIRUS GROWN IN HELA CELLS IN TUBES AND BOTTLES AT DIFFERENT INCUBATION TEMPERATURES
HeLa cells grown in
Tubes Bottles
Days after inoculation
7 15 7 15
TABLE
HI titer when temperature of incubation was (“C) : 22
22-26
37
64 128 512 512
64 128 4096 512
256 64 2048 64
2
HI TITERS OF RUSSIAN SPRING-SUMMER AND CENTRAL EUROPEAN TICK-BORNE ENCEPHALITIS VIRUSES GROWN AT 22" WITH AND WITHOUT PHENOL RED HI Virus
strain
CEE RSSE
Days after inoculation
7 15 7 15
titer
With phenol red
Without phenol red
512 512 1024 256
1024 2048 1024 1024
REPORTS TABLE
3
CONCENTRATION OF DIFFERENT VIRAL HEMAGGLUTININS PRODUCED BY ULTRAFILTRATION
Group
Virus
Titer of hemagglutinin Not enc.
Cont.
Con:entration factor
A
Semliki Sindbis EEE
32 128 32
2048 1024 512
70 15 15
B
CEE RSSE
512 128
4096 8192
10 100
California
Tahyna
<2
8
55
Bunyamwera
Germiston
<2
8
55
extracted twice with acetone using 20 parts of acetone for one part of fluid. Titrations of hemagglutinating antigens were carried out by methods described by Clarke and Casals (4). The infected cell culture fluids were concentrated with an ultrafilter, LKB 6300 A,l a unit used for concentrating aqueous solutions of substances with high molecular weights. Different lots of infected cell culture fluids were pooled at the time of concentration. Ultrafiltration was carried out at 4” overnight or for 2-3 days, depending on the concentration factor. In comparative experiments with Central European tick-borne encephalitis virus, the use of bottles rather than tubes and of incubation temperatures lower than 37” proved generally superior for hemagglutinin production (Table 1). We attribute this result to the presence and survival of larger numbers of cells actively producing hemagglutinin. By omitting phenol red from the maintenance medium, titers of hemagglutinins could be increased two- to fourfold. Table 2 gives the results for tick-borne Russian spring-summer and Central European encephalitis viruses grown at 22” in 160-ml French bottles with and without phenol red in the maintenance medium. A presumably 1 Obtained Rugby Ave.,
from LKB Washington
Instruments, 14, D.C.
Inc.,
4840
DISCUSSION
AND PRELIMINARY
REPORTS
649
maining cells are apparently unaffected in that they gave rise to colonies indistinguishable from those formed by control cells, which have not been exposed to virus. However, Fraser and Gharpure (3) have shown that all cells exposed to the virus dose used (about 1000 PFU per cell) contain virus antigen, which can be detected by specific immunofluorescence in the cytoplasm around the nucleus, a short time after virus adsorption. It follows that the cells which gave rise to colonies of normal appearance, as well as the transformed cells, must take up enough virus to be detectable with fluorescent antibody. This raised the possibility that the cells in the apparently normal colonies had undergone a neoplastic or other inherited change, which was not expressed by an alteration in colonial morphology, at least in the early generations of growth. This communication describes the properREFERENCES ties of three clones showing the normal, ori1. CLARKE, D. H., 1960 Annual Report of The ented, arrangement of cells arising from Rockefeller Foundation Virus Laboratories, BHK21 cells exposed to polyoma virus. New York (mimeographed), pp. 38-42 (1961). These clones were compared with three 2. BUCKLEY, S. M., 1960 Annual Report of The transformed clones with typical random arRockefeller Foundation Virus Laboratories, rangement of cells, arising in the same culNew York (mimeographed), pp. 33-36 (1961). ture. 3. BUCKLEY, S. M., and SRIHONGSE, S., Abstr. 8th Clone 13 of BHK21 cells was exposed to Intern. Congr. Microbial., Montreal, p. 93 virus at a ratio of 2000 PFU per cell as de(1962). scribed elsewhere (.zj. After adsorption, 160 ,i. CLARKE, D. H., and CASALS, J., Am. J. Trap. cells were added to a pet’ri dish containing Med. Hyg. 7, 561-573 (1958). MIHA LIKAI? irradiated mouse feeder cells, and incubated SOKJA M. BUCKLEY at 37” for 7 days to allow development of DELPHINE H. CLARKE colonies. Six colonies were obviously transRockefeller Foundation Virus Laboratories formed out of a total of 129 (the latter New York, New York counted on a duplicate plate). Three wellReceived September 2Y, 1962 separated colonies with normal morphology ’ Institute of Microbiology, University of J,jubland two of the colonies with t,he random jana, Tugoslavia. arrangement of transformed cells were marked. The medium was removed, trypsin was added, and the five colonies were reCharacteristics of Normal and Transformed moved separately under direct vision with Clones Arising from BHK21 Cells a dissecting microscope. Each colony was Exposed to Polyoma Virus grown to a population of about one million cells and cloned by growing isolated single When the BHK21 strain of hamster cells cells in microdrops. An additional clone was (1) is exposed briefly to large dosesof polyisolated from one of the transformed colony oma virus, about 3% of cells undergo neo- cultures. The apparently normal clones plastic transformation as judged by altered were designated A, B, and C. The transcolonial morphology, loss of contact inhibiformed clones were designated Xi, Y, and Z. tion, increased glycolysis, and increased Clones S and Z came from one, and clone transplantability in hamsters (2). The re- Y from the other, original t,ransformed col-
toxic effect of phenol red on cells, causing decreased hemagglutinin titers, is readily demonstrated after a prolonged incubation period. As shown in Table 3, by ultrafiltration of infected fluids prior to extraction with acetone we were able to produce high-titered hemagglutinating antigens for representative strains of arthropod-borne viruses of groups A and B. In addition, hemagglutinins could be demonstrated in HeLa cell culture fluids infected with Tahyna and Germiston viruses, two agents for which hemagglutinins are produced in infected mice with considerable difficulty. We believe that use of the above improved conditions for preparation of hemagglutinating antigens would permit study of a large number of viruses with this valuable serologic tool.
AND
PRELIMINARY
REFERENCES 1. FUJII\IOTO, M., and MURASE, Resins” (M. Honda, H. Yoshino, eds.), pp. 25-28. kawa Publishing Co., Tokyo, 2. MEITFS, L., and Thomas, Analytical Chemistry,” pp. Hill, New York, 1958.
T., “Ion Exchange Kakihana, and Y. In Japanese. Hiro1957. H. C., “Advanced 375-378. McGraw-
TAKESHI
TA;\TIGUCHI
Plant Pathology Laboratory Faculty of Agriculture, Nagoya University Anjyo, Japan Received July 23, 1962
Comment
on “Aphid Vector Electrostatics”
Affected
by
Bradley (1) has recently described experiments in which he exposed the stylets of Myzus persicae to “static” electricity from a charged electroscope, and observed that the insects lost their ability to transmit virus and showed a disinclination to feed. It would seemthat a simple reason for this disinclination to feed exists, which hinges around the necessity of taking dimensions into account when applying physical forces to small objects. It does not seem to be appreciated that electrostatic charges on glass rods, etc., differ only in degree from charges on capacitors which may be extremely dangerous to man. An idea of the magnitude of the charge used by Bradley may be gained from the following considerations. First, the voltage on the electroscope would be about 300-500 volts, values given for a charged gold-leaf electroscope (2). Secondly, the capacity of the electroscope was estimated experimentally on a dummy electroscope having leaves of the same size (10 X 45 mm) set permanently apart at about 60 degrees. It was found to be lo-l1 farad (10 pF) by resonating it with a small inductance and then substituting the capacity of the electroscope by a known capacity.l The resonant frequency was 33 megacycles, both with the electroscope and a capacity of the above value. The capacity of an aphid in ‘This value is about three to four times quoted by Neher (2) for a typical gold-leaf troscope.
that elec-
REPORTS
647
free space was taken as being about that of a sphere 2 mm in diameter, which is about 0.11 pF (5), an estimate which cannot be seriously in error. Consequently, if an aphid were to be charged to the potential of the electroscope, it could only abstract about 1% of the t,otal charge when removed. As the electroscope leaves collapsed “instantly” it follows that a current was flowing through the aphis, the damp brush, and the operator. The approximate magnitude of this current can be estimated if one interprets “instantly” as meaning one-tenth second or less; it is of the order of 5 x lo-* amperes. This may seem to be quite small, but it would probably result in the flow of several amperes per square centimeter at the stylet t,ip. This would undoubtedly cause severe local damage. In addition, the whole current flow would pass along the entire central nervous system of the aphis and might well cause damage there too. It is not altogether surprising that the aphids failed to act as virus vectors after such treatment. REFERENCES R. H. E., ViroEogy 17,95 (1962). H. V., in STRONG, J. “Modern Physical Laboratory Practice,” p. 225. Blackie, London and Glasgow (1949). 3. TERSIAN, F. E. Radio Engineers’ Handbook, p. 113. McGraw-Hill, New York (1950). ROI- MARKHAM Agricultural Research Council Viws Research Unit Huntingdon Rond Cambridge, England
1. BRADLEY, 2. NEHER,
“The “control” experiment used by Bradley in which a hair of a dry brush was applied to the electroscope merely shows that a dry hair is a better insulator than is a damp brush.
Improved Conditions for the Production Arthropod-Borne Viral Hemagglutinins in Infected HeLa Cell Cultures
of
Production of hemagglutinins in HeLa cells infected with arthropod-borne viruses has been reported previously from this laboratory (1-s). In t’he course of further studies, it was found that titers of hemagglu-
648
DISCUSSION
AND
PRELIMINARY
tinins produced in cell cultures could be increased consistently by increasing the number of infected cells, by incubating infected cells at well-defined optimal temperatures, by omitting phenol red from Hanks’ balanced salt solution, a constituent of the maintenance medium, and by concentrating the hemagglutinins in cell culture fluids by ultrafiltration. Large amounts of infected fluids were obtained by propagating various arthropodborne viruses in HeLa cells grown in 16 x 150-mm screw-cap tubes or in 160-ml French square bottles. Following viral inoculat,ion, HeLa cells were maintained in 97% Eagle’s basal medium and 3% fetal bovine serum. Fluids were collected at S-day intervals following inoculation; the infected HeLa cells were refed at the same time and kept for periods of up to 6 weeks after inoculation. Different lots of infected fluids were stored at -70”. They were then thawed and TABLE
1
COMPARISON OF HI TITERS OF CENTRAL EUROPEAN TICK-BORNE ENCEPHALITIS VIRUS GROWN IN HELA CELLS IN TUBES AND BOTTLES AT DIFFERENT INCUBATION TEMPERATURES
HeLa cells grown in
Tubes Bottles
Days after inoculation
7 15 7 15
TABLE
HI titer when temperature of incubation was (“C) : 22
22-26
37
64 128 512 512
64 128 4096 512
256 64 2048 64
2
HI TITERS OF RUSSIAN SPRING-SUMMER AND CENTRAL EUROPEAN TICK-BORNE ENCEPHALITIS VIRUSES GROWN AT 22" WITH AND WITHOUT PHENOL RED HI Virus
strain
CEE RSSE
Days after inoculation
7 15 7 15
titer
With phenol red
Without phenol red
512 512 1024 256
1024 2048 1024 1024
REPORTS TABLE
3
CONCENTRATION OF DIFFERENT VIRAL HEMAGGLUTININS PRODUCED BY ULTRAFILTRATION
Group
Virus
Titer of hemagglutinin Not enc.
Cont.
Con:entration factor
A
Semliki Sindbis EEE
32 128 32
2048 1024 512
70 15 15
B
CEE RSSE
512 128
4096 8192
10 100
California
Tahyna
<2
8
55
Bunyamwera
Germiston
<2
8
55
extracted twice with acetone using 20 parts of acetone for one part of fluid. Titrations of hemagglutinating antigens were carried out by methods described by Clarke and Casals (4). The infected cell culture fluids were concentrated with an ultrafilter, LKB 6300 A,l a unit used for concentrating aqueous solutions of substances with high molecular weights. Different lots of infected cell culture fluids were pooled at the time of concentration. Ultrafiltration was carried out at 4” overnight or for 2-3 days, depending on the concentration factor. In comparative experiments with Central European tick-borne encephalitis virus, the use of bottles rather than tubes and of incubation temperatures lower than 37” proved generally superior for hemagglutinin production (Table 1). We attribute this result to the presence and survival of larger numbers of cells actively producing hemagglutinin. By omitting phenol red from the maintenance medium, titers of hemagglutinins could be increased two- to fourfold. Table 2 gives the results for tick-borne Russian spring-summer and Central European encephalitis viruses grown at 22” in 160-ml French bottles with and without phenol red in the maintenance medium. A presumably 1 Obtained Rugby Ave.,
from LKB Washington
Instruments, 14, D.C.
Inc.,
4840
DISCUSSION
AND PRELIMINARY
REPORTS
649
maining cells are apparently unaffected in that they gave rise to colonies indistinguishable from those formed by control cells, which have not been exposed to virus. However, Fraser and Gharpure (3) have shown that all cells exposed to the virus dose used (about 1000 PFU per cell) contain virus antigen, which can be detected by specific immunofluorescence in the cytoplasm around the nucleus, a short time after virus adsorption. It follows that the cells which gave rise to colonies of normal appearance, as well as the transformed cells, must take up enough virus to be detectable with fluorescent antibody. This raised the possibility that the cells in the apparently normal colonies had undergone a neoplastic or other inherited change, which was not expressed by an alteration in colonial morphology, at least in the early generations of growth. This communication describes the properREFERENCES ties of three clones showing the normal, ori1. CLARKE, D. H., 1960 Annual Report of The ented, arrangement of cells arising from Rockefeller Foundation Virus Laboratories, BHK21 cells exposed to polyoma virus. New York (mimeographed), pp. 38-42 (1961). These clones were compared with three 2. BUCKLEY, S. M., 1960 Annual Report of The transformed clones with typical random arRockefeller Foundation Virus Laboratories, rangement of cells, arising in the same culNew York (mimeographed), pp. 33-36 (1961). ture. 3. BUCKLEY, S. M., and SRIHONGSE, S., Abstr. 8th Clone 13 of BHK21 cells was exposed to Intern. Congr. Microbial., Montreal, p. 93 virus at a ratio of 2000 PFU per cell as de(1962). scribed elsewhere (.zj. After adsorption, 160 ,i. CLARKE, D. H., and CASALS, J., Am. J. Trap. cells were added to a pet’ri dish containing Med. Hyg. 7, 561-573 (1958). MIHA LIKAI? irradiated mouse feeder cells, and incubated SOKJA M. BUCKLEY at 37” for 7 days to allow development of DELPHINE H. CLARKE colonies. Six colonies were obviously transRockefeller Foundation Virus Laboratories formed out of a total of 129 (the latter New York, New York counted on a duplicate plate). Three wellReceived September 2Y, 1962 separated colonies with normal morphology ’ Institute of Microbiology, University of J,jubland two of the colonies with t,he random jana, Tugoslavia. arrangement of transformed cells were marked. The medium was removed, trypsin was added, and the five colonies were reCharacteristics of Normal and Transformed moved separately under direct vision with Clones Arising from BHK21 Cells a dissecting microscope. Each colony was Exposed to Polyoma Virus grown to a population of about one million cells and cloned by growing isolated single When the BHK21 strain of hamster cells cells in microdrops. An additional clone was (1) is exposed briefly to large dosesof polyisolated from one of the transformed colony oma virus, about 3% of cells undergo neo- cultures. The apparently normal clones plastic transformation as judged by altered were designated A, B, and C. The transcolonial morphology, loss of contact inhibiformed clones were designated Xi, Y, and Z. tion, increased glycolysis, and increased Clones S and Z came from one, and clone transplantability in hamsters (2). The re- Y from the other, original t,ransformed col-
toxic effect of phenol red on cells, causing decreased hemagglutinin titers, is readily demonstrated after a prolonged incubation period. As shown in Table 3, by ultrafiltration of infected fluids prior to extraction with acetone we were able to produce high-titered hemagglutinating antigens for representative strains of arthropod-borne viruses of groups A and B. In addition, hemagglutinins could be demonstrated in HeLa cell culture fluids infected with Tahyna and Germiston viruses, two agents for which hemagglutinins are produced in infected mice with considerable difficulty. We believe that use of the above improved conditions for preparation of hemagglutinating antigens would permit study of a large number of viruses with this valuable serologic tool.