Vol. 17, No. 2 Printed in U.S.A.
INFECTION AND IMMUNrrY, Aug. 1977, p. 268-273
Copyright © 1977 American Society for Microbiology
Genetics of Macrophage-Controlled Resistance to Hepatitis Induced by Herpes Simplex Virus Type 2 in Mice S. C. MOGENSEN
Institute of Medical Microbiology, University of Aarhus, 8000 Aarhus C, Denmark Received for publication 22 February 1977
The genetics of innate resistance of mice to hepatitis induced by herpes simplex virus type 2 (HSV-2) was analyzed by crossing resistant male GR to susceptible female BALB/c mice and backcrossing females of this F, generation to susceptible male BALB/c mice. By scoring of macroscopic liver lesions and virus isolation studies from the liver 4 days after intraperitoneal inoculation of HSV-2, it appeared that the resistance was governed by one X-linked dominant gene or closely linked gene complex, as F, female mice were resistant and F1 male mice were susceptible and the trait segregated in a ratio close to 1:1 in the backcross mating. A cellular expression in vitro of virus resistance was found in the macrophage population of the mice as measured by differences in the restriction of HSV-2 replication in macrophage cultures prepared from individual mice. In contrast to what was seen in macrophage cultures, virus replicated equally well in embryonic fibroblast cultures from susceptible and resistant strains of mice. MATERIALS AND METHODS Mice. Inbred specific-pathogen-free BALB/c/A/ BOM mice originated from Gl. Bomholtgaard Laboratory Animal Breeding and Research Center, Ry, Denmark. Inbred specific-pathogen-free GR/AFib mice were obtained from the Fibiger Laboratory, Copenhagen, Denmark. Mice for experimentation were locally bred behind specific-pathogen-free barriers, as were the F, hybrids (GRd x BALB/cy)F, and the first backcross generation [BALB/cd x (GR x BALB/c) Y ]BC1. Virus. HSV-2 strain MS, used throughout this study, has been described previously (15). It is a well-established laboratory strain, which has been passaged five times in this laboratory in human embryonic lung cells. The virus was plaque purified before production of the virus pool used in these experiments. In vivo experiments. The following eight groups of male and female mice were inoculated intraperitoneally with 106 plaque-forming units (PFU) of HSV-2 in 0.2 ml of diluent: BALB/c, GR, (GRc& x BALB/cY)F1, and [BALB/cd x (GR x BALB/ c)YIBC,. Four days after the infection, the mice were killed by exsanguination in ether anesthesia. The livers were examined for macroscopic lesions and were then collected and frozen individually at -70°C until assayed for virus. Assay of livers for virus. Livers were homogenized to a 10% suspension in Eagle minimum essential medium supplemented with 5% calf serum and antibiotics in a homogenizer (model Hannover, Ernst Schutt, Jr.). The suspensions were centrifuged at 4°C for 30 min at 4,000 x g, and the supernatants were tested for virus in human embryonic 268
It has been known for about 40 years that the genetic constitution of a host can play an important role in the outcome of viral infections in animals (24). Since then, a large number of viral infections have been extensively studied for genetically controlled variation in resistance of the host, using mainly the mouse as a model (1, 2, 5, 6, 10-12, 16, 19-22, 25). In some instances, a single gene or gene complex has been found to determine resistance (6, 11, 19, 20) or susceptibility (2, 5, 16); in other cases, the trait has been found to be polygenic (10, 12, 25). Herpes simplex virus type 2 (HSV-2) has previously been shown to induce large focal necrotic lesions of the liver of most mouse strains on intraperitoneal inoculation (14, 15). However, in some of nine inbred mouse strains studied, these characteristic lesions of the liver did not develop. Furthermore, since all mice were reared under identical specific-pathogen-free conditions, it was found likely that the differences observed were genetically determined. In this paper, a further study of susceptible (BALB/c) and resistant (GR) mouse strains is reported. Evidence is presented that the resistance to HSV-2-induced hepatitis is indeed under genetic control, and that a single gene or closely linked gene complex is the major factor responsible for the resistance. Furthermore, a cellular expression of virus resistance was found in the macrophage cell population of the mice.
VOL. 17, 1977
GENETICS OF HSV-2 HEPATITIS IN MICE
lung cell cultures by a plaque method previously described (14). Infectious center assay. The infectious center assay was performed as previously described (13). In brief, peritoneal exudate cells were harvested from the peritoneal cavity of individual 8-week-old female mice by inoculating 3 ml of RPMI-1640 medium containing antibiotics, 20% fetal calf serum, and 10 IU of heat-sterilized heparin without preservative. The number of macrophages and lymphocytes in the suspensions was determined in a fluorescent microscope after cytoplasmic staining with acridine orange as the macrophage marker. The cells were plated on plastic petri dishes (35 mm, Falcon) in a concentration of 5 x 105 macrophages per dish. On the next day, nonadherent cells were removed by washing, and the cultures were inoculated with 5 x 105 PFU of HSV-2. After 60 min of adsorption, the nonadsorbed virus was removed by washing and HSV hyperimmune serum treatment. The macrophages were then overlaid with mouse embryonic cells in methyl cellulose medium, and the plaques appearing in the mouse embryonic cell overlay were counted 2 days later. Adsorption of HSV-2 to macrophages. Assessment of virus adsorption in macrophage cultures was performed as previously described (13). Macrophage cultures from BALB/c and GR mice were infected with 2 x 104 PFU of HSV-2, and after 60 min of adsorption the inoculum was diluted in 20 ml of cold medium and assayed for nonadsorbed virus. Adsorption percentages were calculated as the percentage of virus lost from the inoculum compared to the amount lost from control petri dishes that lacked macrophages. Preparation of single-embryo mouse fibroblast cultures. One male mouse and one female mouse were mated for 24 h. On day 18 of gestation, the embryos were removed aseptically and individual embryos were processed for tissue culture by trypsinization according to standard procedures. The cells were plated on plastic culture flasks (25 cm2, Falcon) in 10 ml of Eagle minimum essential medium containing 15% calf serum. After 7 days of incubation all cultures were confluent, and the cells were passaged to 6-cm plastic petri dishes (7.5 x 105/dish). After 3 days of further growth in a 5% CO2 atmosphere, the secondary monolayers were ready for use. Virus growth in mouse embryonic fibroblasts. The growth of HSV-2 in mouse embryonic fibroblast cultures was examined as previously described (13), using plaquing efficiency, plaque area, and yield of infectious virus from the supernatant as parameters.
RESULTS
269
week-old mice with 106 PFU of virus in 0.2 ml of diluent gave the most clear-cut results: liver lesions as previously described (15) in all BALB/c mice and no such lesions in any of the GR mice. In 4-week-old animals, a few lesions were also seen in some GR mice, which shows that the resistance displayed by the GR mouse strain is relative and age dependent. To further analyze the patterns of inheritance, 8-week-old male and female BALB/c, GR, (GRd x BALB/c9)F1, and [BALB/cc5 x (GR x BALB/c) 9 ]BC1 mice were inoculated intraperitoneally with 106 PFU of HSV-2. The animals were killed 4 days later, and the liver was examined for macroscopic lesions. In Table 1, the results of a series of such experiments are summarized. All BALB/c mice of both sexes were found to be susceptible, whereas all GR mice were resistant. In the F1 generation, all males were susceptible, but the females were resistant. This shows that resistance is the dominant feature, and that the resistance gene(s) is located on the X-chromosome. A few tiny lesions were seen on the liver margins of two GR and three F1 females, but this reflects only the relativity of the GR resistance. The genetic segregation of susceptibility and resistance occurred in mice of both sexes in the first backcross generation (BC1) of resistant F1 females to susceptible BALB/c males. The segregation ratio of about 1:1 in the BC1 generation suggests that resistance is controlled by one major gene or closely linked gene complex. Virus isolation studies. The virus titers of the livers from female mice killed on day 4 are shown in Fig. 1. This particular day was chosen for virus isolation because the virus content of the liver normally reaches a maximum on that day (13). As seen from the figure, virus could be isolated from all mice showing macroscopic TABLE 1. Number of 8-week-old male and female BALB/c, GR, (GR d x BALBEc 9) F1, and [BALB/cd x (GR x BALBIc) Y1BC, mice showing typical type 2 liver lesionsa 4 days after intraperitoneal inoculation of 106 PFU of HSV-2 (summary of experiments) No. of mice with lesions/total inocu-
lated
Mouse strain
BALB/c GR
Male
Female
10/10 0/14 26/26
33/33
0/27 Inheritance of resistance to HSV-2 hepati0/40 F1 tis. In preliminary experiments, the previously 10122t BC1 17/29b in of difference observed (14) susceptibility a A few BALB/c and GR mouse strains to the induction tiny lesions were present on the liver of focal necrotic hepatitis by HSV-2 was exam- margins of two GR and three F1 mice. b Male mice: P = 0.46; female mice: P = 0.83 (not ined, using mice of different ages and varying virus doses. It was found that inoculation of 8- significantly different from the 1:1 segregation).
270
INFECT. IMMUN.
MOGENSEN
whereas the number of infectious centers segregated in a ratio close to 1:1 in the BC1 generation. The difference in the number of infectious centers observed in macrophage cultures pre4 pared from BALB/c and GR mice was not E caused by differences in the adsorption of virus to macrophages. Adsorption experiments showed that the percentage of HSV-2 that was lost from the supernatant medium and could U. not be washed off the cells was 36 and 38% for BALB/c and GR macrophages, respectively. It 0~~~~~~~~~~~~ 0 thus seems that the macrophage population of 0 the mouse represents a cellular expression of 0000OC C~~~~~4O * the virus resistance gene. Growth of HSV-2 in mouse embryonic fibroblasts from susceptible and resistant mice. To rule out that the differences observed in the rate of replication of HSV-2 in macrophages 00 Om OOO (
INFECTION AND IMMUNrrY, Aug. 1977, p. 268-273
Copyright © 1977 American Society for Microbiology
Genetics of Macrophage-Controlled Resistance to Hepatitis Induced by Herpes Simplex Virus Type 2 in Mice S. C. MOGENSEN
Institute of Medical Microbiology, University of Aarhus, 8000 Aarhus C, Denmark Received for publication 22 February 1977
The genetics of innate resistance of mice to hepatitis induced by herpes simplex virus type 2 (HSV-2) was analyzed by crossing resistant male GR to susceptible female BALB/c mice and backcrossing females of this F, generation to susceptible male BALB/c mice. By scoring of macroscopic liver lesions and virus isolation studies from the liver 4 days after intraperitoneal inoculation of HSV-2, it appeared that the resistance was governed by one X-linked dominant gene or closely linked gene complex, as F, female mice were resistant and F1 male mice were susceptible and the trait segregated in a ratio close to 1:1 in the backcross mating. A cellular expression in vitro of virus resistance was found in the macrophage population of the mice as measured by differences in the restriction of HSV-2 replication in macrophage cultures prepared from individual mice. In contrast to what was seen in macrophage cultures, virus replicated equally well in embryonic fibroblast cultures from susceptible and resistant strains of mice. MATERIALS AND METHODS Mice. Inbred specific-pathogen-free BALB/c/A/ BOM mice originated from Gl. Bomholtgaard Laboratory Animal Breeding and Research Center, Ry, Denmark. Inbred specific-pathogen-free GR/AFib mice were obtained from the Fibiger Laboratory, Copenhagen, Denmark. Mice for experimentation were locally bred behind specific-pathogen-free barriers, as were the F, hybrids (GRd x BALB/cy)F, and the first backcross generation [BALB/cd x (GR x BALB/c) Y ]BC1. Virus. HSV-2 strain MS, used throughout this study, has been described previously (15). It is a well-established laboratory strain, which has been passaged five times in this laboratory in human embryonic lung cells. The virus was plaque purified before production of the virus pool used in these experiments. In vivo experiments. The following eight groups of male and female mice were inoculated intraperitoneally with 106 plaque-forming units (PFU) of HSV-2 in 0.2 ml of diluent: BALB/c, GR, (GRc& x BALB/cY)F1, and [BALB/cd x (GR x BALB/ c)YIBC,. Four days after the infection, the mice were killed by exsanguination in ether anesthesia. The livers were examined for macroscopic lesions and were then collected and frozen individually at -70°C until assayed for virus. Assay of livers for virus. Livers were homogenized to a 10% suspension in Eagle minimum essential medium supplemented with 5% calf serum and antibiotics in a homogenizer (model Hannover, Ernst Schutt, Jr.). The suspensions were centrifuged at 4°C for 30 min at 4,000 x g, and the supernatants were tested for virus in human embryonic 268
It has been known for about 40 years that the genetic constitution of a host can play an important role in the outcome of viral infections in animals (24). Since then, a large number of viral infections have been extensively studied for genetically controlled variation in resistance of the host, using mainly the mouse as a model (1, 2, 5, 6, 10-12, 16, 19-22, 25). In some instances, a single gene or gene complex has been found to determine resistance (6, 11, 19, 20) or susceptibility (2, 5, 16); in other cases, the trait has been found to be polygenic (10, 12, 25). Herpes simplex virus type 2 (HSV-2) has previously been shown to induce large focal necrotic lesions of the liver of most mouse strains on intraperitoneal inoculation (14, 15). However, in some of nine inbred mouse strains studied, these characteristic lesions of the liver did not develop. Furthermore, since all mice were reared under identical specific-pathogen-free conditions, it was found likely that the differences observed were genetically determined. In this paper, a further study of susceptible (BALB/c) and resistant (GR) mouse strains is reported. Evidence is presented that the resistance to HSV-2-induced hepatitis is indeed under genetic control, and that a single gene or closely linked gene complex is the major factor responsible for the resistance. Furthermore, a cellular expression of virus resistance was found in the macrophage cell population of the mice.
VOL. 17, 1977
GENETICS OF HSV-2 HEPATITIS IN MICE
lung cell cultures by a plaque method previously described (14). Infectious center assay. The infectious center assay was performed as previously described (13). In brief, peritoneal exudate cells were harvested from the peritoneal cavity of individual 8-week-old female mice by inoculating 3 ml of RPMI-1640 medium containing antibiotics, 20% fetal calf serum, and 10 IU of heat-sterilized heparin without preservative. The number of macrophages and lymphocytes in the suspensions was determined in a fluorescent microscope after cytoplasmic staining with acridine orange as the macrophage marker. The cells were plated on plastic petri dishes (35 mm, Falcon) in a concentration of 5 x 105 macrophages per dish. On the next day, nonadherent cells were removed by washing, and the cultures were inoculated with 5 x 105 PFU of HSV-2. After 60 min of adsorption, the nonadsorbed virus was removed by washing and HSV hyperimmune serum treatment. The macrophages were then overlaid with mouse embryonic cells in methyl cellulose medium, and the plaques appearing in the mouse embryonic cell overlay were counted 2 days later. Adsorption of HSV-2 to macrophages. Assessment of virus adsorption in macrophage cultures was performed as previously described (13). Macrophage cultures from BALB/c and GR mice were infected with 2 x 104 PFU of HSV-2, and after 60 min of adsorption the inoculum was diluted in 20 ml of cold medium and assayed for nonadsorbed virus. Adsorption percentages were calculated as the percentage of virus lost from the inoculum compared to the amount lost from control petri dishes that lacked macrophages. Preparation of single-embryo mouse fibroblast cultures. One male mouse and one female mouse were mated for 24 h. On day 18 of gestation, the embryos were removed aseptically and individual embryos were processed for tissue culture by trypsinization according to standard procedures. The cells were plated on plastic culture flasks (25 cm2, Falcon) in 10 ml of Eagle minimum essential medium containing 15% calf serum. After 7 days of incubation all cultures were confluent, and the cells were passaged to 6-cm plastic petri dishes (7.5 x 105/dish). After 3 days of further growth in a 5% CO2 atmosphere, the secondary monolayers were ready for use. Virus growth in mouse embryonic fibroblasts. The growth of HSV-2 in mouse embryonic fibroblast cultures was examined as previously described (13), using plaquing efficiency, plaque area, and yield of infectious virus from the supernatant as parameters.
RESULTS
269
week-old mice with 106 PFU of virus in 0.2 ml of diluent gave the most clear-cut results: liver lesions as previously described (15) in all BALB/c mice and no such lesions in any of the GR mice. In 4-week-old animals, a few lesions were also seen in some GR mice, which shows that the resistance displayed by the GR mouse strain is relative and age dependent. To further analyze the patterns of inheritance, 8-week-old male and female BALB/c, GR, (GRd x BALB/c9)F1, and [BALB/cc5 x (GR x BALB/c) 9 ]BC1 mice were inoculated intraperitoneally with 106 PFU of HSV-2. The animals were killed 4 days later, and the liver was examined for macroscopic lesions. In Table 1, the results of a series of such experiments are summarized. All BALB/c mice of both sexes were found to be susceptible, whereas all GR mice were resistant. In the F1 generation, all males were susceptible, but the females were resistant. This shows that resistance is the dominant feature, and that the resistance gene(s) is located on the X-chromosome. A few tiny lesions were seen on the liver margins of two GR and three F1 females, but this reflects only the relativity of the GR resistance. The genetic segregation of susceptibility and resistance occurred in mice of both sexes in the first backcross generation (BC1) of resistant F1 females to susceptible BALB/c males. The segregation ratio of about 1:1 in the BC1 generation suggests that resistance is controlled by one major gene or closely linked gene complex. Virus isolation studies. The virus titers of the livers from female mice killed on day 4 are shown in Fig. 1. This particular day was chosen for virus isolation because the virus content of the liver normally reaches a maximum on that day (13). As seen from the figure, virus could be isolated from all mice showing macroscopic TABLE 1. Number of 8-week-old male and female BALB/c, GR, (GR d x BALBEc 9) F1, and [BALB/cd x (GR x BALBIc) Y1BC, mice showing typical type 2 liver lesionsa 4 days after intraperitoneal inoculation of 106 PFU of HSV-2 (summary of experiments) No. of mice with lesions/total inocu-
lated
Mouse strain
BALB/c GR
Male
Female
10/10 0/14 26/26
33/33
0/27 Inheritance of resistance to HSV-2 hepati0/40 F1 tis. In preliminary experiments, the previously 10122t BC1 17/29b in of difference observed (14) susceptibility a A few BALB/c and GR mouse strains to the induction tiny lesions were present on the liver of focal necrotic hepatitis by HSV-2 was exam- margins of two GR and three F1 mice. b Male mice: P = 0.46; female mice: P = 0.83 (not ined, using mice of different ages and varying virus doses. It was found that inoculation of 8- significantly different from the 1:1 segregation).
270
INFECT. IMMUN.
MOGENSEN
whereas the number of infectious centers segregated in a ratio close to 1:1 in the BC1 generation. The difference in the number of infectious centers observed in macrophage cultures pre4 pared from BALB/c and GR mice was not E caused by differences in the adsorption of virus to macrophages. Adsorption experiments showed that the percentage of HSV-2 that was lost from the supernatant medium and could U. not be washed off the cells was 36 and 38% for BALB/c and GR macrophages, respectively. It 0~~~~~~~~~~~~ 0 thus seems that the macrophage population of 0 the mouse represents a cellular expression of 0000OC C~~~~~4O * the virus resistance gene. Growth of HSV-2 in mouse embryonic fibroblasts from susceptible and resistant mice. To rule out that the differences observed in the rate of replication of HSV-2 in macrophages 00 Om OOO (
