Vol. 1, No. 2
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 212-218 Copyright © 1975 American Societv for Microbiology
Printed in U.S.A.
Methods for the Detection of Viruses in Bovine Serum N. S. SWACK, C. K. Y. FONG, G. D. HSIUNG,* AND P. A. GROSS Department of Laboratory Medicine and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, and Veterans Administration Hospital, West Haven, Connecticut 06516*
Received for publication 18 November 1974
An evaluation of selected commonly used procedures for the recovery of endogenous viral contaminants in bovine serum was undertaken. Low speed centrifugation (25,000 x g) was found to be efficient for the recovery of bovine herpesvirus type 1 (BHV-1) and parainfluenza virus type 3 (PI-3) in bovine serum. Decreased infectivity titers were obtained when parainfluenza virus type 3, and to a lesser extent bovine herpesvirus type 1, were concentrated using high speed centrifugation (100,000 x g) for extended time periods. In neither case could infectious virus be recovered from serum containing sufficient titers of homologous neutralizing antibody, although electron microscopy examination revealed the presence of the viruses previously added. In the presence of homologous antibody, virus particles appeared to have a diffuse, poorly defined outer membrane. Neutralizing antibody titers to bovine herpesvirus type 1 and parainfluenza virus types were found in fetal, calf, and adult bovine sera. The prevalence and magnitude of the antibody titers to these viruses increased with the age of the animals examined.
The incorporation of bovine serum as a nutritive supplement into most tissue or cell culture media has resulted in extensive investigation into the prevalence of endogenous bovine viruses. Fetal bovine serum has been implicated in the appearance of bovine virus diarrhea virus in cell cultures previously known to be virus free (4). Bovine herpesvirus type 1 in a concentration of 150 mean tissue culture infective doses (TCID50)/ml has been recovered as an endogenous contaminant in fetal bovine serum by Molander et al. (6). These investigators also isolated bovine virus diarrhea virus from bovine serum collected by cardiac puncture. Recently, a number of investigators (1, 5) have isolated bacteriophage from many lots of bovine serum. The present report is concerned with the evaluation of some of the commonly used procedures for the detection of viruses in bovine serum, together with studies of some of the conditions which might influence virus recovery. MATERIALS AND METHODS Viruses and cell cultures. The viruses used in this study were bovine herpesvirus type 1 (BHV-1), the bovine strain of parainfluenza virus type 3 (PI-3), and the simian virus strain of parainfluenza virus type 5 (PI-5). The BHV-1 and PI-3 viruses were obtained from the American Type Culture Collection and PI-5 was isolated in our laboratories from a primary rhesus monkey kidney cell culture. Primary bovine embry-
onic kidney cells were used for the propagation of BHV-1 and primary monkey kidney cell cultures were used for PI-3 and PI-5. The growth of the latter two viruses was determined by the hemadsorption of
guinea pig erythrocytes onto infected cell culture monolayers, whereas observation of cultures for cytopathic effect was used to determine the infectivity of BHV-1 virus. Source of serum samples for antibody studies. Selected samples consisting of pooled fetal bovine or calf sera were purchased commercially. Single fetal bovine serum samples were obtained from commercial sources and calf and adult sera were drawn from individual animals in Rhode Island. All sera were inactivated at 56 C for 30 min and tested for the presence of neutralizing antibody to selected viruses, including BHV-1, PI-3 and PI-5. Antibody titers were determined by adding 100 TCID5O of virus to each serum dilution, allowing the virus-serum mixture to stand at room temperature for 30 min followed by inoculation onto susceptible cell cultures. The highest dilution of serum which completely inhibited the test dose of virus was considered to be the end point. Sedimentation of virus in serum by centrifugation. A variety of ultracentrifugation speeds and times were employed to evaluate the optimal conditions for sedimentation of known virus previously added to calf serum or fetal bovine serum. Clarification of' serum was done using a GSA rotor at 5,000 rpm for 20 min in a RC-2 Sorvall ultracentrifuge. After this, virus sedimentation was accomplished using a SS-34 rotor at 14,500 rpm (25,000 x g) in the RC-2 ultracentrifuge or a SW27 rotor at 27,000 rpm (100,000 x g) in a Beckman L2-65 ultracentrifuge. All sedimentation procedures were conducted at 4 C.
212
VOL. 1, 1975
213
DETECTION OF VIRUSES IN BOVINE SERUM
The specific time and speed used in each experiment indicated in the results. In certain experiments, pellets obtained by high speed centrifugation were resuspended by brief sonic treatment. Recovery of known virus added to bovine serum. Various concentrations of BHV-1 or PI-3 viruses were added to bovine serum, and their recovery was determined under different conditions. In general, the virus suspension was added to a 100-ml serum sample from which 1 ml was removed for virus assay, and the remainder was divided into 33-ml aliquots and subjected to centrifugation. After careful removal of the supernatant from each tube, the pooled pellets in a volume of approximately 1 ml were considered to represent a 100 times concentration of the added virus as compared to the virus titers in serum before centrifugation. Virus recovery from serum with or without homologous neutralizing antibody was evaluated. Infectivity titrations and examination of specimens in the electron microscope were employed to compare these procedures fordetecting each virus. Detection of virus in bovine sera by virus isolation and by electron microscopy. Virus isolation studies on serum samples were performed as follows: before centrifugation 0.1 ml was inoculated into 10 to 15 culture tubes each of bovine embryonic kidney, monkey kidney (green or rhesus monkey), and rabbit kidney. Pooled serum samples were further tested by inoculating the same primary cell cultures with 0.1 ml of the 100 times concentrated pellets obtained by centrifugation as mentioned above. All inoculated cultures were kept for observation for 30 days without subculturing. At the end of the incubation period the monkey kidney cell cultures were tested for hemadsorption, and bovine embryonic kidney and rabbit kidney cell cultures were fixed and stained for microscopy examination for virus-induced inclusions. Methods for the preparation of serum samples for electron microscopy have been described in detail in the accompanying paper (2). Briefly, clarified sera were pelleted in a Beckman L2-65 ultracentrifuge at 27,000 rpm in SW27 rotor for 90 min. The pellets were fixed with 2% glutaraldehyde and then centrifuged directly into Beem capsules at 30,000 rpm in a SW65 rotor for 60 min. The pellets in Beem capsules were postfixed with osmium tetroxide and embedded in Epon 812. Thin sections were stained with uranyl are
acetate and lead citrate and
were
examined with
a
bly higher than that obtained at 25,000 x g. In those instances where the pellet of BHV-1 obtained from high speed centrifugation was subjected to a brief period of sonic treatment, the resultant virus titers were somewhat higher than those from the untreated sample suggesting some aggregation of virus. No significant amount of infectious virus was obtained from the supernatants. When PI-3 was used in these experiments, titers were observed to be reduced significantly during the prolonged period of high speed centrifugation. This decrease appeared to progress as the time of treatment was increased. The cause of this decrease has not been determined. When smaller amounts of BHV-1 were added to bovine serum samples at concentrations most likely to be encountered in contaminated bovine serum, virus recovery appeared to be almost complete when centrifuged at 100,000 x g for 90 min (Table 2). It was apparent that infectious virus was readily recovered at such high speed centrifugation even when the concentrations of virus in the original serum samples were less than 1 log10 TCID5,Jml of serum. Effect of antibody on recovery of known virus from bovine serum. The presence of homologous neutralizing antibody in the serum markedly reduced the efficiency of the procedure used to recover infectious virus added to the serum samples (Table 3). Serum with an antibody titer of 1:2 masked more than 10-fold BHV-1 as indicated by the decreased infectivity titers, and infectious virus was not recovered when the homologous antibody titer was 1:16. Similar results were obtained with PI-3 in TABLE 1. Recovery of BHV-1 and PI-3 from antibody-free bovine serum after high and low speed centrifugation Virus titers (log TCID,dml of pellet)
Centrifugation
Philips EM 300 electron microscope. RESULTS Effect of speed and time of centrifugation on the recovery of known virus from bovine serum. The effect of centrifugation upon the recovery of known virus from serum at high and
25,000 Oa 90 180 360
PI-3
BHV-1
time gravity (min)
Expt 1
Expt 2
Expt 1
Expt 2
6.4 6.8 8.2 7.4
6.3 7.1 8.0 7.2
5.5 7.0 6.2 6.5
5.5 6.5 6.7 6.0
4.8 6.0 5.9 6.0
5.6 6.1 6.2 6.9
5.4 5.9 5.2
5.5 5.7 4.7 4.2
x
100,000 x low speeds was compared. Representative ex0 periments are shown in Table 1. A sedimenta90 tion procedure using a low speed of centrifuga180 tion (25,000 x g) appeared to be an efficient 360 both BHV1 and of PI-3 method for the recovery virus. After high speed centrifugation at 100,000 aVirus titer x g, recovery of BHV-1 virus was not apprecia- tion.
4.0
in serum samples before centrifuga-
214
J. CLIN. MICROBIOL.
SWACK ET AL.
la illustrates a mixture of BHV-1 and PI-3 virus particles in a fetal bovine serum sample. Each virus type can be identified by its morphology and can be compared with each virus alone in other bovine serum samples (Fig. lb and c and 2). Complete, enveloped, typical herpesvirus particles were easily seen in the thin sections (Fig. lb and c) When PI-3 virus was added to calf serum containing specific homologous antibody, a layer of electron dense fuzzy material, presumably antibody molecules, was seen on the viral envelopes (Fig. 2a and b). These myxovirus particles appeared to be disintegrating when compared to those seen in samples prepared in the absence of neutralizing antibody (Fig. 2c and d). Attempts to isolate endogenous viruses from bovine sera and determination of neutralizing antibody. No virus was isolated in cell cultures inoculated with 21 lots of pooled fetal TABLE 2. Recovery of small concentrations of BHV-1 bovine and eight lots of pooled calf serum by high speed centrifugation obtained from commercial laboratories, and 43 serum samples from bleedings obtained from Infectivity titers of BHV-1 individual calves and 37 adult cattle. There was (log TCID,Jml) Expt no. no evidence of virus-induced cytopathic effect Pelleta Input or hemadsorption, nor were any virus-induced inclusions found in the stained preparations. 1 3.5 5.0 Subcultures and blind passages of inoculated 4.5 1.7 cultures were not done in these studies. Al0.7 2.9 though virus was not isolated from any of the 2.1
JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 1975, p. 212-218 Copyright © 1975 American Societv for Microbiology
Printed in U.S.A.
Methods for the Detection of Viruses in Bovine Serum N. S. SWACK, C. K. Y. FONG, G. D. HSIUNG,* AND P. A. GROSS Department of Laboratory Medicine and Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06510, and Veterans Administration Hospital, West Haven, Connecticut 06516*
Received for publication 18 November 1974
An evaluation of selected commonly used procedures for the recovery of endogenous viral contaminants in bovine serum was undertaken. Low speed centrifugation (25,000 x g) was found to be efficient for the recovery of bovine herpesvirus type 1 (BHV-1) and parainfluenza virus type 3 (PI-3) in bovine serum. Decreased infectivity titers were obtained when parainfluenza virus type 3, and to a lesser extent bovine herpesvirus type 1, were concentrated using high speed centrifugation (100,000 x g) for extended time periods. In neither case could infectious virus be recovered from serum containing sufficient titers of homologous neutralizing antibody, although electron microscopy examination revealed the presence of the viruses previously added. In the presence of homologous antibody, virus particles appeared to have a diffuse, poorly defined outer membrane. Neutralizing antibody titers to bovine herpesvirus type 1 and parainfluenza virus types were found in fetal, calf, and adult bovine sera. The prevalence and magnitude of the antibody titers to these viruses increased with the age of the animals examined.
The incorporation of bovine serum as a nutritive supplement into most tissue or cell culture media has resulted in extensive investigation into the prevalence of endogenous bovine viruses. Fetal bovine serum has been implicated in the appearance of bovine virus diarrhea virus in cell cultures previously known to be virus free (4). Bovine herpesvirus type 1 in a concentration of 150 mean tissue culture infective doses (TCID50)/ml has been recovered as an endogenous contaminant in fetal bovine serum by Molander et al. (6). These investigators also isolated bovine virus diarrhea virus from bovine serum collected by cardiac puncture. Recently, a number of investigators (1, 5) have isolated bacteriophage from many lots of bovine serum. The present report is concerned with the evaluation of some of the commonly used procedures for the detection of viruses in bovine serum, together with studies of some of the conditions which might influence virus recovery. MATERIALS AND METHODS Viruses and cell cultures. The viruses used in this study were bovine herpesvirus type 1 (BHV-1), the bovine strain of parainfluenza virus type 3 (PI-3), and the simian virus strain of parainfluenza virus type 5 (PI-5). The BHV-1 and PI-3 viruses were obtained from the American Type Culture Collection and PI-5 was isolated in our laboratories from a primary rhesus monkey kidney cell culture. Primary bovine embry-
onic kidney cells were used for the propagation of BHV-1 and primary monkey kidney cell cultures were used for PI-3 and PI-5. The growth of the latter two viruses was determined by the hemadsorption of
guinea pig erythrocytes onto infected cell culture monolayers, whereas observation of cultures for cytopathic effect was used to determine the infectivity of BHV-1 virus. Source of serum samples for antibody studies. Selected samples consisting of pooled fetal bovine or calf sera were purchased commercially. Single fetal bovine serum samples were obtained from commercial sources and calf and adult sera were drawn from individual animals in Rhode Island. All sera were inactivated at 56 C for 30 min and tested for the presence of neutralizing antibody to selected viruses, including BHV-1, PI-3 and PI-5. Antibody titers were determined by adding 100 TCID5O of virus to each serum dilution, allowing the virus-serum mixture to stand at room temperature for 30 min followed by inoculation onto susceptible cell cultures. The highest dilution of serum which completely inhibited the test dose of virus was considered to be the end point. Sedimentation of virus in serum by centrifugation. A variety of ultracentrifugation speeds and times were employed to evaluate the optimal conditions for sedimentation of known virus previously added to calf serum or fetal bovine serum. Clarification of' serum was done using a GSA rotor at 5,000 rpm for 20 min in a RC-2 Sorvall ultracentrifuge. After this, virus sedimentation was accomplished using a SS-34 rotor at 14,500 rpm (25,000 x g) in the RC-2 ultracentrifuge or a SW27 rotor at 27,000 rpm (100,000 x g) in a Beckman L2-65 ultracentrifuge. All sedimentation procedures were conducted at 4 C.
212
VOL. 1, 1975
213
DETECTION OF VIRUSES IN BOVINE SERUM
The specific time and speed used in each experiment indicated in the results. In certain experiments, pellets obtained by high speed centrifugation were resuspended by brief sonic treatment. Recovery of known virus added to bovine serum. Various concentrations of BHV-1 or PI-3 viruses were added to bovine serum, and their recovery was determined under different conditions. In general, the virus suspension was added to a 100-ml serum sample from which 1 ml was removed for virus assay, and the remainder was divided into 33-ml aliquots and subjected to centrifugation. After careful removal of the supernatant from each tube, the pooled pellets in a volume of approximately 1 ml were considered to represent a 100 times concentration of the added virus as compared to the virus titers in serum before centrifugation. Virus recovery from serum with or without homologous neutralizing antibody was evaluated. Infectivity titrations and examination of specimens in the electron microscope were employed to compare these procedures fordetecting each virus. Detection of virus in bovine sera by virus isolation and by electron microscopy. Virus isolation studies on serum samples were performed as follows: before centrifugation 0.1 ml was inoculated into 10 to 15 culture tubes each of bovine embryonic kidney, monkey kidney (green or rhesus monkey), and rabbit kidney. Pooled serum samples were further tested by inoculating the same primary cell cultures with 0.1 ml of the 100 times concentrated pellets obtained by centrifugation as mentioned above. All inoculated cultures were kept for observation for 30 days without subculturing. At the end of the incubation period the monkey kidney cell cultures were tested for hemadsorption, and bovine embryonic kidney and rabbit kidney cell cultures were fixed and stained for microscopy examination for virus-induced inclusions. Methods for the preparation of serum samples for electron microscopy have been described in detail in the accompanying paper (2). Briefly, clarified sera were pelleted in a Beckman L2-65 ultracentrifuge at 27,000 rpm in SW27 rotor for 90 min. The pellets were fixed with 2% glutaraldehyde and then centrifuged directly into Beem capsules at 30,000 rpm in a SW65 rotor for 60 min. The pellets in Beem capsules were postfixed with osmium tetroxide and embedded in Epon 812. Thin sections were stained with uranyl are
acetate and lead citrate and
were
examined with
a
bly higher than that obtained at 25,000 x g. In those instances where the pellet of BHV-1 obtained from high speed centrifugation was subjected to a brief period of sonic treatment, the resultant virus titers were somewhat higher than those from the untreated sample suggesting some aggregation of virus. No significant amount of infectious virus was obtained from the supernatants. When PI-3 was used in these experiments, titers were observed to be reduced significantly during the prolonged period of high speed centrifugation. This decrease appeared to progress as the time of treatment was increased. The cause of this decrease has not been determined. When smaller amounts of BHV-1 were added to bovine serum samples at concentrations most likely to be encountered in contaminated bovine serum, virus recovery appeared to be almost complete when centrifuged at 100,000 x g for 90 min (Table 2). It was apparent that infectious virus was readily recovered at such high speed centrifugation even when the concentrations of virus in the original serum samples were less than 1 log10 TCID5,Jml of serum. Effect of antibody on recovery of known virus from bovine serum. The presence of homologous neutralizing antibody in the serum markedly reduced the efficiency of the procedure used to recover infectious virus added to the serum samples (Table 3). Serum with an antibody titer of 1:2 masked more than 10-fold BHV-1 as indicated by the decreased infectivity titers, and infectious virus was not recovered when the homologous antibody titer was 1:16. Similar results were obtained with PI-3 in TABLE 1. Recovery of BHV-1 and PI-3 from antibody-free bovine serum after high and low speed centrifugation Virus titers (log TCID,dml of pellet)
Centrifugation
Philips EM 300 electron microscope. RESULTS Effect of speed and time of centrifugation on the recovery of known virus from bovine serum. The effect of centrifugation upon the recovery of known virus from serum at high and
25,000 Oa 90 180 360
PI-3
BHV-1
time gravity (min)
Expt 1
Expt 2
Expt 1
Expt 2
6.4 6.8 8.2 7.4
6.3 7.1 8.0 7.2
5.5 7.0 6.2 6.5
5.5 6.5 6.7 6.0
4.8 6.0 5.9 6.0
5.6 6.1 6.2 6.9
5.4 5.9 5.2
5.5 5.7 4.7 4.2
x
100,000 x low speeds was compared. Representative ex0 periments are shown in Table 1. A sedimenta90 tion procedure using a low speed of centrifuga180 tion (25,000 x g) appeared to be an efficient 360 both BHV1 and of PI-3 method for the recovery virus. After high speed centrifugation at 100,000 aVirus titer x g, recovery of BHV-1 virus was not apprecia- tion.
4.0
in serum samples before centrifuga-
214
J. CLIN. MICROBIOL.
SWACK ET AL.
la illustrates a mixture of BHV-1 and PI-3 virus particles in a fetal bovine serum sample. Each virus type can be identified by its morphology and can be compared with each virus alone in other bovine serum samples (Fig. lb and c and 2). Complete, enveloped, typical herpesvirus particles were easily seen in the thin sections (Fig. lb and c) When PI-3 virus was added to calf serum containing specific homologous antibody, a layer of electron dense fuzzy material, presumably antibody molecules, was seen on the viral envelopes (Fig. 2a and b). These myxovirus particles appeared to be disintegrating when compared to those seen in samples prepared in the absence of neutralizing antibody (Fig. 2c and d). Attempts to isolate endogenous viruses from bovine sera and determination of neutralizing antibody. No virus was isolated in cell cultures inoculated with 21 lots of pooled fetal TABLE 2. Recovery of small concentrations of BHV-1 bovine and eight lots of pooled calf serum by high speed centrifugation obtained from commercial laboratories, and 43 serum samples from bleedings obtained from Infectivity titers of BHV-1 individual calves and 37 adult cattle. There was (log TCID,Jml) Expt no. no evidence of virus-induced cytopathic effect Pelleta Input or hemadsorption, nor were any virus-induced inclusions found in the stained preparations. 1 3.5 5.0 Subcultures and blind passages of inoculated 4.5 1.7 cultures were not done in these studies. Al0.7 2.9 though virus was not isolated from any of the 2.1
