INFECTION
AND
Vol. 11, No. 1
IMMUNITY, Jan. 1975, p. 159-163
Printed in U.S.A.
Copyright 0 1975 American Society for Microbiology
Characterization of an In Vitro Persistent-State Measles Virus Infection: Species Characterization and Interference in the BGM/MV Cell Line JAY H. MENNA,* ARLENE R. COLLINS, AND THOMAS D. FLANAGAN Department of Microbiology, State University of New York at Buffalo, Buffalo, New York 14214
Received for publication 6 August 1974
Serological methods of mixed agglutination and indirect immunofluorescence showed the BGM/MV cell line to possess only monkey antigens. As a means of further characterizing the species constitution of the BGM/MV cell line, the species specificity of viral-induced interferon from these cells, as well as the response of these cells to exogenous interferons, was determined. Low titers of spontaneously elaborated interferon capable of protecting monkey but not mouse cells were detected in BGM/MV culture fluids. Interferon induced by Newcastle disease virus infection of BGM/MV cells was capable of conferring an antiviral state on monkey and, to a lesser extent, on mouse cells. Exogenous interferons of both homologous (BGM/MV) and heterologous sources failed to confer an antiviral state on BGM/MV cells. BGM/MV cells were found to be partially refractive to superinfection with measles virus but freely replicated mumps and vesicular stomatitis virus. The procedure by which the BGM/MV cell line was established and some of the characteristics of this cell line were described in the preceding paper (6). Data were presented concerning the morphological evolution of this cell line and state of measles virus infection. Infectious measles virus and viral hemagglutinin were not detected in either BGM/MV cell lysates or in culture fluids. By immunofluorescence, intracellular measles antigen was detected in 100% of BGM/MV cells; plasma membrane-associated measles antigen was detected in < 1%. Electron microscopy showed the presence of nucleocapsid-like structures in the cytoplasm of BGM/MV cells. Less than 1% of BGM/MV cells were hemadsorption positive. Enhancement of the hemadsorption capacity of BGM/MV cells was accomplished by treatment of cell monolayers with actinomycin D or enucleation with cytochalasin B. Attempts to induce infectious measles virus using various chemical agents and physical procedures were negative. This paper describes experiments in which the species (monkey and/or mouse) constitution of the BGM/MV cell line was determined. Also presented are data which further characterize the state of measles virus infection of these cells.
MATERIALS AND METHODS Cell cultures. BGM cells, a stable line of African green monkey kidney cells (1), were passaged and maintained as previously described (6). The L-929 line of mouse fibroblasts, provided by C. J. Abeyounis, Buffalo, N.Y., was passaged weekly by scraping confluent monolayers and resuspending the cells to a seeding concentration of 4.5 x 104 cells/ml in medium consisting of Eagle minimal essential medium in Hanks salts supplemented with 10% fetal calf serum (FCS), 100 Ag of streptomycin per ml, and 100 U of penicillin per ml. Primary C3H mouse embryo fibroblasts were prepared from 11- to 13-day-old embryos. Tissues from decapitated embryos were minced and the cells were extracted by trypsinization. Trypsinized cells were sedimented by centrifugation at 450 x g for 10 min at 4 C and resuspended in outgrowth medium consisting of Eagle minimal essential medium in Hanks salts supplemented with 10% FCS, 200 Mg of streptomycin per ml, and 200 U of penicillin per ml. Maintenance medium consisted of Eagle minimal essential medium in Hanks salts supplemented with 5% FCS and antibiotics. BGM/MV cells, a stable measles virus carrier cell line, was initiated as previously described (6). This cell line was passaged weekly by splitting of confluent monolayers 1:3 and seeding in Eagle minimal essential medium supplemented with 10% FCS plus 200 Mg of streptomycin per ml and 200 U of penicillin per ml. Viruses. Mumps virus (ABC strain) was serially
159
160
MENNA, COLLINS, AND FLANAGAN
passaged in BGM cells. Stock virus was prepared from infected monolayers and consisted of clarified cell lysates titering 5 x 107 to 10' plaque-forming units (PFU)/ml in BGM cells. Vesicular stomatitis virus (VSV), Indiana strain, was serially passaged in 10-day-old chicken embryos. Infectious allantoic fluids titered from 2 x 108 to 9 x 10' PFU/ml in L-929 cells and 6 x 108 to 3 x 109 PFU/ml in BGM cells. Measles virus, MV-500E strain (non-mouseadapted), was serially passaged in BGM cells. Stock virus was prepared from infected monolayers and consisted of clarified lysates titering 1.2
x
105 to 1.5 x 106
PFU/ml in BGM cells. Newcastle disease virus (NDV), Victoria strain, was prepared as infectious allantoic fluid and titered from 108 to 109 50% egg lethal doses per ml when assayed in 10-day-old chicken embryos. Infectivity assays. Infectious measles virus and mumps virus were assayed by a plaque formation method in BGM cells as previously described for mumps virus (3). VSV was also titered by plaque assay. In this case, adsorption of virus on either BGM or L-929 cell monolayers was 1 h at 37 C and the staining was done at 24 h. NDV was assayed by inoculation into 10-day-old chicken embryos. Detection of cellular antigen in BGM/MV cells by immunofluorescence. The indirect method of immunofluorescence was employed for the detection of monkey and mouse antigens in 13GM/MV cells. The protocols employed for the detection of intracellular and membrane species antigen were the same as those previously described for the detection of measles antigen (6) except that different antisera were used. Antiserum to mouse antigens was prepared in rabbits by intradermal inoculation of a 10% (wt/vol) mouse spleen cell suspension in Freund complete adjuvant. Rabbits were boostered once and then exsanguinated. The titer of this antiserum using the indirect immunofluorescence assay procedure and acetone-fixed L-929 cells as antigen was 160. Antiserum directed against BGM cell antigens was prepared in rabbits by intradermal and subcutaneous inoculation of BGM cell homogenates emulsified in Freund complete adjuvant and by intravenous inoculation of supernatant fluids collected from clarified BGM cell lysates. By indirect immunofluorescence assay this antiserum had a titer of 160 using acetone-fixed BGM cell monolayers as antigen. Prior to use, BGM antiserum was absorbed with L-929 cells and mouse spleen antiserum with BGM cells. Detection of surface antigen on BGM/MV cells by mixed agglutination. The standard mixed agglutination procedure (2, 5) was employed for the detection of monkey and mouse antigens on BGM/ MV cells. The antisera used were the same as those employed for detection of intracellular species antigen by indirect immunofluorescence. Sheep erythrocytes were used as indicator cells. These cells were sensitized to rabbit antigen by treatment with heat-inactivated rabbit anti-sheep erythrocyte serum followed by treatment with heat-inactivated goat anti-rabbit Fraction II. Duplicate BGM/MV cell monolayers were washed twice with maintenance medium and incu-
INFECT. IMMUN.
bated at room temperature for 90 min with serial 300-fold dilutions of either rabbit anti-mouse, rabbit anti-BGM, or preimmune rabbit serum. Cell monolayers were then washed three times with maintenance medium devoid of FCS and incubated for 60 min at room temperature with sheep erythrocytes sensitized to rabbit antigen and read microscopically for cell adherence. Controls consisted of mouse (mouse embryo fibroblasts) and monkey (BGM) cell monolayers treated as above. Preparation of interferons. Induction of interferon was carried out by inoculation of monolayers of BGM/MV, BGM, and L-929 cell cultures with NDV at approximately 30 50% egg lethal doses/cell. After 24 h of incubation at 37 C, cell culture fluids were collected, adjusted to pH 2 with 1 N HCl, and placed at 4 C for 18 h. The fluids were adjusted to neutrality with 1 N NaOH and centrifuged at 85,000 x g for 1 h. Supernatant fluids were collected and frozen at -20 C in 2-ml volumes. Assay of interferon activity by plaque reduction. Fourfold dilutions of putative interferon preparations were made in Eagle minimal essential medium supplemented with 2% FCS. One milliliter of each dilution plus 4 ml of maintenance medium was added to each of two cell monolayers in plastic tissue culture flasks (Falcon Plastics, Oxnard, Calif.). After incubation at 37 C for 18 h, culture fluids were decanted and monolayers were challenged with 50 PFU of VSV. Interferon titers were expressed as the reciprocal of the dilution which resulted in a 50% reduction in plaque formation. As a means of detecting low levels of interferon activity a modification of this assay procedure was employed. Twofold dilutions of interferon preparations were made in maintenance medium, and each of two cell monolayers was treated with 4 ml per dilution. The titer obtained was then divided by 4 to obtain units of activity per milliliter. Standard laboratory interferon preparations from BGM and L-929 cells were used as controls in each assay.
Assay of interferon activity by microtiter method. BGM/MV, BGM, and L-929 interferon activity in BGM/MV cells was assayed using a microtiter procedure described by Genco et al. (4). Ninetysix welled polystyrene tissue culture microplates (Linbro Chemical Co., Inc., New Haven, Conn.) were seeded with BGM/MV cells at 2 x 104 cells per well and incubated at 37 C in an atmosphere of 5% CO2. Fourfold dilutions of interferon preparations were made in maintenance medium. Eight wells were inoculated per interferon dilution, each receiving 0.1 ml. Cell and virus controls (eight wells per control) received 0.1 ml of maintenance medium. After 24 h of incubation at 37 C in an atmosphere of 5% C02, each well, with the exception of the cell controls, received 3,000 PFU of VSV and incubation at 37 C was continued. When pronounced cytopathic effect was evident in the virus controls (usually 48 to 72 h) the plates were stained with methyl violet (0.5% methylosaniline, 5% formalin, 50% ethanol, and 0.85% NaCl in water). Cell protection was assessed by comparison with uninfected cell controls (4-plus protection) and complete destruction (0 protection in
VOL. 11, 1975
SPECIES CONSTITUTION OF BGM/MV CELL LINE
unprotected virus controls). Interferon titers were expressed as the highest dilution of material which resulted in a calculated protection of 2-plus.
RESULTS Species characterization of the BGM/MV cell line. The BGM/MV cell line was established by cocultivation of measles virus-infected primary mouse brain cells (infected in vivo) with a stable line of African green monkey kidney cells, BGM (6). This cell line evolved from a morphologically mixed cell population consisting of BGM-like (epithelioid) cells and fibroblasts to one of BGM-like homogeneity. The transition from a mixed cell population to a single cell type raised the question as to the species of these cells. The results of mixed agglutination and immunofluorescence studies for detection of monkey and mouse antigens are shown in Table 1. Positive mixed agglutination was observed only when BGM/MV cells were treated with rabbit antiserum directed against BGM cell antigen; the pattern was one of homogeneity and was identical to that observed when BGM cells were treated with this antiserum. No adsorption of indicator cells was observed when BGM/MV cells were treated with rabbit antiserum directed against mouse antigen or with normal rabbit serum. Mouse embryo fibroblasts and BGM cells were reactive only with homologous antisera. TABLE 1. Characterization of species antigen in BGM/MV cells
Antisera Cells tested
Rabbit Rabbit antiantimouse BGM
Control
serum Normal rabbit serum
Mixed agglutinationa
BGM/MV BGM MEF
_ +
+ + -
_ _ -
+
+ +
_
Intracellular and membrane immunofluorescenceb
BGM/MV BGM L-929
-
-
a Cell monolayers were incubated at room temperature for 90 min with rabbit antisera or normal rabbit serum, washed three times with maintenance medium, incubated for 60 min at room temperature with sheep erythrocytes sensitized to rabbit antigen, and read microscopically for cell adherence. bThe indirect immunofluorescence method was employed.
161
Immunofluorescence studies for detection of intracellular monkey and mouse antigens in BGM/MV cells corroborated the findings by mixed agglutination assay. In acetone-fixed preparations, 100% of BGM/MV cells revealed a homogeneous pattern of cytoplasmic fluorescence when treated with rabbit antiserum directed against BGM cell antigen. Rabbit antiserum directed against mouse antigen and normal rabbit serum failed to stain these cells. Membrane immunofluorescence revealed the presence of monkey antigen on 100% of BGM/MV cells; no mouse antigen was detected. BGM and L-929 cell monolayers revealed intracellular and membrane staining only with homologous antisera. Interferon studies. Procedures for determination of species antigen in and on BGM/MV cells revealed the presence of only BGM antigen. It was possible, however, that these cells possessed inducible biological activities which were murine in nature. The detection of such activities would serve as an indirect means of species characterization. Experiments were therefore undertaken to determine the species specificity of spontaneous and virus-induced interferon in these cells as well as the response of these cells to exogenous interferons. Low levels of spontaneously elaborated interferon were detected in BGM/MV culture fluids. Culture fluids taken at various passage levels after 390 days postseeding produced titers of 2 U/ml as assayed in BGM cells; no activity was observed in L-929 cells. This antiviral activity was characterized as one mediated by interferon by its pH stability (stable at pH 2 for 18 h at 4 C), non-sedimentability (85,000 x g for 60 min), and lack of virus specificity (active against measles, mumps, and VSV). Next, experiments were conducted with virus-induced interferon. The results of these experiments are shown in Table 2. Supernatant culture fluids harvested from NDV-infected BGM/MV cultures conferred an antiviral state to both BGM and L-929 cells; titers of 141 and 76 U/ml, respectively, were obtained. BGM interferon gave a titer of 192 U/ml in homologous cells and 16 U/ml in L-929 cells. L-929 cell interferon induced an antiviral state only in homologous cells; a titer of > 1,024 was obtained. BGM/MV, BGM, and L-929 cell interferon failed to confer an antiviral state on BGM/MV cells; titers of less than 4 U/ml were obtained using the microtiter assay procedure. Challenge of BGM/MV cells with exogenous viruses. As a means of further characterizing the BGM/MV cell line, triplicate cultures of BGM/MV cells were challenged with selected
162
MENNA, COLLINS, AND FLANAGAN
viruses at a multiplicity of infection of 0.1 PFU/cell, and the titers (as assayed in BGM cells) recoverable from pooled culture fluids and pooled clarified cell lysates after 24 h incubation at 37 C were compared with similarly treated cultures of BGM cells. The results of these experiments are shown in Table 3. Measles virus replicated to low titers in BGM cells; the titers of infectious virus recovered from the culture fluid and cell lysate was 2.2 x 102 and 9.4 x 102 PFU/ml, respectively. In BGM/MV cells only 3 PFU/ml were detected in the culture fluid, whereas no infectious virus was detected in the cell lysate. Mumps virus replicated in both BGM and BGM/MV cells; the cellassociated and supernatant titers when expressed as PFU per milliliter were greater in TABLE 2. Interferon studies with BGM/MV cultures Interferon sourceb
BGM/MV BGM L-929
Interferon activity in:a
BGMc
L-929c
BGM/MVd
141
76 16 > 1,024
AND
Vol. 11, No. 1
IMMUNITY, Jan. 1975, p. 159-163
Printed in U.S.A.
Copyright 0 1975 American Society for Microbiology
Characterization of an In Vitro Persistent-State Measles Virus Infection: Species Characterization and Interference in the BGM/MV Cell Line JAY H. MENNA,* ARLENE R. COLLINS, AND THOMAS D. FLANAGAN Department of Microbiology, State University of New York at Buffalo, Buffalo, New York 14214
Received for publication 6 August 1974
Serological methods of mixed agglutination and indirect immunofluorescence showed the BGM/MV cell line to possess only monkey antigens. As a means of further characterizing the species constitution of the BGM/MV cell line, the species specificity of viral-induced interferon from these cells, as well as the response of these cells to exogenous interferons, was determined. Low titers of spontaneously elaborated interferon capable of protecting monkey but not mouse cells were detected in BGM/MV culture fluids. Interferon induced by Newcastle disease virus infection of BGM/MV cells was capable of conferring an antiviral state on monkey and, to a lesser extent, on mouse cells. Exogenous interferons of both homologous (BGM/MV) and heterologous sources failed to confer an antiviral state on BGM/MV cells. BGM/MV cells were found to be partially refractive to superinfection with measles virus but freely replicated mumps and vesicular stomatitis virus. The procedure by which the BGM/MV cell line was established and some of the characteristics of this cell line were described in the preceding paper (6). Data were presented concerning the morphological evolution of this cell line and state of measles virus infection. Infectious measles virus and viral hemagglutinin were not detected in either BGM/MV cell lysates or in culture fluids. By immunofluorescence, intracellular measles antigen was detected in 100% of BGM/MV cells; plasma membrane-associated measles antigen was detected in < 1%. Electron microscopy showed the presence of nucleocapsid-like structures in the cytoplasm of BGM/MV cells. Less than 1% of BGM/MV cells were hemadsorption positive. Enhancement of the hemadsorption capacity of BGM/MV cells was accomplished by treatment of cell monolayers with actinomycin D or enucleation with cytochalasin B. Attempts to induce infectious measles virus using various chemical agents and physical procedures were negative. This paper describes experiments in which the species (monkey and/or mouse) constitution of the BGM/MV cell line was determined. Also presented are data which further characterize the state of measles virus infection of these cells.
MATERIALS AND METHODS Cell cultures. BGM cells, a stable line of African green monkey kidney cells (1), were passaged and maintained as previously described (6). The L-929 line of mouse fibroblasts, provided by C. J. Abeyounis, Buffalo, N.Y., was passaged weekly by scraping confluent monolayers and resuspending the cells to a seeding concentration of 4.5 x 104 cells/ml in medium consisting of Eagle minimal essential medium in Hanks salts supplemented with 10% fetal calf serum (FCS), 100 Ag of streptomycin per ml, and 100 U of penicillin per ml. Primary C3H mouse embryo fibroblasts were prepared from 11- to 13-day-old embryos. Tissues from decapitated embryos were minced and the cells were extracted by trypsinization. Trypsinized cells were sedimented by centrifugation at 450 x g for 10 min at 4 C and resuspended in outgrowth medium consisting of Eagle minimal essential medium in Hanks salts supplemented with 10% FCS, 200 Mg of streptomycin per ml, and 200 U of penicillin per ml. Maintenance medium consisted of Eagle minimal essential medium in Hanks salts supplemented with 5% FCS and antibiotics. BGM/MV cells, a stable measles virus carrier cell line, was initiated as previously described (6). This cell line was passaged weekly by splitting of confluent monolayers 1:3 and seeding in Eagle minimal essential medium supplemented with 10% FCS plus 200 Mg of streptomycin per ml and 200 U of penicillin per ml. Viruses. Mumps virus (ABC strain) was serially
159
160
MENNA, COLLINS, AND FLANAGAN
passaged in BGM cells. Stock virus was prepared from infected monolayers and consisted of clarified cell lysates titering 5 x 107 to 10' plaque-forming units (PFU)/ml in BGM cells. Vesicular stomatitis virus (VSV), Indiana strain, was serially passaged in 10-day-old chicken embryos. Infectious allantoic fluids titered from 2 x 108 to 9 x 10' PFU/ml in L-929 cells and 6 x 108 to 3 x 109 PFU/ml in BGM cells. Measles virus, MV-500E strain (non-mouseadapted), was serially passaged in BGM cells. Stock virus was prepared from infected monolayers and consisted of clarified lysates titering 1.2
x
105 to 1.5 x 106
PFU/ml in BGM cells. Newcastle disease virus (NDV), Victoria strain, was prepared as infectious allantoic fluid and titered from 108 to 109 50% egg lethal doses per ml when assayed in 10-day-old chicken embryos. Infectivity assays. Infectious measles virus and mumps virus were assayed by a plaque formation method in BGM cells as previously described for mumps virus (3). VSV was also titered by plaque assay. In this case, adsorption of virus on either BGM or L-929 cell monolayers was 1 h at 37 C and the staining was done at 24 h. NDV was assayed by inoculation into 10-day-old chicken embryos. Detection of cellular antigen in BGM/MV cells by immunofluorescence. The indirect method of immunofluorescence was employed for the detection of monkey and mouse antigens in 13GM/MV cells. The protocols employed for the detection of intracellular and membrane species antigen were the same as those previously described for the detection of measles antigen (6) except that different antisera were used. Antiserum to mouse antigens was prepared in rabbits by intradermal inoculation of a 10% (wt/vol) mouse spleen cell suspension in Freund complete adjuvant. Rabbits were boostered once and then exsanguinated. The titer of this antiserum using the indirect immunofluorescence assay procedure and acetone-fixed L-929 cells as antigen was 160. Antiserum directed against BGM cell antigens was prepared in rabbits by intradermal and subcutaneous inoculation of BGM cell homogenates emulsified in Freund complete adjuvant and by intravenous inoculation of supernatant fluids collected from clarified BGM cell lysates. By indirect immunofluorescence assay this antiserum had a titer of 160 using acetone-fixed BGM cell monolayers as antigen. Prior to use, BGM antiserum was absorbed with L-929 cells and mouse spleen antiserum with BGM cells. Detection of surface antigen on BGM/MV cells by mixed agglutination. The standard mixed agglutination procedure (2, 5) was employed for the detection of monkey and mouse antigens on BGM/ MV cells. The antisera used were the same as those employed for detection of intracellular species antigen by indirect immunofluorescence. Sheep erythrocytes were used as indicator cells. These cells were sensitized to rabbit antigen by treatment with heat-inactivated rabbit anti-sheep erythrocyte serum followed by treatment with heat-inactivated goat anti-rabbit Fraction II. Duplicate BGM/MV cell monolayers were washed twice with maintenance medium and incu-
INFECT. IMMUN.
bated at room temperature for 90 min with serial 300-fold dilutions of either rabbit anti-mouse, rabbit anti-BGM, or preimmune rabbit serum. Cell monolayers were then washed three times with maintenance medium devoid of FCS and incubated for 60 min at room temperature with sheep erythrocytes sensitized to rabbit antigen and read microscopically for cell adherence. Controls consisted of mouse (mouse embryo fibroblasts) and monkey (BGM) cell monolayers treated as above. Preparation of interferons. Induction of interferon was carried out by inoculation of monolayers of BGM/MV, BGM, and L-929 cell cultures with NDV at approximately 30 50% egg lethal doses/cell. After 24 h of incubation at 37 C, cell culture fluids were collected, adjusted to pH 2 with 1 N HCl, and placed at 4 C for 18 h. The fluids were adjusted to neutrality with 1 N NaOH and centrifuged at 85,000 x g for 1 h. Supernatant fluids were collected and frozen at -20 C in 2-ml volumes. Assay of interferon activity by plaque reduction. Fourfold dilutions of putative interferon preparations were made in Eagle minimal essential medium supplemented with 2% FCS. One milliliter of each dilution plus 4 ml of maintenance medium was added to each of two cell monolayers in plastic tissue culture flasks (Falcon Plastics, Oxnard, Calif.). After incubation at 37 C for 18 h, culture fluids were decanted and monolayers were challenged with 50 PFU of VSV. Interferon titers were expressed as the reciprocal of the dilution which resulted in a 50% reduction in plaque formation. As a means of detecting low levels of interferon activity a modification of this assay procedure was employed. Twofold dilutions of interferon preparations were made in maintenance medium, and each of two cell monolayers was treated with 4 ml per dilution. The titer obtained was then divided by 4 to obtain units of activity per milliliter. Standard laboratory interferon preparations from BGM and L-929 cells were used as controls in each assay.
Assay of interferon activity by microtiter method. BGM/MV, BGM, and L-929 interferon activity in BGM/MV cells was assayed using a microtiter procedure described by Genco et al. (4). Ninetysix welled polystyrene tissue culture microplates (Linbro Chemical Co., Inc., New Haven, Conn.) were seeded with BGM/MV cells at 2 x 104 cells per well and incubated at 37 C in an atmosphere of 5% CO2. Fourfold dilutions of interferon preparations were made in maintenance medium. Eight wells were inoculated per interferon dilution, each receiving 0.1 ml. Cell and virus controls (eight wells per control) received 0.1 ml of maintenance medium. After 24 h of incubation at 37 C in an atmosphere of 5% C02, each well, with the exception of the cell controls, received 3,000 PFU of VSV and incubation at 37 C was continued. When pronounced cytopathic effect was evident in the virus controls (usually 48 to 72 h) the plates were stained with methyl violet (0.5% methylosaniline, 5% formalin, 50% ethanol, and 0.85% NaCl in water). Cell protection was assessed by comparison with uninfected cell controls (4-plus protection) and complete destruction (0 protection in
VOL. 11, 1975
SPECIES CONSTITUTION OF BGM/MV CELL LINE
unprotected virus controls). Interferon titers were expressed as the highest dilution of material which resulted in a calculated protection of 2-plus.
RESULTS Species characterization of the BGM/MV cell line. The BGM/MV cell line was established by cocultivation of measles virus-infected primary mouse brain cells (infected in vivo) with a stable line of African green monkey kidney cells, BGM (6). This cell line evolved from a morphologically mixed cell population consisting of BGM-like (epithelioid) cells and fibroblasts to one of BGM-like homogeneity. The transition from a mixed cell population to a single cell type raised the question as to the species of these cells. The results of mixed agglutination and immunofluorescence studies for detection of monkey and mouse antigens are shown in Table 1. Positive mixed agglutination was observed only when BGM/MV cells were treated with rabbit antiserum directed against BGM cell antigen; the pattern was one of homogeneity and was identical to that observed when BGM cells were treated with this antiserum. No adsorption of indicator cells was observed when BGM/MV cells were treated with rabbit antiserum directed against mouse antigen or with normal rabbit serum. Mouse embryo fibroblasts and BGM cells were reactive only with homologous antisera. TABLE 1. Characterization of species antigen in BGM/MV cells
Antisera Cells tested
Rabbit Rabbit antiantimouse BGM
Control
serum Normal rabbit serum
Mixed agglutinationa
BGM/MV BGM MEF
_ +
+ + -
_ _ -
+
+ +
_
Intracellular and membrane immunofluorescenceb
BGM/MV BGM L-929
-
-
a Cell monolayers were incubated at room temperature for 90 min with rabbit antisera or normal rabbit serum, washed three times with maintenance medium, incubated for 60 min at room temperature with sheep erythrocytes sensitized to rabbit antigen, and read microscopically for cell adherence. bThe indirect immunofluorescence method was employed.
161
Immunofluorescence studies for detection of intracellular monkey and mouse antigens in BGM/MV cells corroborated the findings by mixed agglutination assay. In acetone-fixed preparations, 100% of BGM/MV cells revealed a homogeneous pattern of cytoplasmic fluorescence when treated with rabbit antiserum directed against BGM cell antigen. Rabbit antiserum directed against mouse antigen and normal rabbit serum failed to stain these cells. Membrane immunofluorescence revealed the presence of monkey antigen on 100% of BGM/MV cells; no mouse antigen was detected. BGM and L-929 cell monolayers revealed intracellular and membrane staining only with homologous antisera. Interferon studies. Procedures for determination of species antigen in and on BGM/MV cells revealed the presence of only BGM antigen. It was possible, however, that these cells possessed inducible biological activities which were murine in nature. The detection of such activities would serve as an indirect means of species characterization. Experiments were therefore undertaken to determine the species specificity of spontaneous and virus-induced interferon in these cells as well as the response of these cells to exogenous interferons. Low levels of spontaneously elaborated interferon were detected in BGM/MV culture fluids. Culture fluids taken at various passage levels after 390 days postseeding produced titers of 2 U/ml as assayed in BGM cells; no activity was observed in L-929 cells. This antiviral activity was characterized as one mediated by interferon by its pH stability (stable at pH 2 for 18 h at 4 C), non-sedimentability (85,000 x g for 60 min), and lack of virus specificity (active against measles, mumps, and VSV). Next, experiments were conducted with virus-induced interferon. The results of these experiments are shown in Table 2. Supernatant culture fluids harvested from NDV-infected BGM/MV cultures conferred an antiviral state to both BGM and L-929 cells; titers of 141 and 76 U/ml, respectively, were obtained. BGM interferon gave a titer of 192 U/ml in homologous cells and 16 U/ml in L-929 cells. L-929 cell interferon induced an antiviral state only in homologous cells; a titer of > 1,024 was obtained. BGM/MV, BGM, and L-929 cell interferon failed to confer an antiviral state on BGM/MV cells; titers of less than 4 U/ml were obtained using the microtiter assay procedure. Challenge of BGM/MV cells with exogenous viruses. As a means of further characterizing the BGM/MV cell line, triplicate cultures of BGM/MV cells were challenged with selected
162
MENNA, COLLINS, AND FLANAGAN
viruses at a multiplicity of infection of 0.1 PFU/cell, and the titers (as assayed in BGM cells) recoverable from pooled culture fluids and pooled clarified cell lysates after 24 h incubation at 37 C were compared with similarly treated cultures of BGM cells. The results of these experiments are shown in Table 3. Measles virus replicated to low titers in BGM cells; the titers of infectious virus recovered from the culture fluid and cell lysate was 2.2 x 102 and 9.4 x 102 PFU/ml, respectively. In BGM/MV cells only 3 PFU/ml were detected in the culture fluid, whereas no infectious virus was detected in the cell lysate. Mumps virus replicated in both BGM and BGM/MV cells; the cellassociated and supernatant titers when expressed as PFU per milliliter were greater in TABLE 2. Interferon studies with BGM/MV cultures Interferon sourceb
BGM/MV BGM L-929
Interferon activity in:a
BGMc
L-929c
BGM/MVd
141
76 16 > 1,024
