Transmissible Gastroenteritis Virus: Plaques and Plaque Neutralization Test
a
F. C. Thomas and G. C. Dulac*
ABSTRACT A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.
R1SUM: Les auteurs ont titre et neutralise plusieurs souches du virus de la gastro-enterite transmissible et du virus hemagglutinant de l'encephalomyelite du porc, sur cultures de cellules thyroidiennes porcine, par la methode des plages. Les dimensions des plages varierent considerablement d'une souche a l'autre du virus de la gastro-entfrite transmissible, ainsi qu'avec certaines souches particulieres de ce virus. Les souches les plus recemment isol6es produisirent des plages plus petites que celles des souches de laboratoire ou du virus hemagglutinant de l'encephalomyelite porcine. Un imnunserum, specifique au virus de la gastro-ente-
*Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (E), Box 11300, Station H, Ottawa, Ontario K2H 8P9. Submitted July 16, 1975.
Volume 40
-
April, 1976
rite transmissible, en neutralisa les plages, mais non celles du virus hdemagglutinant de l'enc&phalomyelite porcine. La neutralisation des plages par le serum de porcs se rktablissant d'une infection experimentale ou naturelle s'avera aussi sure et plus sensible que la sero-neutralisation conventionnelle en tubes.
I NTRODUCTION Transmissible gastroenteritis v i r u s (TGEV), a coronavirus, continues to cause disease problems in neonatal pigs (2, 3). Reference has been made to different "strains" of the virus and there are apparent differences amongst isolates (3, 5, 8-10). The plaquing system and plaque neutralization (PN) test provide useful tools for cloning viral isolates, measuring their relatedness and measuring antibody responses (6, 7, 9). In the present publication some applications of these techniques to TGEV using pig thyroid cell cultures (9) are reported.
MATERIALS AND METHODS PLAQUING PROCEDURE
Monolayer cell cultures prepared from adult pig thyroids (3) were trypsinized (0.25% trypsin in PBS) and the cells seeded at approximately 3 x 104 cells per well in plastic culture dishes' (Linbro, Model FB-6-TC) using MEM (E)2 and
lLinbro Chemical Co. Ltd., New Haven, Connecticut. 2Mogul-ED, Oshkosh, Wisconsin.
171
10% fetal bovine serum (FBS). These were grown to confluency (usually 48 hours) in a humid C02 incubator and used anytime within the next five days. The inocula (0.1 or 0.2 ml per well) were allowed to absorb for one hour at 370 C and overlay applied. The latter consisted of double ionic strength MEM mixed at 45°C with an equal volume of 1.6% molten agar3 (Ionagar no. 2, batch No. 337, 1973) solution. DEAE-dextran was added to a final concentration of 100 ,ujg/ml and FBS 10% v/v. The overlay (3 ml per well) was applied to the cell sheet after washing and draining and allowed to solidify at room temperature. Plates were inverted and incubated at 37°C for three or four days. Staining was done with a 1:10,000 solution of neutral red in MEM applied to the wells (1 ml per well) three to six hours before reading. Formalin fixation followed by removal of the agar one hour later yielded a permanent record. TABLE I. TGEV Antiserum Neutralizability of TGEV and HEV Isolates
Neutralizability with TGEV Hyperimmune Seruma + + + + + + + + + + + +
Origin Minnesota New York Indiana England Quebec Quebec Quebec Ontario Nova Scotia Ontario Quebec Ontario HEV-1 Ontario HEV-10 -Serum prepared against TGEV-Purdue caused greater than 90%,o reduction of p.f.u. at a 1:100 dilution Isolate
TGEV-Ames TGEV-Diamond TGEV-N.Y. TGEV-Purdue TGEV-British ADRI-1 ADRI-2 ADRI-5 ADRI-6 ADRI-10 ADRI-1I ADRI-12
PLAQUE NEUTRALIZATION TEST
Sequential serum dilutions two, four or tenfold, heat inactivated (56°C 30 minutes) were incubated in 96-well microplates with equal volumes of cloned virus suspension (put through 450 nm filter to remove viral clumps and debris) calculated to produce '30xoid Ltd., London SEI, England.
172
TABLE lI. Plaque Neutralizing Titers (to TGEV-Purdue Isolate) of Pig Sera Following a Field Outbreak of TGEV
Serum
PN titer
1........................ 3.90.................... 2.
....................
4. 5. 6. 7. 8........................ 9........................ 10 .......................
....................
....................
11........................
12 13 14
.................... .................... ....................
< 100 > 5000
9 1700 2000 5000 400 > 5000 1000 1400 400 500 1200
5000
-Mean: 2100
Range: 5000
approximately 60 plaque forming units (p.f.u.) per well. Control inocula consisted of 0.2 ml diluent (phosphate buffered saline + 0.5% bovine serum albumin) mixed and incubated with an equal volume of virus suspension. After one hour at 37°C, 0.2 ml of each serum-virus or control mixture was inoculated onto the drained cell sheet in an FB-6 well. The ratio of serum wells to control wells never exceeded two. One hour after inoculation the cell sheets were drained and washed and the plaquing procedure continued as described above. After plaque formation, endpoints were estimated graphically as described by Russel et al (6).
RESULTS 1) PLAQUE FORMATION BY DIFFERENT ISOLATES
Plaquing with several different isolates, including some with histories of laboratory passage and some recent field isolates, was attempted. All isolates had been identified by their cytopathic effect in thyroid cells and positive fluorescent antibody reaction. All TGEV plaques were neutralizable with known TGEV antiserum. One hemagglutinating encephalomyelitis virus (HEV) (4) was used as well. All produced plaques but the size varied considerably (from approximately 1 to 5 mm). The isolates and their histories are listed in Table I. While some isolates produced
Can. J. comp. Med.
all large and some all small plaques, in- control pig serum (taken from a pig foltermediate sizes were observed and the lowing multiple inoculation with TGEV, size varied somewhat from one assay to Purdue isolate) for 30 minutes at 370C. another. There was a tendency for more These were plaqued with appropriate conrecently isolated field viruses to produce trols. The serum reduced plaque numbers smaller plaques. HEV produced uniformly by more than 90% with the exception of large plaques (>4 mm) by four days. Ex- HEV, which seemed not to be neutralized amples of large plaques (TGEV-Ames and at all (Table I). HEV), small plaques (ADRI-1) and mixed size ranges (ADRI-5) are shown 3) PLAQUE NEUTRALIZING PROFILES in Fig. 1. 2) NEUTRALIZATION OF PLAQUES Several of the TGEV isolates were incubated with a 1 :100 dilution of positive
TG E %--.A m e .lne
-
Using the PN test, antibody titers were determined on serums from several pigs infected orally during the first few days of life by a gut suspension of TGEV.
REV
f.o
T(;GEV -A.DRI-1
TGEV-ADRI-5)
Fig. 1. Example of plaque sizes produeed by various TGRV Isolates and by HEY.
Volume 40
-
April, 1976
173
P 64 72
7_,
.
.
PS1 72
-X
3-
Tb.
~h, T.,
21
o
10
's
21D
?I
X0
S
10
T,
tS
x~
;
30
VWEEKSPO)STINOCULATIOI
Fig. 2. Plaque neutralization and tube neutralization (beta) profiles of serums from TGEV exposed pigs.
Peak titers ranged from 1:1200 to 1:2800. The conventional tube neutralization test (beta system) yielded titer ranges of 1:10 to 1:160. Antibodies were demonstrable by 14 days postinfection and remained at significant levels beyond 162 days (PN test). Four profiles are shown in Fig. 2.
(beta system) tube test endpoints, a feature of obvious advantage as a research tool. The demonstration of the test on convalescent serums from a field outbreak shows its diagnostic utility. Pig kidney (PK) cells have been used for these purposes (2) but titers appear to be lower and the test takes longer to develop (six days as compared to three) (1). In the experience of the authors, the PK plaquing system was not easily reproducible. This may have been partially due to our use of the disposable plastic culture plates rather than closed culture flasks. The former lend themselves much more readily to PN procedures for practical and statistical reasons. Other workers have shown cross neutralization reactions between TGEV isolates (2, 3). In the present study, American and British isolates appeared not to be different from the Canadian isolates. The sensitivity of the PN test would lend itself to more complete examination of different TGEV isolates for small antigenic differences. HEV is obviously antigenically different from the TGEV isolates.
REFERENCES
4) FIELD SERA
To ascertain the usefulness of the PN test in diagnostic or epidemiological role, serums collected from a pig herd following a confirmed TGEV outbreak (virus isolation and serologically) were tested by PN. Thirteen of 14 serums were positive (i.e. >1:100) and ranged from 1:400 to greater than 1:5000 (Table II).
DISCUSSION Pig thyroid cell cultures have been shown to be the system of choice for growing TGEV (3, 9). The present data demonstrate their usefulness in the plaque assay system, both in the PN test as a research and diagnostic tool and as a convenient laboratory marker for recently isolated viruses. PN titers were approximately tenfold higher than conventional
174
1. BOHL, E. H., R. GUPTA, M. V. FERNANDO and L. J. SAIF. Antibody responses in serum, colostrum and milk of swine after infection or vaccination
with transmissi'ble gastroenteritis virus. Infection & Immunity 6: 289-301. 1972. 2. BOHL, E. H. Transmissible gastroenteritis. In Diseases of Swine. H. W. Dunne, Ed. Ames, Iowa: Iowa State University Press. 1970. 3. DULAC, G. C., P. BOULANGER et J. B. PHANEUF. Isolement du virus de la gastro-enterite transmissible du porc sur cultures cellulaires et comparaisons antigeniques avec deux souches ame ricaines. Can. vet. J. 16: 77-81. 1975. 4. GREIG, A. S., D. MITCHELL, A. H. CORNER, G. L. BAINNISTER, E. B. MEADS and R. J. JULIAN. A hemagglutinating virus producing encephalomyelitis in baby pigs. Can. J. comp. Med. 26: 49-56. 1962. 5. MORIN, M., L. G. MOREHOUSE, R. F. SOLORZANO and L. D. OLSON. Transmissible gastroenteritis in feeder swine: Clinical, immunofluorescence and histopathological observations. Can. J. comp. Med. 37: 239-248. 1973. 6. RUSSEL, P. K., A. NISOLAK, P. SUKHAVACHANA and S. VIVONA. A plaque reduction test for dengue virus neutralizing antibodies. J. Immun. 99: 258290. 1967. 7. THOMAS, F. C. and D. 0. TRAINER. Bluetongue virus: Some relationships among North American isolates and further comparisons with EHD virus, Can. J. comp. Med. 35: 187-191. 1971. 8. WILNER, B. I. Transmissible gastroenteritis. In A Classification of the Major Groups of Human and Other Animal Viruses. Burgess Publishing Company. 1969. 9. WITTE, K. H. and B. C. EASTERDAY. Isolation and propagation of the virus of transmissible gastroenteritis of pigs in various pig cell cultures. Arch. ges Virusforsch. 20: 327-350. 1967. 10. WOODE, G. N. Transmissible gastroenteritis of swine. Vet. Bull. 39: 239-248. 1969.
Can. J. comp. Med.
a
F. C. Thomas and G. C. Dulac*
ABSTRACT A plaquing system and plaque neutralization test in porcine thyroid cells were used to study different transmissible gastroenteritis isolates and hemagglutinating encephalomyelitis virus. Among transmissible gastroenteritis virus isolates, plaque size varied considerably and mixed size ranges sometimes occurred. The most recently isolated viruses produced smaller plaques than the laboratory viruses or hemagglutinating encephalomyelitis virus. All transmissible gastroenteritis virus isolates reacted in the plaque neutralization test with a transmissible gastroenteritis virus antiserum which showed no activity against hemagglutinating encephalomyelitis virus. Plaque neutralization results both from experimentally infected pigs and following a field outbreak demonstrated the reliability of this test and its greater sensitivity than the conventional tube test.
R1SUM: Les auteurs ont titre et neutralise plusieurs souches du virus de la gastro-enterite transmissible et du virus hemagglutinant de l'encephalomyelite du porc, sur cultures de cellules thyroidiennes porcine, par la methode des plages. Les dimensions des plages varierent considerablement d'une souche a l'autre du virus de la gastro-entfrite transmissible, ainsi qu'avec certaines souches particulieres de ce virus. Les souches les plus recemment isol6es produisirent des plages plus petites que celles des souches de laboratoire ou du virus hemagglutinant de l'encephalomyelite porcine. Un imnunserum, specifique au virus de la gastro-ente-
*Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (E), Box 11300, Station H, Ottawa, Ontario K2H 8P9. Submitted July 16, 1975.
Volume 40
-
April, 1976
rite transmissible, en neutralisa les plages, mais non celles du virus hdemagglutinant de l'enc&phalomyelite porcine. La neutralisation des plages par le serum de porcs se rktablissant d'une infection experimentale ou naturelle s'avera aussi sure et plus sensible que la sero-neutralisation conventionnelle en tubes.
I NTRODUCTION Transmissible gastroenteritis v i r u s (TGEV), a coronavirus, continues to cause disease problems in neonatal pigs (2, 3). Reference has been made to different "strains" of the virus and there are apparent differences amongst isolates (3, 5, 8-10). The plaquing system and plaque neutralization (PN) test provide useful tools for cloning viral isolates, measuring their relatedness and measuring antibody responses (6, 7, 9). In the present publication some applications of these techniques to TGEV using pig thyroid cell cultures (9) are reported.
MATERIALS AND METHODS PLAQUING PROCEDURE
Monolayer cell cultures prepared from adult pig thyroids (3) were trypsinized (0.25% trypsin in PBS) and the cells seeded at approximately 3 x 104 cells per well in plastic culture dishes' (Linbro, Model FB-6-TC) using MEM (E)2 and
lLinbro Chemical Co. Ltd., New Haven, Connecticut. 2Mogul-ED, Oshkosh, Wisconsin.
171
10% fetal bovine serum (FBS). These were grown to confluency (usually 48 hours) in a humid C02 incubator and used anytime within the next five days. The inocula (0.1 or 0.2 ml per well) were allowed to absorb for one hour at 370 C and overlay applied. The latter consisted of double ionic strength MEM mixed at 45°C with an equal volume of 1.6% molten agar3 (Ionagar no. 2, batch No. 337, 1973) solution. DEAE-dextran was added to a final concentration of 100 ,ujg/ml and FBS 10% v/v. The overlay (3 ml per well) was applied to the cell sheet after washing and draining and allowed to solidify at room temperature. Plates were inverted and incubated at 37°C for three or four days. Staining was done with a 1:10,000 solution of neutral red in MEM applied to the wells (1 ml per well) three to six hours before reading. Formalin fixation followed by removal of the agar one hour later yielded a permanent record. TABLE I. TGEV Antiserum Neutralizability of TGEV and HEV Isolates
Neutralizability with TGEV Hyperimmune Seruma + + + + + + + + + + + +
Origin Minnesota New York Indiana England Quebec Quebec Quebec Ontario Nova Scotia Ontario Quebec Ontario HEV-1 Ontario HEV-10 -Serum prepared against TGEV-Purdue caused greater than 90%,o reduction of p.f.u. at a 1:100 dilution Isolate
TGEV-Ames TGEV-Diamond TGEV-N.Y. TGEV-Purdue TGEV-British ADRI-1 ADRI-2 ADRI-5 ADRI-6 ADRI-10 ADRI-1I ADRI-12
PLAQUE NEUTRALIZATION TEST
Sequential serum dilutions two, four or tenfold, heat inactivated (56°C 30 minutes) were incubated in 96-well microplates with equal volumes of cloned virus suspension (put through 450 nm filter to remove viral clumps and debris) calculated to produce '30xoid Ltd., London SEI, England.
172
TABLE lI. Plaque Neutralizing Titers (to TGEV-Purdue Isolate) of Pig Sera Following a Field Outbreak of TGEV
Serum
PN titer
1........................ 3.90.................... 2.
....................
4. 5. 6. 7. 8........................ 9........................ 10 .......................
....................
....................
11........................
12 13 14
.................... .................... ....................
< 100 > 5000
9 1700 2000 5000 400 > 5000 1000 1400 400 500 1200
5000
-Mean: 2100
Range: 5000
approximately 60 plaque forming units (p.f.u.) per well. Control inocula consisted of 0.2 ml diluent (phosphate buffered saline + 0.5% bovine serum albumin) mixed and incubated with an equal volume of virus suspension. After one hour at 37°C, 0.2 ml of each serum-virus or control mixture was inoculated onto the drained cell sheet in an FB-6 well. The ratio of serum wells to control wells never exceeded two. One hour after inoculation the cell sheets were drained and washed and the plaquing procedure continued as described above. After plaque formation, endpoints were estimated graphically as described by Russel et al (6).
RESULTS 1) PLAQUE FORMATION BY DIFFERENT ISOLATES
Plaquing with several different isolates, including some with histories of laboratory passage and some recent field isolates, was attempted. All isolates had been identified by their cytopathic effect in thyroid cells and positive fluorescent antibody reaction. All TGEV plaques were neutralizable with known TGEV antiserum. One hemagglutinating encephalomyelitis virus (HEV) (4) was used as well. All produced plaques but the size varied considerably (from approximately 1 to 5 mm). The isolates and their histories are listed in Table I. While some isolates produced
Can. J. comp. Med.
all large and some all small plaques, in- control pig serum (taken from a pig foltermediate sizes were observed and the lowing multiple inoculation with TGEV, size varied somewhat from one assay to Purdue isolate) for 30 minutes at 370C. another. There was a tendency for more These were plaqued with appropriate conrecently isolated field viruses to produce trols. The serum reduced plaque numbers smaller plaques. HEV produced uniformly by more than 90% with the exception of large plaques (>4 mm) by four days. Ex- HEV, which seemed not to be neutralized amples of large plaques (TGEV-Ames and at all (Table I). HEV), small plaques (ADRI-1) and mixed size ranges (ADRI-5) are shown 3) PLAQUE NEUTRALIZING PROFILES in Fig. 1. 2) NEUTRALIZATION OF PLAQUES Several of the TGEV isolates were incubated with a 1 :100 dilution of positive
TG E %--.A m e .lne
-
Using the PN test, antibody titers were determined on serums from several pigs infected orally during the first few days of life by a gut suspension of TGEV.
REV
f.o
T(;GEV -A.DRI-1
TGEV-ADRI-5)
Fig. 1. Example of plaque sizes produeed by various TGRV Isolates and by HEY.
Volume 40
-
April, 1976
173
P 64 72
7_,
.
.
PS1 72
-X
3-
Tb.
~h, T.,
21
o
10
's
21D
?I
X0
S
10
T,
tS
x~
;
30
VWEEKSPO)STINOCULATIOI
Fig. 2. Plaque neutralization and tube neutralization (beta) profiles of serums from TGEV exposed pigs.
Peak titers ranged from 1:1200 to 1:2800. The conventional tube neutralization test (beta system) yielded titer ranges of 1:10 to 1:160. Antibodies were demonstrable by 14 days postinfection and remained at significant levels beyond 162 days (PN test). Four profiles are shown in Fig. 2.
(beta system) tube test endpoints, a feature of obvious advantage as a research tool. The demonstration of the test on convalescent serums from a field outbreak shows its diagnostic utility. Pig kidney (PK) cells have been used for these purposes (2) but titers appear to be lower and the test takes longer to develop (six days as compared to three) (1). In the experience of the authors, the PK plaquing system was not easily reproducible. This may have been partially due to our use of the disposable plastic culture plates rather than closed culture flasks. The former lend themselves much more readily to PN procedures for practical and statistical reasons. Other workers have shown cross neutralization reactions between TGEV isolates (2, 3). In the present study, American and British isolates appeared not to be different from the Canadian isolates. The sensitivity of the PN test would lend itself to more complete examination of different TGEV isolates for small antigenic differences. HEV is obviously antigenically different from the TGEV isolates.
REFERENCES
4) FIELD SERA
To ascertain the usefulness of the PN test in diagnostic or epidemiological role, serums collected from a pig herd following a confirmed TGEV outbreak (virus isolation and serologically) were tested by PN. Thirteen of 14 serums were positive (i.e. >1:100) and ranged from 1:400 to greater than 1:5000 (Table II).
DISCUSSION Pig thyroid cell cultures have been shown to be the system of choice for growing TGEV (3, 9). The present data demonstrate their usefulness in the plaque assay system, both in the PN test as a research and diagnostic tool and as a convenient laboratory marker for recently isolated viruses. PN titers were approximately tenfold higher than conventional
174
1. BOHL, E. H., R. GUPTA, M. V. FERNANDO and L. J. SAIF. Antibody responses in serum, colostrum and milk of swine after infection or vaccination
with transmissi'ble gastroenteritis virus. Infection & Immunity 6: 289-301. 1972. 2. BOHL, E. H. Transmissible gastroenteritis. In Diseases of Swine. H. W. Dunne, Ed. Ames, Iowa: Iowa State University Press. 1970. 3. DULAC, G. C., P. BOULANGER et J. B. PHANEUF. Isolement du virus de la gastro-enterite transmissible du porc sur cultures cellulaires et comparaisons antigeniques avec deux souches ame ricaines. Can. vet. J. 16: 77-81. 1975. 4. GREIG, A. S., D. MITCHELL, A. H. CORNER, G. L. BAINNISTER, E. B. MEADS and R. J. JULIAN. A hemagglutinating virus producing encephalomyelitis in baby pigs. Can. J. comp. Med. 26: 49-56. 1962. 5. MORIN, M., L. G. MOREHOUSE, R. F. SOLORZANO and L. D. OLSON. Transmissible gastroenteritis in feeder swine: Clinical, immunofluorescence and histopathological observations. Can. J. comp. Med. 37: 239-248. 1973. 6. RUSSEL, P. K., A. NISOLAK, P. SUKHAVACHANA and S. VIVONA. A plaque reduction test for dengue virus neutralizing antibodies. J. Immun. 99: 258290. 1967. 7. THOMAS, F. C. and D. 0. TRAINER. Bluetongue virus: Some relationships among North American isolates and further comparisons with EHD virus, Can. J. comp. Med. 35: 187-191. 1971. 8. WILNER, B. I. Transmissible gastroenteritis. In A Classification of the Major Groups of Human and Other Animal Viruses. Burgess Publishing Company. 1969. 9. WITTE, K. H. and B. C. EASTERDAY. Isolation and propagation of the virus of transmissible gastroenteritis of pigs in various pig cell cultures. Arch. ges Virusforsch. 20: 327-350. 1967. 10. WOODE, G. N. Transmissible gastroenteritis of swine. Vet. Bull. 39: 239-248. 1969.
Can. J. comp. Med.
