CLINICAL RESEARCH NOTE PRECIPITINS IN BOVINE SERA TO INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS ANTIGENS C. LE Q. DARCEL*
Precipitins to antigens present in the virions of the virus of infectious bovine rhinotracheitis of cattle (IBR virus) occur in sera of naturally infected cattle (1), but the antigen used for this study is very complex, containing all the virus proteins (at least 26, P. P. Pastoret, Faculty of Veterinary Medicine, Liege, Belgium, personal communication) and also proteins from the tissue culture system used to grow the virus. Of the several virus proteins, the envelope proteins situated on the outside of the virus particle can be expected to be the most likely to be involved in the early immune response of cattle to this virus. Studies of these envelope proteins are described in the present paper. From other work (4) it was expected that the envelope antigens of the IBR virus would have a specific gravity of 1.09 in sucrose density gradients. In fact, at this level in such gradients of ultracentrifuged concentrates of IBR virus-infected tissue cultures, a band occurs which when it is not visible to the eye can still be detected by spectrophotometry at 280 nm. Fluorocarbon treated material from this zone, provisionally termed "envelope" (Ev) antigens, has been used in the tests to be described. The antigens have been obtained from sequential passages of cells from different fetuses infected by either the Colorado strain of IBR virus (strain #34) or strain #108 (3). The infected tissue cultures were harvested when maximum cytopathic effects were visible. The harvested material was frozen at -65°C, thawed, inactivated (checked by passage of an aliquot in tissue culture) by heating in a water bath to 560C for 30 minutes, subjected to two more cycles of freezing and thawing and then centrifuged at low speed, 830 x g for 10 minutes, to remove cellular debris. The supernatants were concen'Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta, Canada TIJ 3Z4.
trated with polyethylene glycol (PEG)1 approximately 50 fold, to 2 ml. This was layered over 3 ml 0.05M, pH 7.4 Tris buffer containing sucrose to give a 0-60% sucrose gradient and centrifuged at 6 x 104 x g for 22 hours. When the tubes were taken from the centrifuge they were marked against a scale so that the contents (12 ml) could be removed from the top in 1 ml aliquots. A drop from each aliquot was taken for determination of refractive index2 from which the density was calculated. The fraction corresponding to a density of 1.08-1.09 was collected and shaken with an equal amount of fluorocarbon.3 The aqueous supernatant was concentrated twofold with PEG for serological studies. Control samples were obtained from uninfected tissue culture cells in each case. In addition a "top" fraction, i.e. material lighter than density 1.08-1.09 was collected from each tube. These Freon extracted concentrates were first tested against 80 bovine sera, of which 40 were positive and 40 were negative for IBR virus neutralizing (SVN) antibodies (3). These serum samples were obtained from cattle raised at ADRI(W) and from samples submitted for diagnostic serology. For further studies of the immunodiffusion reaction between bovine sera and Ev antigens, blood was collected from 14 animals in the ADRI(W) herd (#1-#14) with SVN antibodies and from five (#16-#21) without detectable SN titres. Sera were also collected from four bulls infected with the two laboratory strains of IBR virus. Two bulls were inoculated with 109 TCID3,, of strain #34 (sera #21 and #22) intramuscularly and the other two bulls (sera #23 and #24) were given the same dose of strain #108. Booster 1Polyethylene glycol M.W. 15-20,000, MC/B, North American Scientific Chemical Ltd., Calgary,
Alberta. 2Determined with a hand refractometer (Bellingham & Stanley, London, England). 3Freon 113, Dupont, Maitland, Ontario.
'59 CAN. VET. JOUR., vol. 18, no. 9, September, 1977
CANADIAN VETERINARY JOURNAL
d
e
FIGuRE 1. Gel-immunodiffusion tests of cattle sera against Ev antigens prepared from virus infected bovine fetal kidney tissue cultures: la. Ev antigens from cultures infected with virus strain #108 antigen in centre well, with alternation in the outer wells of sera obtained from cattle infected with strain #108 (21) and with strain #34 (23). lb. Ibid., with Ev antigen #34 in centre well. Ic. Sera #21 in centre well, Ev antigens #108E and #34E and their controls #108C and #34C, prepared from uninfected cells obtained from the same fetus, in the outer wells. Only antigens from the infected cultures give the reaction. ld. Sera #2, from naturally infected cow, in centre well, "top" fractions #108T and #34T as well as Ev antigens #108E and #34E in the outer wells. Shows similarity of antigen in "top" and Ev fractions. le. "Top" antigen #108T in the centre well, sera from naturally infected cattle #1-8 in outer wells. Shows lack of identity and doubling of precipitin arcs with certain sera.
injections of the same dose of the two viruses were given three weeks later and the serum samples were taken at three and six weeks postinfection. The 1% agarose gels used for immunodif-
fusion (GID) (2) were made with 0.015M veronal buffer4 containing 0.1% merthiolate. The serum and virus extracts were placed in wells punched in these gels covering the bottom of plastic Petri dishes or the surface of glass slides. The gels were left in a humidified chamber for 36-60 hours at 37°C and were examined at intervals during this time for the appearance of precipitin arcs. The Ev antigens reacted in GID test with bovine sera containing SVN antibodies (Figure la, b). Some lots of antigen produced by the sequential passages required further concentration to produce the expected positive reaction. Forty of the 80 sera tested were positive to the SVN and GID tests and the other 40 were negative to both tests. Only antigens from infected cultures gave these reactions (Figure lc). When antigens from these two strains were alternated around a central well containing positive serum a continuous ring was obtained with no spurs or cross-overs (Figure ld). This suggests that there is at least one major antigen present and that it is identical in the two virus strains. Figure ld also shows that this major protein present in the "top zone" is the same as that in the S.G. 1.08-1.09 band. Figure le shows that more than one precipitin arc may occur with different antisera when reacting with the "top" fraction. Contamination of the cell culture with another virus is unlikely to be responsible for the positive GID reactions, since antigens extracted from control cell cultures not infected with IBR virus gave negative reactions. As a further check the Freon extracted antigens were examined by electron microscopy using a Hitachi H4-12A microscope. One ml of the antigen was diluted to 10 ml with water, centrifuged at 60,000 x g for 90 minutes, the pellet taken up in 0.5 ml distilled water and the resulting suspension clarified by low speed (230 x g) centrifugation for one minute. A drop of the clarified material was placed on a grid which was then negatively stained with pH 7.2, 2% sodium phosphotungstate (including a trace of Fraction V bovine serum albumin). Occasional damaged herpesvirus particles as well as cellular membranes and viral envelopes could be seen among the mass of protein precipitates present (Figure 2). No other virus particles could be seen, although one field showed "non-virus" particles (Figure 2c) of the type seen in vesicular fluid from lesions of human patients with herpes (5).
4B-2 buffer, Spinco Division of Beckman Instruments, Palo Alto, California. 260
PRECIPITINS
a
FIGURE 2. Electron microscopy of #108 Ev antigen, bar is equivalent to 100 nm: 2a. Amorphous proteinaceous material. 2b. Membranous material probably coming from both the host cell membranes as well envelopes. 2c. Empty capsids with their envelopes as well as "non-virus" particles (arrows). 2d. Empty capsids with amorphous proteinaceous material.
The Ev antigen is obviously not a pure preparation and the source of this virus antigen remains to be determined. This study confirms the observation of Charton et al. (1) that IBR infected cattle carry precipitins in their sera to IBR virus antigens and that these are demonstrable by GID techniques. There was, in fact, complete agreement between results obtained with the GID test and SVN test. Further, it has been shown that at least one of the viral antigens
reacting with bovine
sera is present in the density gradient fraction, with a specific gravity that would be anticipated if the antigen was derived from the virus envelopes, and this antigen appears to be identical in the two strains of virus tested. Additional antigen-antibody precipitin arcs are sometimes demonstrable in certain bovine sera. These reactions appear to be related to antigenic material present in the "top"
(
Precipitins to antigens present in the virions of the virus of infectious bovine rhinotracheitis of cattle (IBR virus) occur in sera of naturally infected cattle (1), but the antigen used for this study is very complex, containing all the virus proteins (at least 26, P. P. Pastoret, Faculty of Veterinary Medicine, Liege, Belgium, personal communication) and also proteins from the tissue culture system used to grow the virus. Of the several virus proteins, the envelope proteins situated on the outside of the virus particle can be expected to be the most likely to be involved in the early immune response of cattle to this virus. Studies of these envelope proteins are described in the present paper. From other work (4) it was expected that the envelope antigens of the IBR virus would have a specific gravity of 1.09 in sucrose density gradients. In fact, at this level in such gradients of ultracentrifuged concentrates of IBR virus-infected tissue cultures, a band occurs which when it is not visible to the eye can still be detected by spectrophotometry at 280 nm. Fluorocarbon treated material from this zone, provisionally termed "envelope" (Ev) antigens, has been used in the tests to be described. The antigens have been obtained from sequential passages of cells from different fetuses infected by either the Colorado strain of IBR virus (strain #34) or strain #108 (3). The infected tissue cultures were harvested when maximum cytopathic effects were visible. The harvested material was frozen at -65°C, thawed, inactivated (checked by passage of an aliquot in tissue culture) by heating in a water bath to 560C for 30 minutes, subjected to two more cycles of freezing and thawing and then centrifuged at low speed, 830 x g for 10 minutes, to remove cellular debris. The supernatants were concen'Animal Pathology Division, Health of Animals Branch, Agriculture Canada, Animal Diseases Research Institute (Western), P.O. Box 640, Lethbridge, Alberta, Canada TIJ 3Z4.
trated with polyethylene glycol (PEG)1 approximately 50 fold, to 2 ml. This was layered over 3 ml 0.05M, pH 7.4 Tris buffer containing sucrose to give a 0-60% sucrose gradient and centrifuged at 6 x 104 x g for 22 hours. When the tubes were taken from the centrifuge they were marked against a scale so that the contents (12 ml) could be removed from the top in 1 ml aliquots. A drop from each aliquot was taken for determination of refractive index2 from which the density was calculated. The fraction corresponding to a density of 1.08-1.09 was collected and shaken with an equal amount of fluorocarbon.3 The aqueous supernatant was concentrated twofold with PEG for serological studies. Control samples were obtained from uninfected tissue culture cells in each case. In addition a "top" fraction, i.e. material lighter than density 1.08-1.09 was collected from each tube. These Freon extracted concentrates were first tested against 80 bovine sera, of which 40 were positive and 40 were negative for IBR virus neutralizing (SVN) antibodies (3). These serum samples were obtained from cattle raised at ADRI(W) and from samples submitted for diagnostic serology. For further studies of the immunodiffusion reaction between bovine sera and Ev antigens, blood was collected from 14 animals in the ADRI(W) herd (#1-#14) with SVN antibodies and from five (#16-#21) without detectable SN titres. Sera were also collected from four bulls infected with the two laboratory strains of IBR virus. Two bulls were inoculated with 109 TCID3,, of strain #34 (sera #21 and #22) intramuscularly and the other two bulls (sera #23 and #24) were given the same dose of strain #108. Booster 1Polyethylene glycol M.W. 15-20,000, MC/B, North American Scientific Chemical Ltd., Calgary,
Alberta. 2Determined with a hand refractometer (Bellingham & Stanley, London, England). 3Freon 113, Dupont, Maitland, Ontario.
'59 CAN. VET. JOUR., vol. 18, no. 9, September, 1977
CANADIAN VETERINARY JOURNAL
d
e
FIGuRE 1. Gel-immunodiffusion tests of cattle sera against Ev antigens prepared from virus infected bovine fetal kidney tissue cultures: la. Ev antigens from cultures infected with virus strain #108 antigen in centre well, with alternation in the outer wells of sera obtained from cattle infected with strain #108 (21) and with strain #34 (23). lb. Ibid., with Ev antigen #34 in centre well. Ic. Sera #21 in centre well, Ev antigens #108E and #34E and their controls #108C and #34C, prepared from uninfected cells obtained from the same fetus, in the outer wells. Only antigens from the infected cultures give the reaction. ld. Sera #2, from naturally infected cow, in centre well, "top" fractions #108T and #34T as well as Ev antigens #108E and #34E in the outer wells. Shows similarity of antigen in "top" and Ev fractions. le. "Top" antigen #108T in the centre well, sera from naturally infected cattle #1-8 in outer wells. Shows lack of identity and doubling of precipitin arcs with certain sera.
injections of the same dose of the two viruses were given three weeks later and the serum samples were taken at three and six weeks postinfection. The 1% agarose gels used for immunodif-
fusion (GID) (2) were made with 0.015M veronal buffer4 containing 0.1% merthiolate. The serum and virus extracts were placed in wells punched in these gels covering the bottom of plastic Petri dishes or the surface of glass slides. The gels were left in a humidified chamber for 36-60 hours at 37°C and were examined at intervals during this time for the appearance of precipitin arcs. The Ev antigens reacted in GID test with bovine sera containing SVN antibodies (Figure la, b). Some lots of antigen produced by the sequential passages required further concentration to produce the expected positive reaction. Forty of the 80 sera tested were positive to the SVN and GID tests and the other 40 were negative to both tests. Only antigens from infected cultures gave these reactions (Figure lc). When antigens from these two strains were alternated around a central well containing positive serum a continuous ring was obtained with no spurs or cross-overs (Figure ld). This suggests that there is at least one major antigen present and that it is identical in the two virus strains. Figure ld also shows that this major protein present in the "top zone" is the same as that in the S.G. 1.08-1.09 band. Figure le shows that more than one precipitin arc may occur with different antisera when reacting with the "top" fraction. Contamination of the cell culture with another virus is unlikely to be responsible for the positive GID reactions, since antigens extracted from control cell cultures not infected with IBR virus gave negative reactions. As a further check the Freon extracted antigens were examined by electron microscopy using a Hitachi H4-12A microscope. One ml of the antigen was diluted to 10 ml with water, centrifuged at 60,000 x g for 90 minutes, the pellet taken up in 0.5 ml distilled water and the resulting suspension clarified by low speed (230 x g) centrifugation for one minute. A drop of the clarified material was placed on a grid which was then negatively stained with pH 7.2, 2% sodium phosphotungstate (including a trace of Fraction V bovine serum albumin). Occasional damaged herpesvirus particles as well as cellular membranes and viral envelopes could be seen among the mass of protein precipitates present (Figure 2). No other virus particles could be seen, although one field showed "non-virus" particles (Figure 2c) of the type seen in vesicular fluid from lesions of human patients with herpes (5).
4B-2 buffer, Spinco Division of Beckman Instruments, Palo Alto, California. 260
PRECIPITINS
a
FIGURE 2. Electron microscopy of #108 Ev antigen, bar is equivalent to 100 nm: 2a. Amorphous proteinaceous material. 2b. Membranous material probably coming from both the host cell membranes as well envelopes. 2c. Empty capsids with their envelopes as well as "non-virus" particles (arrows). 2d. Empty capsids with amorphous proteinaceous material.
The Ev antigen is obviously not a pure preparation and the source of this virus antigen remains to be determined. This study confirms the observation of Charton et al. (1) that IBR infected cattle carry precipitins in their sera to IBR virus antigens and that these are demonstrable by GID techniques. There was, in fact, complete agreement between results obtained with the GID test and SVN test. Further, it has been shown that at least one of the viral antigens
reacting with bovine
sera is present in the density gradient fraction, with a specific gravity that would be anticipated if the antigen was derived from the virus envelopes, and this antigen appears to be identical in the two strains of virus tested. Additional antigen-antibody precipitin arcs are sometimes demonstrable in certain bovine sera. These reactions appear to be related to antigenic material present in the "top"
(
