INFECTION AND IMMUNrrY, Jan. 1975, p. 152-158 Copyright 0 1975 American Society for Microbiology
Vol. 11, No. 1 Printed in U.S.A.
Characterization of an In Vitro Persistent-State Measles Virus Infection: Establishment and Virological Characterization of 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
The parameters of a persistent-state measles virus infection in BGM/MV cells examined. The BGM/MV cell line was established by cocultivation of measles virus-infected primary C3H mouse brain cells with a stable line of African green monkey kidney cells (BGM). Initially, a morphologically mixed population of cells existed: BGM-like (epithelioid) and fibroblasts. Gradually the fibroblasts were replaced by BGM-like cells, resulting in a morphologically homogeneous population. Measles cytopathic effect was noted 2 days after initiation of this culture and persisted for approximately 290 days. The time of disappearance of viral cytopathic effect corresponded to the time at which morphological homogeneity was reached. Low titers of infectious measles virus were detected in the BGM/MV culture up to 20 days postseeding; thereafter none was observed. After 440 days in culture, 100% of BGM/MV cells demonstrated intractyoplasmic measles antigen by immunofluorescence. Nuclear fluorescence was never observed. Electron microscopy revealed the presence of measles virus nucleocapsid within and almost completely filling the cytoplasm of BGM/MV cells. The plasma membrane of these cells appeared normal; no maturing or budding particles were observed. Measles virus hemagglutinin was not detected in either clarified cell lysates or in supernatant culture fluids. Cell membrane alteration by measles virus was detected in less than 1% of these cells by hemadsorption and by membrane immunofluorescence. The hemadsorption activity of the cells could be enhanced (30 to 70%) by treatment with actinomycin D or enucleation with cytochalasin B; these treatments, however, were unsuccessful in inducing detectable levels of measles hemagglutinin. Treatment of BGM/MV cells with 5-bromo-2'-deoxyuridine (BUdR) at 5 to 50 Ag/ml and cytosine arabinoside at 1 to 50 gg/ml failed to enhance hemadsorption activity. Doses of 5bromo-2'-deoxyuridine ranging from 5 to 200 ,ug/ml and of actinomycin D ranging from 0.1 to 10 Ag/ml were ineffective in inducing the synthesis of infectious virus. Various physical methods of induction of infectious virus was also unsuccessful. were
Persistent-state measles virus infections have been established in various cell lines, and the parameters characterizing the virus-host cell relationship were investigated (2, 4, 10). These phenomena are of interest in regard to certain in vivo correlates, such as subacute sclerosing panencephalitis and possibly multiple sclerosis. The nature of the virus-host cell relationship in an in vitro persistent-state infection may be dependent on many variables: the strain of infecting virus, the species of cell in which the virus is grown, the temperature of incubation, and the capacity of the cell to produce interferon in response to the virus infection, as well as superimposed factors such as addition of virus-specific antibody and various metabolic
inhibitors to cell culture fluids. Walker (14) has classified persistent-state virus infections or viral carrier states of animal cell cultures into four types: (i) persistent infections of genetically resistant cells, (ii) persistent infections of genetically susceptible cells protected by antiviral factors in the medium, -(iii) persistent infections of genetically susceptible cells protected by interference or interferon, and (iv) regulated virus infections of cells in culture. Regulated virus infections of cells in culture appear to be under some form of intracellular control. Many of the cells in the population are infected and will divide and grow into infected clones. The replicative cycle of the virus does not proceed in the normal fashion, leading to
152
VOL- 11, 1975
EVOLUTION OF BGM/MV CELL LINE
153
gate. Cover slips were washed for 30 min in PBS, mounted in buffered glycerol, and observed using an American Optical Fluorolume microscope. Detection of measles virus membrane antigen. Detection of measles virus membrane antigen on BGM/MV cells was accomplished using unfixed cell monolayers and the same protocol as for detection of intracellular viral antigen. HAD assay. A 0.5% suspension of rhesus monkey erythrocytes in maintenance medium was used in hemadsorption (HAD) studies. Only those lots of erythrocytes capable of being agglutinated by measles hemagglutinin were used. Cell monolayers were MATERIALS AND METHODS washed three times with maintenance medium, incuCell culture. BGM cells, a stable line of African bated with rhesus monkey erythrocytes for 60 min at green monkey kidney cells (1), were passaged weekly room temperature, washed three times with PBS, and by trypsinization and seeded at 9 x 104 cells/ml using observed microscopically. HA assay. Hemagglutination (HA) assays for deEagle minimal essential medium supplemented with 10% fetal calf serum (FCS), 100 ug of streptomycin tection of measles hemagglutinin were performed per ml, and 100 U of penicillin per ml. Maintenance using a microtiter procedure (11). Serial twofold medium consisted of Eagle minimal essential medium dilutions of sonified samples and standard measles hemagglutinin (Flow Laboratories Co. Inc., Rockville, supplemented with 5% FCS and antibiotics. Viruses. The Edmonston strain of measles virus, Md.) were made in 96-welled polystyrene plates adapted to growth in mice by Imagawa and Adams (Linbro Chemical Co., New Haven, Conn.) using a (5), was provided by Fred Rapp, Hershey, Pa. In our sodium citrate buffer (pH 7.4) containing 0.1% gelatin laboratory the virus was serially passaged in suckling as diluent. Fresh rhesus monkey erythrocytes were C3H mice using an intracerebral route of inoculation added, and specimens were incubated at 37 C for 60 (0.01 ml of undiluted stock virus per mouse). Stock min and read. Induction studies. Various physical and chemical virus was prepared from the brains of moribund mice as 10% (wt/vol) homogenates in Medium 199 supple- agents were used in attempts to induce the synthesis mented with 2% FCS, antibiotics as above, and 10% of infectious measles virus or enhance the HAD dimethyl sulfoxide. Preparations titered 5 x 10' to 2.3 activity of BGM/MV cultures. Partially confluent x 105 plaque-forming units/ml in BGM cells. monolayers of BGM/MV cells were exposed to BUdR Chemicals. Actinomycin D, cytosine arabinoside at 5, 25, 50, 100, and 200 ug/ml and confluent (1-p-D-arabinofuranosyl cytosine), BUdR (5-bromo- monolayers were exposed to actinomycin D at 0.1, 1.0, 2'-deoxyuridine), and colchicine were purchased 5.0, and 10 ug/ml, and incubation at 37 C for 24 h was from Sigma Chemical Co., Inc., St. Louis, Mo. Cyto- carried out. Infectivity assays were then performed on chalasin B was purchased from Aldrich Chemical Co., clarified cell lysates. In attempts to enhance the HAD Inc., Milwaukee, Wis. activity of BGM/MV cells, monolayers were incuInfectivity assays. Infectious measles virus was bated at 37 C for 24 h with the following agents in assayed by a plaque formation method in BGM cells, maintenance medium: actinomycin D at 1.0 jg/ml; as previously described for mumps virus (3). BUdR at 5, 25, and 50 Ag/ml; cytosine arabinoside at Cocultivation procedure. As a means of detecting 1, 10, and 50 ,g/ml; and colchicine at 1.0 jig/ml. HAD infectious measles virus in the BGM/MV cell line assays were then performed. (established by cocultivation of measles virusOther procedures used in attempts to induce the infected primary mouse brain cells with BGM cells), formation of infectious measles virus in BGM/MV BGM/MV cells were cocultivated with BGM cells. cultures included incubation at 30 C for 36 h and Confluent monolayers of BGM/MV and BGM cells ultraviolet irradiation. Duplicate cultures of were trypsinized, and the cell concentrations were BGM/MV cells grown in plastic tissue culture flasks adjusted to 106 cells/ml in Eagle minimal essential (35 by 10 mm; Falcon Plastics, Oxnard, Calif.) at 37 C medium supplemented with 10% FCS and antibiotics. in an atmosphere of 5% CO2 were exposed to ultravioCocultivation was accomplished by mixing these cells let irradiation for periods of 5, 10, and 15 s at a in a proportion of 3 volumes of BGM to 1 volume of distance of 35 cm using a Westinghouse Sterilamp AMP 782 L-30. In these experiments supernatant BGM/MV and seeding the cell mixture. Detection of intracellular measles antigen in culture fluids and cell lysates prepared from duplicate BGM/MV cells by immunofluorescence. Immuno- cultures after ultraviolet irradiation or low-temperafluorescence procedures for detection of measles virus ture incubation were pooled, centrifuged at 900 x g antigen in BGM/MV cells were performed by the for 15 min at 4 C, and assayed for infectious virus in indirect method. Cell monolayers were acetone fixed BGM cells. for 10 min at 4 C, treated with rabbit antiserum Enucleation of BGM/MV cells with cytochalasin prepared against measles antigen for 30 min at 37 C, B. The method of enucleation employed was that washed in phosphate-buffered saline (PBS) for 15 described by Prescott et al. (8). Cytochalasin B was min, and then incubated at 37 C for 30 min with a dissolved as a 0.1% (wt/vol) solution in dimethyl fluorescein-labeled goat anti-rabbit globulin conju- sulfoxide. BGM/MV cells were grown on round cover
virus release and viral cytopathic effect (CPE). Resistance to superinfection with the same virus is frequently observed, whereas little or no resistance is seen with related or unrelated viruses. This paper and the accompanying paper (6) describe a regulated type of persistent-state measles virus infection of a cell line established in our laboratory. The evolution of this cell line is described with respect to cellular morphology and state of virus infection.
154
MENNA, COLLINS, AND FLANAGAN
INFECT. IMMUN.
slips 18 mm in diameter. Cover slips with confluent TABLE 1. Evolutionary characteristics of the monolayers were inserted into 30-ml glass centrifuge BGM/MV cell line tubes (Corex no. 8445) containing 5 ml of maintePassage Viral Cellular nance medium (37 C) plus cytochalasin B at 5 to 50 Days PS level morphology CPEa ,ug/ml; the cell monolayers were oriented towards the bottom of the tube. Tubes were centrifuged for 40 min 1-4 1-288 Mixedb + at 3,000 x g in a Sorvall RC2-B centrifuge using an 289-750 5-71 BGM-like SS-34 rotor. After centrifugation, the cover slips were placed in maintenance medium and incubated at aViral CPE consisted of "spindle-cells" and syn37 C in 5% CO2. The percent of enucleated cells was cytia. approximated by staining with methylene blue. As a 'Both fibroblasts and BGM-like (epithelioid) cells control, BGM/MV monolayers were centrifuged as were noted. above in maintenance medium devoid of cytochalasin B and containing dimethyl sulfoxide equal in concen- sisted morphologically of two cell types: fibrotration to the dimethyl sulfoxide present in the stock blasts and BGM-like cells (epithelioid). With cytochalasin B added to the enucleation medium. time, a change in the relative proportion of Preparation of specimens for electron these cell types occurred, and by approximately microscopy. BGM/MV cell monolayers were removed from the surface of tissue culture flasks by scraping 290 days PS only BGM-like cells were observed. into PBS. Cell suspensions were centrifuged for 10 Cytopathological changes typical of those inmin at 300 x g and washed twice in PBS, and the final duced by measles ("spindle-cells" and syncytia) pellet was fixed in 2.5% glutaraldehyde for 15 min at were observed in the BGM/MV culture on day 2 4 C. Cell pellets were rinsed once in buffer and fixed PS. The extent of CPE decreased with time and in 1% osmic acid in a Millonig buffer (pH 7.4) at 4 C by 289 days PS none was observed. The time of for 2 h. The fixed materials were then dehydrated in disappearance of viral CPE closely approxgraded dilutions of ethanol, embedded in Epon aral- imated the time at which the morphological dite, and sectioned. Preparations were stained with transition was complete. uranyl acetate and examined with a Siemens 101 Production of infectious virus. Early in the electron microscope. evolution of the BGM/MV cell line, low levels of
1I
RESULTS Establishment of the BGM/MV cell line. The BGM/MV cell line was established by cocultivation of primary mouse (C3H strain) brain cells infected (in vivo) with the mouse brain-adapted Edmonston strain of measles virus with BGM cells. Newborn C3H mice (less than 24 h old) were inoculated intracerebrally with the mouse brain-adapted Edmonston strain measles virus (0.01 ml of undiluted stock virus per mouse), and a brain cell suspension was prepared by trypsinization of brains removed from moribund mice. Extracted mouse brain 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 ,g of streptomycin per ml, and 200 U of penicillin per ml. Equal cell concentrations of BGM and mouse brain cells were mixed in a proportion of 3 volumes of BGM to 1 volume of mouse and the mixture was seeded. The passage history, evolution of cell morphology, and viral CPE are shown in Table
infectious virus were detected. Supernatant culture fluids harvested on days 7 and 20 PS produced titers of 10 and 200 plaque-forming units/ml, respectively. Infectivity assays performed on culture fluids after 20 days PS failed to demonstrate the presence of infectious virus. BGM/MV cell lysates prepared between day 242 and day 750 PS by a freeze-thaw procedure were also negative for infectious virus. HAD and HA assays. HA assays performed on sonified BGM/MV culture fluids and sonified cell lysates prepared after 290 days PS failed to demonstrate the presence of measles hemagglutinin. HAD assays performed after 290 days PS resulted in positive adsorption on less than 1% of the BGM/MV cells in the monolayer. HA and HAD assays were not performed prior to 290 days PS. Detection of measles antigen by immunofluorescence. The status of measles virus infection in BGM/MV cells was further elucidated by immunofluorescence studies. The results of these studies are shown in Table 2. Immunofluorescence studies for detection of intracellular measles antigen were performed at 1. BGM/MV cells were fed weekly with mainte- passage levels 2, 7, 15, and 26 and at various nance medium consisting of Eagle minimal passages up to and including passage 70. At essential medium supplemented with 5% FCS passage levels 2 and 7, 60 to 70% of the cells reand passaged for the first time at 260 days vealed cytoplasmic fluorescent staining with postseeding (PS). Since the first passage, measles antiserum. Fluorescence was found BGM/MV cells have been passaged weekly. throughout the cytoplasm of BGM/MV cells When initiated, the BGM/MV cell line con- and was coarse-granular to globular in appear-
VOL. 11, 1975
EVOLUTION OF BGM/MV CELL LINE
ance. At passage level 2, foci of fluorescence consisted mainly of giant cells, whereas singlenucleated fluorescent cells predominated at passage 7 and all subsequent passages. At passage level 15, 80 to 90% of BGM/MV cells contained measles antigen. By passage 26, 100% of these cells contained measles antigen (Fig. 1); the cellular location and pattern of fluorescence was the same as that seen at earlier passages. Nuclear fluorescence was never observed. Plasma membrane-associated measles antigen was detected in less than 1% of BGM/MV cells by immunofluorescence. Studies for the detection of plasma membrane-associated measles antigen were performed after morphological homogeneity had been reached (290 days PS). Electron microscopic studies of BGM/MV cells. Electron microscopic examination of TABLE 2. Detection of intracellular measles virus antigen in BGM/MV cells by immunofluorescencea Passage level
with measles Cellsantigen (%)
2 7 15 26-70
60-70 60-70 80-90 100
Cells were acetone fixed, and an indirect immunofluorescence assay for measles virus antigen was performed. a
155
BGM/MV cells after 290 days PS revealed the presence of granular and filamentous structures within and almost completely filling the cytoplasm of the cells (Fig. 2). These structures were not observed in close association with the cytoplasmic membrane of BGM/MV cells nor were budding virus particles observed. The outer diameter of the granular structures corresponded to the width of the filamentous structures (approximately 30 to 40 nm), suggesting that the granular structures represented crosssections of the filamentous structures. The inner diameter of the granular structures measured approximately 10 to 15 nm. Attempts to induce infectious virus in BGM/MV cells. Induction of infectious measles virus in BGM/MV cultures was attempted using various physical and chemical agents. No infectious measles virus was detected in supernatant culture fluids or in clarified cell lysates when BGM/MV cultures were treated with BUdR at 5, 25, 50, 100, and 200 ug/ml. Actinomycin D at 0.1, 1.0, 5, and 10 Ag/ml also failed to induce detectable levels of infectious virus. Ultraviolet irradiation of BGM/MV cells, as well as incubation at 30 C for 36 h, also proved ineffective. Cocultivation of BGM/MV cells at various passage levels with BGM cells failed to elicit infectious virus or
CPE. Experiments were also carried out to determine the effect of various agents on the HAD activity of BGM/MV cell monolayers. The re-
FIG. 1. Immunofluorescence staining of BGM/MV cells for measles antigen. x450. Measles antigen was observed in the cytoplasm of 100% of BGM/MV cells. Nuclear staining was never observed. Note the granular and globular appearance of measles antigen staining.
1566
INFECT. IMMUN.
MENNA, COLLINS, AND FLANAGAN
cell lysates from cytochalasin B enucleated cultures were negative. The deoxyribonucleic acid inhibitors BUdR and cytosine arabinoside failed to enhance HAD activity. Colchicine increased HAD activity to a low degree (approximately 10%).
sults of these studies are shown in Table 3. Duplicate cultures of BGM/MV cells were incubated with these agents at stated concentrations in maintenance medium for 24 h at 37 C, at which time an HAD assay was performed. As a control, duplicate BGM/MV cell monolayers were treated with maintenance medium alone. Less then 1% of the cells treated with maintenance medium were HAD positive. Actinomycin D at 1.0 Ag/ml enhanced the HAD activity of BGM/MV cells significantly; approximately 70% of the cells were HAD positive. Concentrations of actinomycin D greater than or less than 1.0 ,ug/ml were less effective. Although actinomycin D enhanced the HAD activity of BGM/MV cells, comparable doses of this drug (1 and 2 ug/ml) failed to induce detectable levels of HA activity. HAD assays were also performed on BGM/MV monolayers enucleated with cytochalasin B at 10, 25, and 50 gg/ml. Cells were enucleated with cytochalasin B and incubated for 24 h, and an HAD assay was performed. Enucleation of BGM/MV cells using cytochalasin B at 10 ,ug/ml increased HAD activity 30 to 40%. Concentrations of cytochalasin B greater than 10 Ag/ml failed to further enhance enucleation and HAD activity. HA tests on sonified supernatants and sonified
DISCUSSION The BGM/MV cell line was established in our laboratory by cocultivation of measles virusTABLE 3. Enhancement of HAD activity of BGM/MV cells Treatment
~
1.0
-70 -30-40
10, 25, & 50 5, 10, 15, 20, 25, & 50 1, 5, 10, 20, &
Cytosine arabinosidea
50 1.0
Colchicinea
Vol. 11, No. 1 Printed in U.S.A.
Characterization of an In Vitro Persistent-State Measles Virus Infection: Establishment and Virological Characterization of 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
The parameters of a persistent-state measles virus infection in BGM/MV cells examined. The BGM/MV cell line was established by cocultivation of measles virus-infected primary C3H mouse brain cells with a stable line of African green monkey kidney cells (BGM). Initially, a morphologically mixed population of cells existed: BGM-like (epithelioid) and fibroblasts. Gradually the fibroblasts were replaced by BGM-like cells, resulting in a morphologically homogeneous population. Measles cytopathic effect was noted 2 days after initiation of this culture and persisted for approximately 290 days. The time of disappearance of viral cytopathic effect corresponded to the time at which morphological homogeneity was reached. Low titers of infectious measles virus were detected in the BGM/MV culture up to 20 days postseeding; thereafter none was observed. After 440 days in culture, 100% of BGM/MV cells demonstrated intractyoplasmic measles antigen by immunofluorescence. Nuclear fluorescence was never observed. Electron microscopy revealed the presence of measles virus nucleocapsid within and almost completely filling the cytoplasm of BGM/MV cells. The plasma membrane of these cells appeared normal; no maturing or budding particles were observed. Measles virus hemagglutinin was not detected in either clarified cell lysates or in supernatant culture fluids. Cell membrane alteration by measles virus was detected in less than 1% of these cells by hemadsorption and by membrane immunofluorescence. The hemadsorption activity of the cells could be enhanced (30 to 70%) by treatment with actinomycin D or enucleation with cytochalasin B; these treatments, however, were unsuccessful in inducing detectable levels of measles hemagglutinin. Treatment of BGM/MV cells with 5-bromo-2'-deoxyuridine (BUdR) at 5 to 50 Ag/ml and cytosine arabinoside at 1 to 50 gg/ml failed to enhance hemadsorption activity. Doses of 5bromo-2'-deoxyuridine ranging from 5 to 200 ,ug/ml and of actinomycin D ranging from 0.1 to 10 Ag/ml were ineffective in inducing the synthesis of infectious virus. Various physical methods of induction of infectious virus was also unsuccessful. were
Persistent-state measles virus infections have been established in various cell lines, and the parameters characterizing the virus-host cell relationship were investigated (2, 4, 10). These phenomena are of interest in regard to certain in vivo correlates, such as subacute sclerosing panencephalitis and possibly multiple sclerosis. The nature of the virus-host cell relationship in an in vitro persistent-state infection may be dependent on many variables: the strain of infecting virus, the species of cell in which the virus is grown, the temperature of incubation, and the capacity of the cell to produce interferon in response to the virus infection, as well as superimposed factors such as addition of virus-specific antibody and various metabolic
inhibitors to cell culture fluids. Walker (14) has classified persistent-state virus infections or viral carrier states of animal cell cultures into four types: (i) persistent infections of genetically resistant cells, (ii) persistent infections of genetically susceptible cells protected by antiviral factors in the medium, -(iii) persistent infections of genetically susceptible cells protected by interference or interferon, and (iv) regulated virus infections of cells in culture. Regulated virus infections of cells in culture appear to be under some form of intracellular control. Many of the cells in the population are infected and will divide and grow into infected clones. The replicative cycle of the virus does not proceed in the normal fashion, leading to
152
VOL- 11, 1975
EVOLUTION OF BGM/MV CELL LINE
153
gate. Cover slips were washed for 30 min in PBS, mounted in buffered glycerol, and observed using an American Optical Fluorolume microscope. Detection of measles virus membrane antigen. Detection of measles virus membrane antigen on BGM/MV cells was accomplished using unfixed cell monolayers and the same protocol as for detection of intracellular viral antigen. HAD assay. A 0.5% suspension of rhesus monkey erythrocytes in maintenance medium was used in hemadsorption (HAD) studies. Only those lots of erythrocytes capable of being agglutinated by measles hemagglutinin were used. Cell monolayers were MATERIALS AND METHODS washed three times with maintenance medium, incuCell culture. BGM cells, a stable line of African bated with rhesus monkey erythrocytes for 60 min at green monkey kidney cells (1), were passaged weekly room temperature, washed three times with PBS, and by trypsinization and seeded at 9 x 104 cells/ml using observed microscopically. HA assay. Hemagglutination (HA) assays for deEagle minimal essential medium supplemented with 10% fetal calf serum (FCS), 100 ug of streptomycin tection of measles hemagglutinin were performed per ml, and 100 U of penicillin per ml. Maintenance using a microtiter procedure (11). Serial twofold medium consisted of Eagle minimal essential medium dilutions of sonified samples and standard measles hemagglutinin (Flow Laboratories Co. Inc., Rockville, supplemented with 5% FCS and antibiotics. Viruses. The Edmonston strain of measles virus, Md.) were made in 96-welled polystyrene plates adapted to growth in mice by Imagawa and Adams (Linbro Chemical Co., New Haven, Conn.) using a (5), was provided by Fred Rapp, Hershey, Pa. In our sodium citrate buffer (pH 7.4) containing 0.1% gelatin laboratory the virus was serially passaged in suckling as diluent. Fresh rhesus monkey erythrocytes were C3H mice using an intracerebral route of inoculation added, and specimens were incubated at 37 C for 60 (0.01 ml of undiluted stock virus per mouse). Stock min and read. Induction studies. Various physical and chemical virus was prepared from the brains of moribund mice as 10% (wt/vol) homogenates in Medium 199 supple- agents were used in attempts to induce the synthesis mented with 2% FCS, antibiotics as above, and 10% of infectious measles virus or enhance the HAD dimethyl sulfoxide. Preparations titered 5 x 10' to 2.3 activity of BGM/MV cultures. Partially confluent x 105 plaque-forming units/ml in BGM cells. monolayers of BGM/MV cells were exposed to BUdR Chemicals. Actinomycin D, cytosine arabinoside at 5, 25, 50, 100, and 200 ug/ml and confluent (1-p-D-arabinofuranosyl cytosine), BUdR (5-bromo- monolayers were exposed to actinomycin D at 0.1, 1.0, 2'-deoxyuridine), and colchicine were purchased 5.0, and 10 ug/ml, and incubation at 37 C for 24 h was from Sigma Chemical Co., Inc., St. Louis, Mo. Cyto- carried out. Infectivity assays were then performed on chalasin B was purchased from Aldrich Chemical Co., clarified cell lysates. In attempts to enhance the HAD Inc., Milwaukee, Wis. activity of BGM/MV cells, monolayers were incuInfectivity assays. Infectious measles virus was bated at 37 C for 24 h with the following agents in assayed by a plaque formation method in BGM cells, maintenance medium: actinomycin D at 1.0 jg/ml; as previously described for mumps virus (3). BUdR at 5, 25, and 50 Ag/ml; cytosine arabinoside at Cocultivation procedure. As a means of detecting 1, 10, and 50 ,g/ml; and colchicine at 1.0 jig/ml. HAD infectious measles virus in the BGM/MV cell line assays were then performed. (established by cocultivation of measles virusOther procedures used in attempts to induce the infected primary mouse brain cells with BGM cells), formation of infectious measles virus in BGM/MV BGM/MV cells were cocultivated with BGM cells. cultures included incubation at 30 C for 36 h and Confluent monolayers of BGM/MV and BGM cells ultraviolet irradiation. Duplicate cultures of were trypsinized, and the cell concentrations were BGM/MV cells grown in plastic tissue culture flasks adjusted to 106 cells/ml in Eagle minimal essential (35 by 10 mm; Falcon Plastics, Oxnard, Calif.) at 37 C medium supplemented with 10% FCS and antibiotics. in an atmosphere of 5% CO2 were exposed to ultravioCocultivation was accomplished by mixing these cells let irradiation for periods of 5, 10, and 15 s at a in a proportion of 3 volumes of BGM to 1 volume of distance of 35 cm using a Westinghouse Sterilamp AMP 782 L-30. In these experiments supernatant BGM/MV and seeding the cell mixture. Detection of intracellular measles antigen in culture fluids and cell lysates prepared from duplicate BGM/MV cells by immunofluorescence. Immuno- cultures after ultraviolet irradiation or low-temperafluorescence procedures for detection of measles virus ture incubation were pooled, centrifuged at 900 x g antigen in BGM/MV cells were performed by the for 15 min at 4 C, and assayed for infectious virus in indirect method. Cell monolayers were acetone fixed BGM cells. for 10 min at 4 C, treated with rabbit antiserum Enucleation of BGM/MV cells with cytochalasin prepared against measles antigen for 30 min at 37 C, B. The method of enucleation employed was that washed in phosphate-buffered saline (PBS) for 15 described by Prescott et al. (8). Cytochalasin B was min, and then incubated at 37 C for 30 min with a dissolved as a 0.1% (wt/vol) solution in dimethyl fluorescein-labeled goat anti-rabbit globulin conju- sulfoxide. BGM/MV cells were grown on round cover
virus release and viral cytopathic effect (CPE). Resistance to superinfection with the same virus is frequently observed, whereas little or no resistance is seen with related or unrelated viruses. This paper and the accompanying paper (6) describe a regulated type of persistent-state measles virus infection of a cell line established in our laboratory. The evolution of this cell line is described with respect to cellular morphology and state of virus infection.
154
MENNA, COLLINS, AND FLANAGAN
INFECT. IMMUN.
slips 18 mm in diameter. Cover slips with confluent TABLE 1. Evolutionary characteristics of the monolayers were inserted into 30-ml glass centrifuge BGM/MV cell line tubes (Corex no. 8445) containing 5 ml of maintePassage Viral Cellular nance medium (37 C) plus cytochalasin B at 5 to 50 Days PS level morphology CPEa ,ug/ml; the cell monolayers were oriented towards the bottom of the tube. Tubes were centrifuged for 40 min 1-4 1-288 Mixedb + at 3,000 x g in a Sorvall RC2-B centrifuge using an 289-750 5-71 BGM-like SS-34 rotor. After centrifugation, the cover slips were placed in maintenance medium and incubated at aViral CPE consisted of "spindle-cells" and syn37 C in 5% CO2. The percent of enucleated cells was cytia. approximated by staining with methylene blue. As a 'Both fibroblasts and BGM-like (epithelioid) cells control, BGM/MV monolayers were centrifuged as were noted. above in maintenance medium devoid of cytochalasin B and containing dimethyl sulfoxide equal in concen- sisted morphologically of two cell types: fibrotration to the dimethyl sulfoxide present in the stock blasts and BGM-like cells (epithelioid). With cytochalasin B added to the enucleation medium. time, a change in the relative proportion of Preparation of specimens for electron these cell types occurred, and by approximately microscopy. BGM/MV cell monolayers were removed from the surface of tissue culture flasks by scraping 290 days PS only BGM-like cells were observed. into PBS. Cell suspensions were centrifuged for 10 Cytopathological changes typical of those inmin at 300 x g and washed twice in PBS, and the final duced by measles ("spindle-cells" and syncytia) pellet was fixed in 2.5% glutaraldehyde for 15 min at were observed in the BGM/MV culture on day 2 4 C. Cell pellets were rinsed once in buffer and fixed PS. The extent of CPE decreased with time and in 1% osmic acid in a Millonig buffer (pH 7.4) at 4 C by 289 days PS none was observed. The time of for 2 h. The fixed materials were then dehydrated in disappearance of viral CPE closely approxgraded dilutions of ethanol, embedded in Epon aral- imated the time at which the morphological dite, and sectioned. Preparations were stained with transition was complete. uranyl acetate and examined with a Siemens 101 Production of infectious virus. Early in the electron microscope. evolution of the BGM/MV cell line, low levels of
1I
RESULTS Establishment of the BGM/MV cell line. The BGM/MV cell line was established by cocultivation of primary mouse (C3H strain) brain cells infected (in vivo) with the mouse brain-adapted Edmonston strain of measles virus with BGM cells. Newborn C3H mice (less than 24 h old) were inoculated intracerebrally with the mouse brain-adapted Edmonston strain measles virus (0.01 ml of undiluted stock virus per mouse), and a brain cell suspension was prepared by trypsinization of brains removed from moribund mice. Extracted mouse brain 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 ,g of streptomycin per ml, and 200 U of penicillin per ml. Equal cell concentrations of BGM and mouse brain cells were mixed in a proportion of 3 volumes of BGM to 1 volume of mouse and the mixture was seeded. The passage history, evolution of cell morphology, and viral CPE are shown in Table
infectious virus were detected. Supernatant culture fluids harvested on days 7 and 20 PS produced titers of 10 and 200 plaque-forming units/ml, respectively. Infectivity assays performed on culture fluids after 20 days PS failed to demonstrate the presence of infectious virus. BGM/MV cell lysates prepared between day 242 and day 750 PS by a freeze-thaw procedure were also negative for infectious virus. HAD and HA assays. HA assays performed on sonified BGM/MV culture fluids and sonified cell lysates prepared after 290 days PS failed to demonstrate the presence of measles hemagglutinin. HAD assays performed after 290 days PS resulted in positive adsorption on less than 1% of the BGM/MV cells in the monolayer. HA and HAD assays were not performed prior to 290 days PS. Detection of measles antigen by immunofluorescence. The status of measles virus infection in BGM/MV cells was further elucidated by immunofluorescence studies. The results of these studies are shown in Table 2. Immunofluorescence studies for detection of intracellular measles antigen were performed at 1. BGM/MV cells were fed weekly with mainte- passage levels 2, 7, 15, and 26 and at various nance medium consisting of Eagle minimal passages up to and including passage 70. At essential medium supplemented with 5% FCS passage levels 2 and 7, 60 to 70% of the cells reand passaged for the first time at 260 days vealed cytoplasmic fluorescent staining with postseeding (PS). Since the first passage, measles antiserum. Fluorescence was found BGM/MV cells have been passaged weekly. throughout the cytoplasm of BGM/MV cells When initiated, the BGM/MV cell line con- and was coarse-granular to globular in appear-
VOL. 11, 1975
EVOLUTION OF BGM/MV CELL LINE
ance. At passage level 2, foci of fluorescence consisted mainly of giant cells, whereas singlenucleated fluorescent cells predominated at passage 7 and all subsequent passages. At passage level 15, 80 to 90% of BGM/MV cells contained measles antigen. By passage 26, 100% of these cells contained measles antigen (Fig. 1); the cellular location and pattern of fluorescence was the same as that seen at earlier passages. Nuclear fluorescence was never observed. Plasma membrane-associated measles antigen was detected in less than 1% of BGM/MV cells by immunofluorescence. Studies for the detection of plasma membrane-associated measles antigen were performed after morphological homogeneity had been reached (290 days PS). Electron microscopic studies of BGM/MV cells. Electron microscopic examination of TABLE 2. Detection of intracellular measles virus antigen in BGM/MV cells by immunofluorescencea Passage level
with measles Cellsantigen (%)
2 7 15 26-70
60-70 60-70 80-90 100
Cells were acetone fixed, and an indirect immunofluorescence assay for measles virus antigen was performed. a
155
BGM/MV cells after 290 days PS revealed the presence of granular and filamentous structures within and almost completely filling the cytoplasm of the cells (Fig. 2). These structures were not observed in close association with the cytoplasmic membrane of BGM/MV cells nor were budding virus particles observed. The outer diameter of the granular structures corresponded to the width of the filamentous structures (approximately 30 to 40 nm), suggesting that the granular structures represented crosssections of the filamentous structures. The inner diameter of the granular structures measured approximately 10 to 15 nm. Attempts to induce infectious virus in BGM/MV cells. Induction of infectious measles virus in BGM/MV cultures was attempted using various physical and chemical agents. No infectious measles virus was detected in supernatant culture fluids or in clarified cell lysates when BGM/MV cultures were treated with BUdR at 5, 25, 50, 100, and 200 ug/ml. Actinomycin D at 0.1, 1.0, 5, and 10 Ag/ml also failed to induce detectable levels of infectious virus. Ultraviolet irradiation of BGM/MV cells, as well as incubation at 30 C for 36 h, also proved ineffective. Cocultivation of BGM/MV cells at various passage levels with BGM cells failed to elicit infectious virus or
CPE. Experiments were also carried out to determine the effect of various agents on the HAD activity of BGM/MV cell monolayers. The re-
FIG. 1. Immunofluorescence staining of BGM/MV cells for measles antigen. x450. Measles antigen was observed in the cytoplasm of 100% of BGM/MV cells. Nuclear staining was never observed. Note the granular and globular appearance of measles antigen staining.
1566
INFECT. IMMUN.
MENNA, COLLINS, AND FLANAGAN
cell lysates from cytochalasin B enucleated cultures were negative. The deoxyribonucleic acid inhibitors BUdR and cytosine arabinoside failed to enhance HAD activity. Colchicine increased HAD activity to a low degree (approximately 10%).
sults of these studies are shown in Table 3. Duplicate cultures of BGM/MV cells were incubated with these agents at stated concentrations in maintenance medium for 24 h at 37 C, at which time an HAD assay was performed. As a control, duplicate BGM/MV cell monolayers were treated with maintenance medium alone. Less then 1% of the cells treated with maintenance medium were HAD positive. Actinomycin D at 1.0 Ag/ml enhanced the HAD activity of BGM/MV cells significantly; approximately 70% of the cells were HAD positive. Concentrations of actinomycin D greater than or less than 1.0 ,ug/ml were less effective. Although actinomycin D enhanced the HAD activity of BGM/MV cells, comparable doses of this drug (1 and 2 ug/ml) failed to induce detectable levels of HA activity. HAD assays were also performed on BGM/MV monolayers enucleated with cytochalasin B at 10, 25, and 50 gg/ml. Cells were enucleated with cytochalasin B and incubated for 24 h, and an HAD assay was performed. Enucleation of BGM/MV cells using cytochalasin B at 10 ,ug/ml increased HAD activity 30 to 40%. Concentrations of cytochalasin B greater than 10 Ag/ml failed to further enhance enucleation and HAD activity. HA tests on sonified supernatants and sonified
DISCUSSION The BGM/MV cell line was established in our laboratory by cocultivation of measles virusTABLE 3. Enhancement of HAD activity of BGM/MV cells Treatment
~
1.0
-70 -30-40
10, 25, & 50 5, 10, 15, 20, 25, & 50 1, 5, 10, 20, &
Cytosine arabinosidea
50 1.0
Colchicinea
