INFECON AND IMMUNMTY, Sept. 1976, p. 660-666 Copyright © 1976 American Society for Microbiology
Vol. 14, No. 3 Printed in U.S.A.
Fetal and Adult Bovine Interferon Production During Bovine Viral Diarrhea Virus Infection C. R. RINALDO, JR.,'* D. W. ISACKSON, J. C. OVERALL, JR., L. A. GLASGOW, T. T. BROWN,2 S. I. BISTNER, J. H. GILLESPIE, AND F. W. SCOTT Departments ofPediatrics and Microbiology, University of Utah College of Medicine, Salt Lake City, Utah 84132*; and Departments of Microbiology, Pathology, and Large Animal Medicine, Obstetrics and Surgery, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Received for publication 15 April 1976
Levels of interferon in adult bovine serum and in fetal bovine serum and tissues were examined during the course of transplacental bovine viral diarrhea virus infection. The cows produced circulating interferon between 2 and 9 days after viral inoculation, with mean peak levels in the serum on day 4. Interferon could be routinely detected in fetal tissues (e.g., thymus, spleen, and kidney) between days 4 and 21 after viral inoculation of the cows at 149 to 150 days of gestation (mid-second trimester) and in fetal serum from day 13 through day 21. Interferon was also detectable in the serum and tissues of fetuses from dams infected at day 95 of gestation (the beginning of the second trimester). In general, no differences were found between the ability of the adult and fetus to produce interferon. Fetal lamb kidney cells were more sensitive to the antiviral effects of bovine interferon than were fetal bovine kidney cells. The antiviral substance from the fetal and adult animals was characterized as interferon by standard criteria. There are many examples both in man and experimental animals of enhanced susceptibility of the fetus to viral infection (3, 9, 30, 34). Congenital rubella virus and cytomegalovirus infections are representative of such diseases of the human fetus. It has been well documented that infection of the fetus with rubella virus or cytomegalovirus during pregnancy can result in severe congenital defects of the central nervous system (CNS), eye, ear, heart, and skeleton (14, 18). In contrast, infection of children or adults with either ofthese viruses usually leads to a relatively mild or asymptomatic illness
(25).
Bluetongue virus (21), hog cholera virus (15), and bovine viral diarrhea (BVD) virus (8, 23, 49) are examples of agents which may cause severe disease in the fetuses of domestic animals. In the cow, which has a 9-month gestation period, infection with BVD virus during the first trimester of pregnancy can result in fetal mummification, stillbirth, or abortion (4, 8, 24). Cerebellar and ocular lesions may develop in the fetuses of cows infected with BVD virus during the second trimester of gestation ' Present address: Harvard Medical School, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114. 2 Present address: Department of Pathology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74074. 660
(4-6, 41). The BVD virus has little or no effect on the fetuses of cows infected during the last trimester of pregnancy (8, 24). The precise mechanisms involved in the agerelated resistance of the fetus to viral infection have not been well defined. Several investigations have suggested that the inability of the fetal and newborn animal to control viral infection may be related to an inadequate interferon response (1, 2, 13, 19, 22, 27, 32, 40). In contrast to these results, other studies have shown that the capacity to produce interferon is not reduced in the immature host (10, 11, 31, 36, 44, 48). Studies from this laboratory have shown that the fetal lamb is as competent as the adult sheep to produce interferon in response to viral infection either after direct inoculation in utero (33, 35) or in cell cultures derived from fetuses of various gestational ages (38). To more fully define the role of interferon in age-related host resistance, the present studies were undertaken to evaluate interferon production in the pregnant heifer and in the bovine fetus after transplacental infection with BVD virus between 65 and 150 days of gestation. MATERIALS AND METHODS Cells and media. Fetal bovine kidney (FBK) cells were prepared from kidneys removed aseptically from bovine fetuses obtained from the Midvale Packing Co., Midvale, Utah. The renal capsule was
VOL. 14, 1976
INTERFERON IN BVD VIRUS INFECTION
661
removed, and the cortical and medullary regions RESULTS were minced and washed with phosphate-buffered Interferon production in the adult bovine. saline. Cells were extracted in a 0.25% (wt/vol) trypsin sodium citrate-potassium chloride solution The levels of circulating interferon in nine susfor 1 h, centrifuged, and resuspended in Eagle mini- ceptible pregnant heifers during the course of mum essential medium consisting of Earle bal- BVD virus infection (phase II studies) are preanced salt solution supplemented with minimum es- sented in Fig. 1. As assayed in FBK cells, intersential medium vitamins and amino acids (Grand feron was detectable from day 3 through day 6 Island Biological Co., Grand Island, N.Y.), 1% (volV postviral inoculation and reached mean peak vol) of a 300 ,ug/liter solution of glutamine, 100 U of titers penicillin per ml, 100 ,ug of streptomycin per ml, 10% titers of 36 U/ml on day 4. Interferon (vol/vol) fetal calf serum (Grand Island Biological), ranged from undetectable levels to 100 U/ml in and NaHCO3 as a buffer. The FBK cells were main- the separate heifers. The serum interferon retained in 32-oz glass prescription bottles (Brockway sponse of the heifers used in phase I and III Glass Co., Brockway, Pa.) and used in the second to experiments was equivalent to that shown for fifth passage. Procedures have previously been de- phase II. scribed (37) for the culturing of fetal lamb kidney During the course of the phase II studies, it (FLK) and mouse L cells. was discovered that FLK cells were much more Viruses. The cytopathogenic Holmes strain of sensitive to the antiviral effects of bovine interBVD virus used in these experiments was originally feron than were FBK cells. Therefore, phase II isolated from the maternal and fetal tissues of a III and samples were assayed for interferon pregnant cow that died of natural infection (42). The stock virus was prepared and assayed in fetal bovine activity in both bovine (FBK) and sheep (FLK) spleen cells, and the median tissue culture infective cells. The titers of interferon in the serum ofthe dose was calculated by the Karber method (28). The nine phase II BVD virus-infected heifers were viral pools used in these experiments had a titer of consistently several-fold higher in the sheep approximately 104 median tissue culture infective than in the bovine cells (Fig. 1). The mean peak doses per 0.1 ml. A detailed description has been titer in FLK cells was 590 U/ml, approximately reported (37) for the preparation and assay ofvesicu- 16-fold higher than in the FBK cells. The peak lar stomatitis virus (VSV) and Sindbis virus pools. titers reached levels greater than 1,000 U/ml in Experimental animals and procedures. The study was divided into three phases. Pregnant Holstein- several animals. Although the kinetics of the Friesian heifers with known breeding dates were 10,000 housed in an isolated barn in Ithaca, N.Y. There were 11 heifers used in phase I of the experiment, 10 A INTERFERON ASSAYED ON heifers in phase II, and 6 heifers in phase III. All of FLK CELLS the heifers, with the exception of one heifer in phase 0 INTERFERON II, did not have serum-neutralizing antibody titers ASSAYED ON against BVD virus. The animal with neutralizing FBK CELLS antibody was used as a control. The heifers were serologically surveyed each month to ensure that the animals remained susceptible to the virus. Be1,000 fore inoculation with virus, the pregnant heifers were moved into environmentally controlled individual isolation units. Heifers in phases I and II of the study were intravenously inoculated with 250,000 median tissue culture infective doses of the Holmes strain of BVD virus at either 149 or 150 days of gestation. Heifers in phase III of the study were T inoculated similarly with the same amount of the virus at either 65, 75, 85, 95, or 115 days ofgestation. At sequential times after inoculation of the heifers with BVD virus, the fetuses were surgically removed by cesarean section from their dams. Immediately thereafter they were exsanguinated, and fetal serum and tissues were stored at -70°C for interferon assay. Interferon assay. The fetal and adult bovine samples were transferred in dry ice by air freight to the University of Utah College of Medicine. The sam9 10 7 8 O 2 3 4 5 6 1 ples were assayed by the 50% plaque reduction techDAYS POST- INOCULATION nique in FBK and FLK cells using VSV as the FIG. 1. Mean levels and range of interferon titers challenge virus (37). An internal laboratory reference bovine interferon was included with each as- induced in the sera of nine dams inoculated intravesay; titers varied from two- to threefold between nously with BVD virus and assayed in FLK and in FBK cells. different assays on FLK cells.
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interferon response followed a similar pattern with both assay systems, the enhanced sensitivity of the FLK cells allowed detection of serum interferon from day 2 through day 9. In comparing the sensitivity of the sheep and bovine cells, an interferon standard kindly donated by J. D. Todd (Jensen-Salsbery Labs, Kansas City, Mo.) was assayed in both cell lines. The standard consisted of nasal secretions from calves which had been inoculated with infectious bovine rhinotracheitis virus and was known to contain antiviral activity which had been characterized as interferon (46). The bovine interferon sample titered 200 U/ml in FBK cells and 715 U/ml in FLK cells, i.e., nearly a fourfold increase in the sheep cells. Interferon production in the bovine fetus: phase I studies. Data on the production of interferon in the bovine fetus after inoculation of their dams with BVD virus during the phase I studies are presented in Table 1. Interferon was present in fetal serum from 13 to 21 days after inoculation of the cows with virus and ranged from 4 to 70 U/ml as assayed in FBK cells. Low levels of interfering activity were also noted in the serum of fetuses taken at 27 and 56 days postviral inoculation. Interferon was detected in fetal tissues between 4 and 21 days after viral infection of the dams with highest levels consistently present in the liver, thymus, and kidney. Interferon was found irregularly and at lower levels in spleen, lung, mesenteric lymph node, and cotyledon. One possible explanation for the low or undetectable levels of interferon in fetuses removed from 21 to 70 days was that they were not infected with BVD virus. However, these fetuses were shown to be infected as evidenced by the presence of elevated levels of serum immunoglobulins M and G1 (T. T. Brown et al., unpublished observations), and cerebellar and ocular lesions typical of BVD virus infection (5,
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Vol. 14, No. 3 Printed in U.S.A.
Fetal and Adult Bovine Interferon Production During Bovine Viral Diarrhea Virus Infection C. R. RINALDO, JR.,'* D. W. ISACKSON, J. C. OVERALL, JR., L. A. GLASGOW, T. T. BROWN,2 S. I. BISTNER, J. H. GILLESPIE, AND F. W. SCOTT Departments ofPediatrics and Microbiology, University of Utah College of Medicine, Salt Lake City, Utah 84132*; and Departments of Microbiology, Pathology, and Large Animal Medicine, Obstetrics and Surgery, New York State College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Received for publication 15 April 1976
Levels of interferon in adult bovine serum and in fetal bovine serum and tissues were examined during the course of transplacental bovine viral diarrhea virus infection. The cows produced circulating interferon between 2 and 9 days after viral inoculation, with mean peak levels in the serum on day 4. Interferon could be routinely detected in fetal tissues (e.g., thymus, spleen, and kidney) between days 4 and 21 after viral inoculation of the cows at 149 to 150 days of gestation (mid-second trimester) and in fetal serum from day 13 through day 21. Interferon was also detectable in the serum and tissues of fetuses from dams infected at day 95 of gestation (the beginning of the second trimester). In general, no differences were found between the ability of the adult and fetus to produce interferon. Fetal lamb kidney cells were more sensitive to the antiviral effects of bovine interferon than were fetal bovine kidney cells. The antiviral substance from the fetal and adult animals was characterized as interferon by standard criteria. There are many examples both in man and experimental animals of enhanced susceptibility of the fetus to viral infection (3, 9, 30, 34). Congenital rubella virus and cytomegalovirus infections are representative of such diseases of the human fetus. It has been well documented that infection of the fetus with rubella virus or cytomegalovirus during pregnancy can result in severe congenital defects of the central nervous system (CNS), eye, ear, heart, and skeleton (14, 18). In contrast, infection of children or adults with either ofthese viruses usually leads to a relatively mild or asymptomatic illness
(25).
Bluetongue virus (21), hog cholera virus (15), and bovine viral diarrhea (BVD) virus (8, 23, 49) are examples of agents which may cause severe disease in the fetuses of domestic animals. In the cow, which has a 9-month gestation period, infection with BVD virus during the first trimester of pregnancy can result in fetal mummification, stillbirth, or abortion (4, 8, 24). Cerebellar and ocular lesions may develop in the fetuses of cows infected with BVD virus during the second trimester of gestation ' Present address: Harvard Medical School, Department of Medicine, Massachusetts General Hospital, Boston, MA 02114. 2 Present address: Department of Pathology, College of Veterinary Medicine, Oklahoma State University, Stillwater, OK 74074. 660
(4-6, 41). The BVD virus has little or no effect on the fetuses of cows infected during the last trimester of pregnancy (8, 24). The precise mechanisms involved in the agerelated resistance of the fetus to viral infection have not been well defined. Several investigations have suggested that the inability of the fetal and newborn animal to control viral infection may be related to an inadequate interferon response (1, 2, 13, 19, 22, 27, 32, 40). In contrast to these results, other studies have shown that the capacity to produce interferon is not reduced in the immature host (10, 11, 31, 36, 44, 48). Studies from this laboratory have shown that the fetal lamb is as competent as the adult sheep to produce interferon in response to viral infection either after direct inoculation in utero (33, 35) or in cell cultures derived from fetuses of various gestational ages (38). To more fully define the role of interferon in age-related host resistance, the present studies were undertaken to evaluate interferon production in the pregnant heifer and in the bovine fetus after transplacental infection with BVD virus between 65 and 150 days of gestation. MATERIALS AND METHODS Cells and media. Fetal bovine kidney (FBK) cells were prepared from kidneys removed aseptically from bovine fetuses obtained from the Midvale Packing Co., Midvale, Utah. The renal capsule was
VOL. 14, 1976
INTERFERON IN BVD VIRUS INFECTION
661
removed, and the cortical and medullary regions RESULTS were minced and washed with phosphate-buffered Interferon production in the adult bovine. saline. Cells were extracted in a 0.25% (wt/vol) trypsin sodium citrate-potassium chloride solution The levels of circulating interferon in nine susfor 1 h, centrifuged, and resuspended in Eagle mini- ceptible pregnant heifers during the course of mum essential medium consisting of Earle bal- BVD virus infection (phase II studies) are preanced salt solution supplemented with minimum es- sented in Fig. 1. As assayed in FBK cells, intersential medium vitamins and amino acids (Grand feron was detectable from day 3 through day 6 Island Biological Co., Grand Island, N.Y.), 1% (volV postviral inoculation and reached mean peak vol) of a 300 ,ug/liter solution of glutamine, 100 U of titers penicillin per ml, 100 ,ug of streptomycin per ml, 10% titers of 36 U/ml on day 4. Interferon (vol/vol) fetal calf serum (Grand Island Biological), ranged from undetectable levels to 100 U/ml in and NaHCO3 as a buffer. The FBK cells were main- the separate heifers. The serum interferon retained in 32-oz glass prescription bottles (Brockway sponse of the heifers used in phase I and III Glass Co., Brockway, Pa.) and used in the second to experiments was equivalent to that shown for fifth passage. Procedures have previously been de- phase II. scribed (37) for the culturing of fetal lamb kidney During the course of the phase II studies, it (FLK) and mouse L cells. was discovered that FLK cells were much more Viruses. The cytopathogenic Holmes strain of sensitive to the antiviral effects of bovine interBVD virus used in these experiments was originally feron than were FBK cells. Therefore, phase II isolated from the maternal and fetal tissues of a III and samples were assayed for interferon pregnant cow that died of natural infection (42). The stock virus was prepared and assayed in fetal bovine activity in both bovine (FBK) and sheep (FLK) spleen cells, and the median tissue culture infective cells. The titers of interferon in the serum ofthe dose was calculated by the Karber method (28). The nine phase II BVD virus-infected heifers were viral pools used in these experiments had a titer of consistently several-fold higher in the sheep approximately 104 median tissue culture infective than in the bovine cells (Fig. 1). The mean peak doses per 0.1 ml. A detailed description has been titer in FLK cells was 590 U/ml, approximately reported (37) for the preparation and assay ofvesicu- 16-fold higher than in the FBK cells. The peak lar stomatitis virus (VSV) and Sindbis virus pools. titers reached levels greater than 1,000 U/ml in Experimental animals and procedures. The study was divided into three phases. Pregnant Holstein- several animals. Although the kinetics of the Friesian heifers with known breeding dates were 10,000 housed in an isolated barn in Ithaca, N.Y. There were 11 heifers used in phase I of the experiment, 10 A INTERFERON ASSAYED ON heifers in phase II, and 6 heifers in phase III. All of FLK CELLS the heifers, with the exception of one heifer in phase 0 INTERFERON II, did not have serum-neutralizing antibody titers ASSAYED ON against BVD virus. The animal with neutralizing FBK CELLS antibody was used as a control. The heifers were serologically surveyed each month to ensure that the animals remained susceptible to the virus. Be1,000 fore inoculation with virus, the pregnant heifers were moved into environmentally controlled individual isolation units. Heifers in phases I and II of the study were intravenously inoculated with 250,000 median tissue culture infective doses of the Holmes strain of BVD virus at either 149 or 150 days of gestation. Heifers in phase III of the study were T inoculated similarly with the same amount of the virus at either 65, 75, 85, 95, or 115 days ofgestation. At sequential times after inoculation of the heifers with BVD virus, the fetuses were surgically removed by cesarean section from their dams. Immediately thereafter they were exsanguinated, and fetal serum and tissues were stored at -70°C for interferon assay. Interferon assay. The fetal and adult bovine samples were transferred in dry ice by air freight to the University of Utah College of Medicine. The sam9 10 7 8 O 2 3 4 5 6 1 ples were assayed by the 50% plaque reduction techDAYS POST- INOCULATION nique in FBK and FLK cells using VSV as the FIG. 1. Mean levels and range of interferon titers challenge virus (37). An internal laboratory reference bovine interferon was included with each as- induced in the sera of nine dams inoculated intravesay; titers varied from two- to threefold between nously with BVD virus and assayed in FLK and in FBK cells. different assays on FLK cells.
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RINALDO ET AL.
INFECT. IMMUN.
interferon response followed a similar pattern with both assay systems, the enhanced sensitivity of the FLK cells allowed detection of serum interferon from day 2 through day 9. In comparing the sensitivity of the sheep and bovine cells, an interferon standard kindly donated by J. D. Todd (Jensen-Salsbery Labs, Kansas City, Mo.) was assayed in both cell lines. The standard consisted of nasal secretions from calves which had been inoculated with infectious bovine rhinotracheitis virus and was known to contain antiviral activity which had been characterized as interferon (46). The bovine interferon sample titered 200 U/ml in FBK cells and 715 U/ml in FLK cells, i.e., nearly a fourfold increase in the sheep cells. Interferon production in the bovine fetus: phase I studies. Data on the production of interferon in the bovine fetus after inoculation of their dams with BVD virus during the phase I studies are presented in Table 1. Interferon was present in fetal serum from 13 to 21 days after inoculation of the cows with virus and ranged from 4 to 70 U/ml as assayed in FBK cells. Low levels of interfering activity were also noted in the serum of fetuses taken at 27 and 56 days postviral inoculation. Interferon was detected in fetal tissues between 4 and 21 days after viral infection of the dams with highest levels consistently present in the liver, thymus, and kidney. Interferon was found irregularly and at lower levels in spleen, lung, mesenteric lymph node, and cotyledon. One possible explanation for the low or undetectable levels of interferon in fetuses removed from 21 to 70 days was that they were not infected with BVD virus. However, these fetuses were shown to be infected as evidenced by the presence of elevated levels of serum immunoglobulins M and G1 (T. T. Brown et al., unpublished observations), and cerebellar and ocular lesions typical of BVD virus infection (5,
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