THE JOURNAL OF INFECTIOUS DISEASES • VOL. 132, NO.2· © 1975 by the University of Chicago. All rights reserved.
AUGUST 1975
MAJOR ARTICLES In Vitro Analysis of Experimental Avian Viral Myocarditis Catherine W. C. Davis, Joseph W. St. Geme, Jr., Frank D. Dufour, and Hawley L. Martin
From the Laboratory for Microbiologic and Immunologic Research, Department of Pediatrics, Harbor General Hospital, UCLA School of Medicine, Torrance, California
Myocarditis represents the only histopathological abnormality in chickens hatched from eggs inoculated with mumps virus during early embryonic development [1]. Titers of virus in the heart are higher than those in other organs throughout and at the completion of embryonic development (authors' unpublished observations) [2]. Initial studies of replication of mumps virus in primary cells cultivated from seven organs of II-day-old chicken embryos showed that cultures of heart cells produced 10-100 times as
much virus during a 72-hr period as did cultures of lung, liver, bursa, brain, kidney, and spleen cells [3]. Studies with heart, lung, and liver cell cultures from 11- to 19-day-old chicken embryos substantiated the initial findings, and we pursued a more detailed analysis of the cellular determinants of mumps virus cardiotropism. The lung and liver were chosen for comparison with the heart because the data from mumps virus infection in vivo have indicated that these organs have intermediate and low susceptibility to the virus, respectively [2].
Received for publication August 27, 1973, and in revised form March 24, 1975. This investigation was supported by a grant from the Los Angeles County Heart Association, by general research support grant no. RR05551 from the National Institutes of Health, and by training grant no. 1TOI AI 00 367 from the V.S. Public Health Service. This paper was presented in part at the 71st annual meeting of the American Society for Microbiology, May 2-7, 1971, Minneapolis, Minnesota. We thank Miriam Arce and Robert Dufau for technical assistance; Drs. David Imagawa and John Holland for constructive review of this work; and Lynn Thompson and Judy Liston for careful preparation of the manuscript. Please address requests for reprints to Dr. Catherine W. C. Davis, Department of Pediatrics, Harbor General Hospital, VCLA School of Medicine, 1000 West Carson Street, Torrance, California 90509.
Materials and Methods
Pathogen-free fertile white leghorn eggs were purchased from Kimber Farms, Fremont, Calif. Cell cultures. Embryonic organs were removed, minced, rinsed free of blood, and trypsinized with several changes of 0.1 %-0.2% trypsin in a solution of glucose, potassium, and sodium for 45 min. The trypsin solution was discarded after sedimentation of the cells by lowspeed centrifugation, and the cells were resuspended in growth medium consisting of Eagle's basal medium and Hanks' balanced salt solution (HBSS) supplemented with 10% fetal bovine Eggs.
125
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The cardiotropism of mumps virus in ovo was reproduced in vitro in a comparative study of the replication of mumps virus in cultivated heart, lung, and liver cells from 11- to 19-day-old chicken embryos. The increased proportion of virus-producing cells in the heart was shown not to be due to greater adsorption of virus to heart celIs. Neither interferon nor other cellular inhibitory products were responsible for the lower level of production of virus by lung and liver cells. The proportion of cells producing virus in relation to the amount of virus adsorbed and the proportion of hemadsorbing cells that released infectious virus were greater in heart cells; this finding suggested more efficient viral release in the heart than at the other sites. Inhibition of protein synthesis by puromycin after infection rapidly stimulated the production of mumps virus only in liver cells. Thus a relatively late stage in viral synthesis may be attenuated in the less susceptible liver cells, and events late in viral synthesis may determine organ-related differences in the multiplication of mumps virus in ovo.
126
Measurement of viral adsorption. Viral adsorption to primary cells and organs was estimated from differences between the titer of virus in the inoculum before adsorption at 35 C and that afterward. Thermoinactivation of virus in MM at this temperature was negligible. Virus was inoculated into cultures in small (O.I-ml) or large (1.0-ml) volumes. Input multiplicities ranged from 0.004 to 4 pfu/cell. Adsorption of virus was measured after 1-3 hr or at 15-min intervals for 2 hr. Fresh heart, lung, and liver samples were prepared by trituration of rinsed organs from 11- or 19-day-old embryos. The organ homogenates were rinsed twice in MM, centrifuged twice, and resuspended. The final pellet was resuspended in 10' times its own volume of undiluted stock virus for adsorption. Measurement of viral penetration. The decrease in the amount of trypsin-releasable virus with time after completion of viral adsorption was used to estimate the rate of viral penetration into cultured heart, lung, and liver cells. After adsorption for 3 hr the residual viral inoculum was removed with a thorough rinse. Thereafter, at 60-min intervals during a 5-hr period, cells were treated with trypsin-EDTA for 15 min. The detached cells were removed by low-speed centrifugation, and the supernatant medium was assayed for virus. In HeLa cells a decrease in amount of trypsin-releasable virus after adsorption was paralleled by an increase in the number of infected cells (authors' unpublished observation). Other experiments showed that the infectivity of mumps virus was affected minimally by trypsin in the presence of cells; in contrast, virus was markedly degraded when treated with trypsin alone. Interferon assay. Culture fluids from cells infected with mumps virus were assayed for interferon on monolayers of primary chicken embryonic fibroblasts obtained from the eviscerated torsos of ll-day-old embryos. The New Jersey strain of vesicular stomatitis virus, propagated once in the chicken embryonic allantois and twice in HeLa cells, was used as the challenge virus. Culture fluids were adjusted to pH 2 by addition of 1 N HCI, incubated overnight at 4 C, and readjusted to pH 7.4 with 1 N NaOH. From an initial 1: 10 dilution, serial twofold dilutions were made in Eagle's basal medium and 2%
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serum, 100 units of penicillinlml, and 100 JLg of streptomycin/mI. Singly dispersed cells were diluted so that 1-2 x 106 cells/ml could be added to glass culture tubes. Confluent monolayers of approximately 5 x 105 cells Per tube were obtained after two to three days in culture; at this time the cultures were infected with mumps virus. Cells were removed' from cultures for enumeration and estimation of percentages of virogenic or hemadsorbing cells (see below) by incubation for 15 min with 0.2 ml of trypsin-EDTA (0.05% trypsin:0.02% EDTA, Grand Island Biological, Grand Island, N.Y.); 0.8 ml of growth medium was then added. Organotypic reaggregation of the freshly cultured embryonic cells imparted the histological characteristics of the corresponding intact organs. Evidence of continued functioning of differentiated cells for the duration of the experiments was provided by rhythmic contraction of heart cells and by the presence of glycogen granules stained with Best's carmine in liver cells. Since overgrowth by fibroblasts was noted in some cultures after seven days, experiments were completed soon after initial cultivation. Virus and assay. A strain of mumps virus (Amaris), isolated from the saliva of a pregnant woman with parotitis and propagated 22 times in calf serum-adapted HeLa cells, was used. Stock virus was rendered cell-free by two cycles of low-speed centrifugation; this virus had a titer of 106 -10 7 pfu/ml. Virus was stored at -70 C in maintenance medium (MM: Eagle's basal medium with HBSS, 5% fetal bovine serum, and antibiotics). Free virus and suspensions of trypsinized, virus-producing cells (virogenic inf~c tious centers) were assayed by a hemadsorption plaque assay in HeLa cell monolayers grown in polystyrene flasks [4]. Infected cells were also assayed by the direct hemadsorption technique [5], whereby trypsinized cells were mixed with a 1% suspension of guinea pig erythrocytes in MM and incubated overnight at 4 C before being read. The percentage of hemadsorbing cells was calculated from the ratio of hemadsorbing cells to total cells counted on a hemocytometer grid. The possible deleterious effect of trypsin-EDTA on hemadsorption was minimized and standardized among cultures for the shortest time that allowed complete separation of cells (15 min).
Davis et al.
Experimental Avian Myocarditis
50 mg of 2-p-phenylene-bis-(5-phenyloxazole) benzenelliter, Amersham/Searle). The samples were counted in a liquid scintillation counter system (model 6848, Nuclear Chicago, Chicago, 111.). Metabolic inhibition studies. The short-t~rm effect of actinomycin D and puromycin dihydrocWoride (Sigma Chemical, St. Louis, Mo.) on production of mumps virus was tested by treatment of cultured heart, lung, and liver cells that had been infected with virus two to four days previously. Cells were exposed for 30 min to 0.001-20 ILg of actinomycin Dlml, rinsed with HBSS, and fed with 1 ml of MM containing 2.5 or 5 IL Ci of [5- 3H]uridine. The protocol for puromycin treatment differed in that after an initial 30- to 60-min treatment with puromycin (0.01-200 ILg/ml), 0.5 IL Ci of [l4C]amino acids in 0.1 ml of MM was added to the medium already present. In experiments with both actinomycin D and puromycin, media and cells were harvested 4-6 hr after treatment for determination of the amount of virus released, the percentage of hemadsorbing cells, and the incorporation of radioactive precursors into cells. Long-term effects of actinomycin D on the production of mumps virus were tested by exposure (either before or after infection) of cells for 30 min to 0.1, 1.0, or lOlLg of actinomycin Dlml, rinsing of cells twice with HBSS, addition of MM, and harvest of virus and cells daily thereafter. In these experiments, RNA synthesis was measured at various intervals after treatment with actinomycin D by exposure of treated and control cultures to 2.5 IL Ci of [5_ 3H]uridine for 1 hr and processing of the samples as described above. Results
Viral replication. The multiplication of virus in primary heart, lung, and liver cells was ascertained by harvest of culture fluid and cells on successive days after infection. CPE did not appear in any of the mumps virus-infected cell cultures. The population of cells in the different cultures remained essentially constant for at least four days after infection, after which it decreased in some experiments. No organ-related differences in protein or RNA synthesis (counts per min of incorporated [14C]amino acids and [5-
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bovine fetal serum (MM-2). Aliquots (i5 ml) were incubated overnight at 35 C with polystyrene flask cultures of chicken embryonic fibroblasts and were rinsed off before challenge with 50 pfu of vesicular stomatitis virus. After adsorption for 2 hr, residual viral inoculum was rinsed off with HBSS and 3 ml of 1.5% nutrient agar (MM-2 with 1.5% Bacto agar [Difco, Detroit, Mich.]) was added. After further incubation for two days at 35 C, 1 ml of 0.05% neutral red in HBSS was added to each flask and allowed to diffuse through the agar overnight at room temperature (about 24 C) to stain viable cells. The number of plaques in flasks pretreated with the test samples was compared with the number in control flasks that had been incubated either with MM-2 or with corresponding dilutions of fluids harvested at appropriate intervals from uninfected .chicken embryonic cells. The titer of interferon was expressed as the reciprocal of the dilution that produced a 50% reduction in the number of plaques. Reference chicken embryonic interferon (National Institutes of Health) was used as a control to confirm the sensitivity of our assay. Isotope procedures. Synthesis of RN A was measured by addition of 2.5 or 5.0 IL Ci of [53H]uridine (specific activity, 24-30 Ci/mmol, Amersham/Searle, Arlington Heights, Ill.) in 1 ml of MM to each culture. For measurement of protein synthesis, 0.5 IL Ci of [l4C]amino acid (specific activity, 52-54 mCi/milliatom of carbon, Amersham/Searle) in MM was added to each culture. At harvest, the cells were rinsed twice in 1 ml of cold phosphate-buffered saline containing Ca and Mg, and 1 ml of 0.05 M Tris aminomethane-hydrochloride buffer (p H 7.4) containing 0.05 M KCI and 0.0015 M MgCl2 was added to each culture. Cultures were frozen and thawed in a dry ice-alcohol bath for removal ofthe monolayers from the glass, an equal volume of 1 M trichloroacetic acid was added, and cultures were refrigerated overnight. The next day, precipitates were rinsed twice in 0.5 M trichloroacetic acid and extracted with two rinses of ether-alcohol (l: 1) and one rinse of ether. The final precipitates were dissolved in 0.5 ml of NCS tissue solubilizer (Amersham/Searle) and were added to 15 ml of scintillation fluid (Spectrafluor, ® diluted with toluene to give 4 mg of 2,5-diphenyloxazole and
127
Davis
128
3H]uridine, respectively, per cell) were seen, whether or not the cells were infected. During the first 24 hr of viral replication, the percentage of virogenic cells was significantly higher in heart than in lung or liver cell cultures (table 1), but the difference among organs in percentage of hemadsorbing cells was minimal. Two or three days after infection percentages of virogenic heart cells were higher than percentages of virogenic liver cells in all of five experiments and than percentages of virogenic lung cells in four of five experiments. Differences among organs in percentages of hemadsorbing cells were also remarkable; the results from a typical experiment are given in figure 1. Between two and eight days after infection, percentages of hemadsorbing heart cells were higher than percentages of hemadsorbing liver cells in 36 of 37 determinations and than percentages of hemadsorbing lung cells in 27 of 30 readings. Between two and eight days after infection, titers of virus in fluids from heart cell cultures were higher than those in fluids from corresponding liver cells in 33 of 34 harvests (differences of 0.7- to 140-fold) and from corresponding lung cells in 26 of 28 harvests (differences of 0.6- to 400-fold). In figure 2 are the results from two experiments in which cumulative titers of released virus on successive days were compared for the three organs. The age of embryos (11-19 days) from which cells were cultivated did not affect the results. Viral adsorption and penetration. There were no significant differences among eight experiments in the percentage of mumps virus adsorbed to heart, lung, and liver cells in 3 hr. Mean percentages (±SE) of adsorbed virus were: heart, 41.4 (±4.6); lung, 46.3 (±3.2); and liver 43.8 (±4.5). Adsorption constants were calculated from the results of five kinetic experiments. Mean values (±SE) were: heart, 8.7 x 10- 9 (±1.2);
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DAYS AFTER INFECTION· Figure 1. Percentage of hemadsorbing cells in cultures derived from 18-day-old chicken embryos on successive days after infection with mumps virus.
lung, 7.5 x 10- 9 (±O.82); and liver, 7.7 x 10- 9 (±O.37) cm 3 /min/cell. The differences among organs were not significant. Experiments with fresh organ homogenates also failed to show enhanced adsorption of mumps virus by heart tissue. Rate constants for the -decrease of amount of trypsin-releasable virus, as a reflection of the rate of viral penetration, were calculated for heart, lung, and liver cells in four experiments (table 2). In two of these experiments, the rate of viral penetration was highest for heart cells, but the mean values for heart, lung, and liver cells were not significantly different. We conclude
Percentage of virogenic cells at the end of the first cycle of mumps virus replication. Experiment
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3.9 2.3 0.6
1.3 0.4 0.3
3.3 0.8 0.6
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7.1 2.7 1.2
1.7 1.2 0.3
2.65 1.01 0.87
(±0.75) (±0.35) (±O.33)
* Difference between values for cells from the heart and those for cells from the lungs and the liver was significant (Student's I-test, P < 0.05).
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Table 1.
el
Experimental A vian Myocarditis
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Figure 2. Cumulative growth curves of mumps virus in medium from cultures of heart. lung, and liver cells from ll-day-old (left) and 16-day-old (right) chicken embryos.
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that the phase of viral penetration measured by the trypsin release technique does J?ot occur significantly faster in heart cells than in lung or liver cells. Viral release. The efficiency of production of virogenic cells during the first cycle of viral replication was calculated for heart, lung, and liver cells from the results of five experiments in which both viral adsorption multiplicity and the number of virogenic cells at the end of 20 hi were known. The adsorption mUltiplicities were used for calculation of expected numbers of infected Table 2. Rate constants for decrease in amount of trypsin-releasable mumps virus after adsorption to cultured heart, lung, and liver cells. Tissue Experiment no. 1 2 3 4 Mean (±SE)*
Heart
Lung
Liver
32.0 14.0 8.4 5.6 15.0(± 5.93)
22.0 11.0 7.2 5.9 11.5(± 3.65)
17.0 6.6 11.0 3.6 9.55(± 2.91)
NOTE. Data were calculated from the formula KTR = 2.3 log (Vo/V.), where KTR is the rate constant (x 10- 3 min-I) for decrease in amount of trypsin-releasable virus, and V o and V. are the concentrations of virus at zero-time and time (t), respectively. * The difference between values for cells from the heart and those for cells from the lungs and liver was not significant by Student's t-test.
cells, and the efficiencies were estimated by division of observed by expected numbers of infected cells. Mean percentage efficiencies (±SE) were: heart, 5.4% (±1.22); lung, 1.5% (±O.31); and liver, 1.6% (±O.66). By Student's t-test, the value for heart cells was significantly higher than that for lung cells (P < 0.01) or that for liver cells (P < 0.05). Heart cells are therefore more efficient in releasing virus in relation to the amount of virus adsorbed than are lung and liver cells. This observation is also supported by the higher ratio of virogenic to hemadsorbing cells one day (three experiments) and five days (one experiment) after infection in heart than in lung or liver cultures. A ratio of virogenic to hemadsorbing cells of < 1 implies that more cells synthesized viral hemagglutinin than were capable of releasing infectious virus during the four days of the infectious center assay. These observations indicate more efficient release of virus in heart cells, since a higher proportion of hemadsorbing lung and liver cells failed to release measurable infectious virus. Extracellular inhibitors of viral replication. Medium harvested from heart, lung, and liver cell cultures from 11- and I8-day-old embryos 12, 24, and 48 hr after infection with mumps virus was assayed for interferon. Interferon was not detected, and, since the initial dilution of 2.5-ml samples tested was 1: 10, the titers
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Davis et al.
130
cells (figure 3). Virus produced by all cells treated with concentrations of puromycin of> 10 JL g/ml showed a greater than normal decrease in titer when assayed again after freezing at -70 C; this decrease may have indicated structural defectiveness. However, the percentage of hemadsorbing cells in these cultures was not altered l5y puromycin treatment. Puromycin-induced stimulation of virus production by liver cells was therefore not dependent on an increase in the number of hemadsorbing cells. Discussion
Experimentally produced mumps virus myocarditis in the chicken embryo is associated with a higher percentage of infected cells and a higher concentration of free virus in the heart than in other organs [1, 2]. Infection of differentiated heart, lung, and liver cells cultivated from chicken embryos 11-19 days old also showed that heart cells produce far more mumps virus in vitro than do cells from lung and fiver. The age of the embryo from which cells were deri ved did not affect cellular susceptibility to mumps virus. Present results have shown that organ-related differences in susceptibility to mumps virus are not attributable to differences in viral receptor density or affinity. This system contrasts with that of picornaviruses, where tissue tropisms appear to be determined predominantly by the presence or absence of viral receptors [6, 7]. The rate of viral penetration into cultivated cells, discerned by quantitation of the decline of trypsinreleasable mumps virus after adsorption, was similar for heart, lung, and liver. Both the efficiency of virus production in relation to viral adsorption and the ratio of virogenic to hemadsorbing cells suggest that defects in later stages of viral replication, assembly, or release may contribute to lower levels of virus production in lung and liver cells than in heart cells. Experiments with puromycin revealed that infected chicken embryonic liver cells could be induced to increase their production of mumps virus a short time after protein synthesis was inhibited. Increased production of virus by puromycin-treated liver cells was not dependent upon an increase in the number of hemadsorbing cells, an observation suggesting that newly pro-
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were 99% inhibition with 20 JLg/ mI). Tests of the long-term effect of actinomycin D on mumps virus infection showed a stimulation of production of virus (up to 14-fold) and percentages of hemadsorbing cells (up to fourfold) in heart and liver cell cultures between one and three days after treatment with 0.1 or 1.0 JLg/ml of the antibiotic. Less stimulation by actinomycin D was seen in lung cultures. Results from two experiments in which virusproducing heart, lung, and liver cells were treated with puromycin showed a concentration-dependent increase in production of mumps virus by liver cells within 4-5 hr but no stimulation of virus production by heart or lung
Experimental Avian Myocarditis
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duced virus evolved from cells already capable of hemadsorbtion. Indeed, the viral precursor proteins were probably synthesized before treatment with puromycin to permit production of virus during the time at which amino acid incorporation was >98% inhibited. Neither lung nor heart cells showed an increase in production of mumps virus after puromycin treatment, but the fact that they continued to produce virus for 4-5 hr after protein synthesis had been almost completely inhibited suggests that adequate stores of viral structural proteins were available within the cells. The lability to freeze-thawing of virus produced by all cultures in the presence of higher concentrations of puromycin may reflect a deficiency of certain structural components of the virus or faulty assembly under these conditions. Enhancement of production of virus in liver cells by puromycin may provide a key to control mechanisms that limit mumps virus production in these cells. Northrop [8] showed a rapid increase in production of mumps virus in a persistently infected human conjunctival cell line after treatment with puromycin and actinomycin D. However, several differences between Northrop's system and that described herein are apparent. Firstly, in the persistently infected human conjunctival cells, increased production of virus after treatment with puromycin was accompanied by a dramatic increase in the percentage
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of hemadsorbing cells; this fact suggested that production of virus was blocked at a stage before incorporation of hemagglutinin into the cell membrane. In mumps virus-infected chicken embryonic liver cells, on the other hand, the increased amount of virus was derived from cells that were already producing viral hemagglutinin and were capable of hemadsorbtion. Secondly, persistently infected human conjunctival cells could be stimulated to produce more mumps virus than controls within a short time after treatment with actinomycin D, whereas any stimulation of virus production by actinomycin D in chicken embryonic heart, lung, or liver cells was delayed for 24-72 hr. The rather general stimulation of production of virus and percentages of hemadsorbing cells in chicken embryonic heart, lung, and liver cells after treatment with low doses of actinomycin D may have resulted from reduced competition for ribosomes with cellular messenger RNA. An effect via inhibition of interferon induction or action is an unlikely explanation si-nce interferon has not been detected in culture fluids overlaying these cells, nor were the cultures resistant to challenge by vesicular stomatitis virus. Puromycin may affect the balance among transcription, translation, and replication of mumps virus, as do other antibiotics for related viruses [9-11]. Variations in the efficiency of late stages of assembly or release in different types
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Figure 3. Effect of puromycin on incorporation of [14C]amino acids and production of mumps virus. Cultures -of heart, lung, and liver cells from 12-day-old chicken .embryos were infected with mumps virus; three days later 1 ml of maintenance medium (see text) containing a specific puromycin concentration was added to replicate tubes. After 1 hr 0.1· ml of maintenance medium containing 0.51L Ci of [I4C]amino acids was added to each tube. After a further 4 hr the medium was recovered for immediate virus titration, and cells from replicate tubes were processed for determination of percentages of hemadsorbing cells and incorporation of [ 14 C]amino acids into protein.
131
132
References
I. S1. Geme, J. W., Jr., Peralta, H., Farias, E., Davis, C. W. C. Noren. G. R. Experimental gestational mumps virus infection and endocardial fibroe1astosis. Pediatrics 48:821-826, 1971. 2. St. Geme, J. W., Jr., Davis, C. W. C, Peralta, H. J., Farias. N. E., Yamauchi. T., Cooper, M. D. The biologic perturbations of persistent embryonic mumps virus infection. Pediatr. Res. 7:541-552, 1973. 3. Davis, C W. C, S1. Geme, J. W., Jr. Differential sus-
4.
5.
6.
7.
8.
9.
10.
I I.
12.
13.
14.
15.
ceptibility of chick embryo cells to mumps virus. Fed. Proc. 28:697, 1969. Davis, C W. C, S1. Geme, J. W., Jr. A rapid hemadsorption plaque assay for mumps virus. Proc. Soc. Exp. BioI. Med. 136: 1319-1322, 1971. Durand, D. P., Borland, R. Effect of input multiplicity on infection of cells with myxoviruses as studied by hemadsorption. Proc. Soc. Exp. BioI. Med. 130:4447. 1969. McLaren. L. C, Holland. J. J., Syverton, J. T. The mammalian cell-virus relationship. I. Attachment of poliovirus to cultivated cells of primate and nonprimate origin. J. Exp. Med. 109:475-485, 1959. Holland. J. J. Receptor affinities as major determinants of enterovirus tissue tropisms in humans. Virology 15:312-326, 1961. Northrop, R. L. Effect of puromycin and actinomycin D on a persistent mumps virus infection in vitro. J. Viro!. 4: 133-140, 1969. Bratt,' M. A. RNA synthesis in N DV-infected chick embryo cells treated with different concentrations of actinomycin D. Virology 39: 141-145, 1969. East, J. L., Kingsbury, D. W. Mumps virus replication in chick embryo lung cells: properties of ribonucleic acids in virions and infected cells. J. Virol. 8:161-173, 1971. Scholtissek, C., Rott, R. Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40:989-996, 1970. Holmes, K. V., Choppin, P. W. On the role of the response of the cell membrane in determining virus virulence. Contrasting effects of the parainfluenza virus SV5 in two cell types. J. Exp. Med. 124:501-520, 1966. Compans, R. W., Holmes, K. V., Dales, S., Choppin, P. W. An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5. Virology 30:411-426, 1966. Darlington, R. W., Portner, A~, Kingsbury, D. W. Sendai virus replication: an ultrastructural comparison of productive and abortive infections in avian cells. J. Gen. Viro!. 9: 169-177, 1970. Knight, P., Duff, R., Rapp, F. Latency of human measles virus in hamster cells. J. Virol. 10:995-1001, 1972.
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of cell have been documented for other paramyxoviruses [12-15]. By analogy with these studies, we are presently investigating the possible role that the lipid composition of plasma membranes or the formation of incomplete virus may play in favoring production of mumps virus in chicken embryonic heart cells. Formation of more incomplete mumps virus by lung and liver cells than by heart cells would certainly explain the smaller virogenic-to-hemadsorbing cell ratios in lung and liver cell cultures [5]. It may seem surprising that primary chicken embryonic cell cultures, which contain several different types of cells of which the relative proportions may vary between experiments, have yielded such con~istent results in terms of organ-related differences in production of mumps virus, and that these differences correspond to those seen in ovo. In spite of the low overall level of mumps virus production in these cultures, it should be possible to elucidate further the mechanisms controlling production of mumps virus, especially when more is known about the synthesis of viral intermediate and structural components in the different types of chicken embryonic cell.
Davis et al.
AUGUST 1975
MAJOR ARTICLES In Vitro Analysis of Experimental Avian Viral Myocarditis Catherine W. C. Davis, Joseph W. St. Geme, Jr., Frank D. Dufour, and Hawley L. Martin
From the Laboratory for Microbiologic and Immunologic Research, Department of Pediatrics, Harbor General Hospital, UCLA School of Medicine, Torrance, California
Myocarditis represents the only histopathological abnormality in chickens hatched from eggs inoculated with mumps virus during early embryonic development [1]. Titers of virus in the heart are higher than those in other organs throughout and at the completion of embryonic development (authors' unpublished observations) [2]. Initial studies of replication of mumps virus in primary cells cultivated from seven organs of II-day-old chicken embryos showed that cultures of heart cells produced 10-100 times as
much virus during a 72-hr period as did cultures of lung, liver, bursa, brain, kidney, and spleen cells [3]. Studies with heart, lung, and liver cell cultures from 11- to 19-day-old chicken embryos substantiated the initial findings, and we pursued a more detailed analysis of the cellular determinants of mumps virus cardiotropism. The lung and liver were chosen for comparison with the heart because the data from mumps virus infection in vivo have indicated that these organs have intermediate and low susceptibility to the virus, respectively [2].
Received for publication August 27, 1973, and in revised form March 24, 1975. This investigation was supported by a grant from the Los Angeles County Heart Association, by general research support grant no. RR05551 from the National Institutes of Health, and by training grant no. 1TOI AI 00 367 from the V.S. Public Health Service. This paper was presented in part at the 71st annual meeting of the American Society for Microbiology, May 2-7, 1971, Minneapolis, Minnesota. We thank Miriam Arce and Robert Dufau for technical assistance; Drs. David Imagawa and John Holland for constructive review of this work; and Lynn Thompson and Judy Liston for careful preparation of the manuscript. Please address requests for reprints to Dr. Catherine W. C. Davis, Department of Pediatrics, Harbor General Hospital, VCLA School of Medicine, 1000 West Carson Street, Torrance, California 90509.
Materials and Methods
Pathogen-free fertile white leghorn eggs were purchased from Kimber Farms, Fremont, Calif. Cell cultures. Embryonic organs were removed, minced, rinsed free of blood, and trypsinized with several changes of 0.1 %-0.2% trypsin in a solution of glucose, potassium, and sodium for 45 min. The trypsin solution was discarded after sedimentation of the cells by lowspeed centrifugation, and the cells were resuspended in growth medium consisting of Eagle's basal medium and Hanks' balanced salt solution (HBSS) supplemented with 10% fetal bovine Eggs.
125
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The cardiotropism of mumps virus in ovo was reproduced in vitro in a comparative study of the replication of mumps virus in cultivated heart, lung, and liver cells from 11- to 19-day-old chicken embryos. The increased proportion of virus-producing cells in the heart was shown not to be due to greater adsorption of virus to heart celIs. Neither interferon nor other cellular inhibitory products were responsible for the lower level of production of virus by lung and liver cells. The proportion of cells producing virus in relation to the amount of virus adsorbed and the proportion of hemadsorbing cells that released infectious virus were greater in heart cells; this finding suggested more efficient viral release in the heart than at the other sites. Inhibition of protein synthesis by puromycin after infection rapidly stimulated the production of mumps virus only in liver cells. Thus a relatively late stage in viral synthesis may be attenuated in the less susceptible liver cells, and events late in viral synthesis may determine organ-related differences in the multiplication of mumps virus in ovo.
126
Measurement of viral adsorption. Viral adsorption to primary cells and organs was estimated from differences between the titer of virus in the inoculum before adsorption at 35 C and that afterward. Thermoinactivation of virus in MM at this temperature was negligible. Virus was inoculated into cultures in small (O.I-ml) or large (1.0-ml) volumes. Input multiplicities ranged from 0.004 to 4 pfu/cell. Adsorption of virus was measured after 1-3 hr or at 15-min intervals for 2 hr. Fresh heart, lung, and liver samples were prepared by trituration of rinsed organs from 11- or 19-day-old embryos. The organ homogenates were rinsed twice in MM, centrifuged twice, and resuspended. The final pellet was resuspended in 10' times its own volume of undiluted stock virus for adsorption. Measurement of viral penetration. The decrease in the amount of trypsin-releasable virus with time after completion of viral adsorption was used to estimate the rate of viral penetration into cultured heart, lung, and liver cells. After adsorption for 3 hr the residual viral inoculum was removed with a thorough rinse. Thereafter, at 60-min intervals during a 5-hr period, cells were treated with trypsin-EDTA for 15 min. The detached cells were removed by low-speed centrifugation, and the supernatant medium was assayed for virus. In HeLa cells a decrease in amount of trypsin-releasable virus after adsorption was paralleled by an increase in the number of infected cells (authors' unpublished observation). Other experiments showed that the infectivity of mumps virus was affected minimally by trypsin in the presence of cells; in contrast, virus was markedly degraded when treated with trypsin alone. Interferon assay. Culture fluids from cells infected with mumps virus were assayed for interferon on monolayers of primary chicken embryonic fibroblasts obtained from the eviscerated torsos of ll-day-old embryos. The New Jersey strain of vesicular stomatitis virus, propagated once in the chicken embryonic allantois and twice in HeLa cells, was used as the challenge virus. Culture fluids were adjusted to pH 2 by addition of 1 N HCI, incubated overnight at 4 C, and readjusted to pH 7.4 with 1 N NaOH. From an initial 1: 10 dilution, serial twofold dilutions were made in Eagle's basal medium and 2%
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serum, 100 units of penicillinlml, and 100 JLg of streptomycin/mI. Singly dispersed cells were diluted so that 1-2 x 106 cells/ml could be added to glass culture tubes. Confluent monolayers of approximately 5 x 105 cells Per tube were obtained after two to three days in culture; at this time the cultures were infected with mumps virus. Cells were removed' from cultures for enumeration and estimation of percentages of virogenic or hemadsorbing cells (see below) by incubation for 15 min with 0.2 ml of trypsin-EDTA (0.05% trypsin:0.02% EDTA, Grand Island Biological, Grand Island, N.Y.); 0.8 ml of growth medium was then added. Organotypic reaggregation of the freshly cultured embryonic cells imparted the histological characteristics of the corresponding intact organs. Evidence of continued functioning of differentiated cells for the duration of the experiments was provided by rhythmic contraction of heart cells and by the presence of glycogen granules stained with Best's carmine in liver cells. Since overgrowth by fibroblasts was noted in some cultures after seven days, experiments were completed soon after initial cultivation. Virus and assay. A strain of mumps virus (Amaris), isolated from the saliva of a pregnant woman with parotitis and propagated 22 times in calf serum-adapted HeLa cells, was used. Stock virus was rendered cell-free by two cycles of low-speed centrifugation; this virus had a titer of 106 -10 7 pfu/ml. Virus was stored at -70 C in maintenance medium (MM: Eagle's basal medium with HBSS, 5% fetal bovine serum, and antibiotics). Free virus and suspensions of trypsinized, virus-producing cells (virogenic inf~c tious centers) were assayed by a hemadsorption plaque assay in HeLa cell monolayers grown in polystyrene flasks [4]. Infected cells were also assayed by the direct hemadsorption technique [5], whereby trypsinized cells were mixed with a 1% suspension of guinea pig erythrocytes in MM and incubated overnight at 4 C before being read. The percentage of hemadsorbing cells was calculated from the ratio of hemadsorbing cells to total cells counted on a hemocytometer grid. The possible deleterious effect of trypsin-EDTA on hemadsorption was minimized and standardized among cultures for the shortest time that allowed complete separation of cells (15 min).
Davis et al.
Experimental Avian Myocarditis
50 mg of 2-p-phenylene-bis-(5-phenyloxazole) benzenelliter, Amersham/Searle). The samples were counted in a liquid scintillation counter system (model 6848, Nuclear Chicago, Chicago, 111.). Metabolic inhibition studies. The short-t~rm effect of actinomycin D and puromycin dihydrocWoride (Sigma Chemical, St. Louis, Mo.) on production of mumps virus was tested by treatment of cultured heart, lung, and liver cells that had been infected with virus two to four days previously. Cells were exposed for 30 min to 0.001-20 ILg of actinomycin Dlml, rinsed with HBSS, and fed with 1 ml of MM containing 2.5 or 5 IL Ci of [5- 3H]uridine. The protocol for puromycin treatment differed in that after an initial 30- to 60-min treatment with puromycin (0.01-200 ILg/ml), 0.5 IL Ci of [l4C]amino acids in 0.1 ml of MM was added to the medium already present. In experiments with both actinomycin D and puromycin, media and cells were harvested 4-6 hr after treatment for determination of the amount of virus released, the percentage of hemadsorbing cells, and the incorporation of radioactive precursors into cells. Long-term effects of actinomycin D on the production of mumps virus were tested by exposure (either before or after infection) of cells for 30 min to 0.1, 1.0, or lOlLg of actinomycin Dlml, rinsing of cells twice with HBSS, addition of MM, and harvest of virus and cells daily thereafter. In these experiments, RNA synthesis was measured at various intervals after treatment with actinomycin D by exposure of treated and control cultures to 2.5 IL Ci of [5_ 3H]uridine for 1 hr and processing of the samples as described above. Results
Viral replication. The multiplication of virus in primary heart, lung, and liver cells was ascertained by harvest of culture fluid and cells on successive days after infection. CPE did not appear in any of the mumps virus-infected cell cultures. The population of cells in the different cultures remained essentially constant for at least four days after infection, after which it decreased in some experiments. No organ-related differences in protein or RNA synthesis (counts per min of incorporated [14C]amino acids and [5-
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bovine fetal serum (MM-2). Aliquots (i5 ml) were incubated overnight at 35 C with polystyrene flask cultures of chicken embryonic fibroblasts and were rinsed off before challenge with 50 pfu of vesicular stomatitis virus. After adsorption for 2 hr, residual viral inoculum was rinsed off with HBSS and 3 ml of 1.5% nutrient agar (MM-2 with 1.5% Bacto agar [Difco, Detroit, Mich.]) was added. After further incubation for two days at 35 C, 1 ml of 0.05% neutral red in HBSS was added to each flask and allowed to diffuse through the agar overnight at room temperature (about 24 C) to stain viable cells. The number of plaques in flasks pretreated with the test samples was compared with the number in control flasks that had been incubated either with MM-2 or with corresponding dilutions of fluids harvested at appropriate intervals from uninfected .chicken embryonic cells. The titer of interferon was expressed as the reciprocal of the dilution that produced a 50% reduction in the number of plaques. Reference chicken embryonic interferon (National Institutes of Health) was used as a control to confirm the sensitivity of our assay. Isotope procedures. Synthesis of RN A was measured by addition of 2.5 or 5.0 IL Ci of [53H]uridine (specific activity, 24-30 Ci/mmol, Amersham/Searle, Arlington Heights, Ill.) in 1 ml of MM to each culture. For measurement of protein synthesis, 0.5 IL Ci of [l4C]amino acid (specific activity, 52-54 mCi/milliatom of carbon, Amersham/Searle) in MM was added to each culture. At harvest, the cells were rinsed twice in 1 ml of cold phosphate-buffered saline containing Ca and Mg, and 1 ml of 0.05 M Tris aminomethane-hydrochloride buffer (p H 7.4) containing 0.05 M KCI and 0.0015 M MgCl2 was added to each culture. Cultures were frozen and thawed in a dry ice-alcohol bath for removal ofthe monolayers from the glass, an equal volume of 1 M trichloroacetic acid was added, and cultures were refrigerated overnight. The next day, precipitates were rinsed twice in 0.5 M trichloroacetic acid and extracted with two rinses of ether-alcohol (l: 1) and one rinse of ether. The final precipitates were dissolved in 0.5 ml of NCS tissue solubilizer (Amersham/Searle) and were added to 15 ml of scintillation fluid (Spectrafluor, ® diluted with toluene to give 4 mg of 2,5-diphenyloxazole and
127
Davis
128
3H]uridine, respectively, per cell) were seen, whether or not the cells were infected. During the first 24 hr of viral replication, the percentage of virogenic cells was significantly higher in heart than in lung or liver cell cultures (table 1), but the difference among organs in percentage of hemadsorbing cells was minimal. Two or three days after infection percentages of virogenic heart cells were higher than percentages of virogenic liver cells in all of five experiments and than percentages of virogenic lung cells in four of five experiments. Differences among organs in percentages of hemadsorbing cells were also remarkable; the results from a typical experiment are given in figure 1. Between two and eight days after infection, percentages of hemadsorbing heart cells were higher than percentages of hemadsorbing liver cells in 36 of 37 determinations and than percentages of hemadsorbing lung cells in 27 of 30 readings. Between two and eight days after infection, titers of virus in fluids from heart cell cultures were higher than those in fluids from corresponding liver cells in 33 of 34 harvests (differences of 0.7- to 140-fold) and from corresponding lung cells in 26 of 28 harvests (differences of 0.6- to 400-fold). In figure 2 are the results from two experiments in which cumulative titers of released virus on successive days were compared for the three organs. The age of embryos (11-19 days) from which cells were cultivated did not affect the results. Viral adsorption and penetration. There were no significant differences among eight experiments in the percentage of mumps virus adsorbed to heart, lung, and liver cells in 3 hr. Mean percentages (±SE) of adsorbed virus were: heart, 41.4 (±4.6); lung, 46.3 (±3.2); and liver 43.8 (±4.5). Adsorption constants were calculated from the results of five kinetic experiments. Mean values (±SE) were: heart, 8.7 x 10- 9 (±1.2);
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lung, 7.5 x 10- 9 (±O.82); and liver, 7.7 x 10- 9 (±O.37) cm 3 /min/cell. The differences among organs were not significant. Experiments with fresh organ homogenates also failed to show enhanced adsorption of mumps virus by heart tissue. Rate constants for the -decrease of amount of trypsin-releasable virus, as a reflection of the rate of viral penetration, were calculated for heart, lung, and liver cells in four experiments (table 2). In two of these experiments, the rate of viral penetration was highest for heart cells, but the mean values for heart, lung, and liver cells were not significantly different. We conclude
Percentage of virogenic cells at the end of the first cycle of mumps virus replication. Experiment
Organ Heart Lung Liver
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2
3
4
5
6
7
8
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(±SE)*
0.4 0.1 0.07
3.9 2.3 0.6
1.3 0.4 0.3
3.3 0.8 0.6
2.1 0.4 3.0
7.1 2.7 1.2
1.7 1.2 0.3
2.65 1.01 0.87
(±0.75) (±0.35) (±O.33)
* Difference between values for cells from the heart and those for cells from the lungs and the liver was significant (Student's I-test, P < 0.05).
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Table 1.
el
Experimental A vian Myocarditis
129
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Figure 2. Cumulative growth curves of mumps virus in medium from cultures of heart. lung, and liver cells from ll-day-old (left) and 16-day-old (right) chicken embryos.
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DAYS AFTER INFECTION
that the phase of viral penetration measured by the trypsin release technique does J?ot occur significantly faster in heart cells than in lung or liver cells. Viral release. The efficiency of production of virogenic cells during the first cycle of viral replication was calculated for heart, lung, and liver cells from the results of five experiments in which both viral adsorption multiplicity and the number of virogenic cells at the end of 20 hi were known. The adsorption mUltiplicities were used for calculation of expected numbers of infected Table 2. Rate constants for decrease in amount of trypsin-releasable mumps virus after adsorption to cultured heart, lung, and liver cells. Tissue Experiment no. 1 2 3 4 Mean (±SE)*
Heart
Lung
Liver
32.0 14.0 8.4 5.6 15.0(± 5.93)
22.0 11.0 7.2 5.9 11.5(± 3.65)
17.0 6.6 11.0 3.6 9.55(± 2.91)
NOTE. Data were calculated from the formula KTR = 2.3 log (Vo/V.), where KTR is the rate constant (x 10- 3 min-I) for decrease in amount of trypsin-releasable virus, and V o and V. are the concentrations of virus at zero-time and time (t), respectively. * The difference between values for cells from the heart and those for cells from the lungs and liver was not significant by Student's t-test.
cells, and the efficiencies were estimated by division of observed by expected numbers of infected cells. Mean percentage efficiencies (±SE) were: heart, 5.4% (±1.22); lung, 1.5% (±O.31); and liver, 1.6% (±O.66). By Student's t-test, the value for heart cells was significantly higher than that for lung cells (P < 0.01) or that for liver cells (P < 0.05). Heart cells are therefore more efficient in releasing virus in relation to the amount of virus adsorbed than are lung and liver cells. This observation is also supported by the higher ratio of virogenic to hemadsorbing cells one day (three experiments) and five days (one experiment) after infection in heart than in lung or liver cultures. A ratio of virogenic to hemadsorbing cells of < 1 implies that more cells synthesized viral hemagglutinin than were capable of releasing infectious virus during the four days of the infectious center assay. These observations indicate more efficient release of virus in heart cells, since a higher proportion of hemadsorbing lung and liver cells failed to release measurable infectious virus. Extracellular inhibitors of viral replication. Medium harvested from heart, lung, and liver cell cultures from 11- and I8-day-old embryos 12, 24, and 48 hr after infection with mumps virus was assayed for interferon. Interferon was not detected, and, since the initial dilution of 2.5-ml samples tested was 1: 10, the titers
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Davis et al.
130
cells (figure 3). Virus produced by all cells treated with concentrations of puromycin of> 10 JL g/ml showed a greater than normal decrease in titer when assayed again after freezing at -70 C; this decrease may have indicated structural defectiveness. However, the percentage of hemadsorbing cells in these cultures was not altered l5y puromycin treatment. Puromycin-induced stimulation of virus production by liver cells was therefore not dependent on an increase in the number of hemadsorbing cells. Discussion
Experimentally produced mumps virus myocarditis in the chicken embryo is associated with a higher percentage of infected cells and a higher concentration of free virus in the heart than in other organs [1, 2]. Infection of differentiated heart, lung, and liver cells cultivated from chicken embryos 11-19 days old also showed that heart cells produce far more mumps virus in vitro than do cells from lung and fiver. The age of the embryo from which cells were deri ved did not affect cellular susceptibility to mumps virus. Present results have shown that organ-related differences in susceptibility to mumps virus are not attributable to differences in viral receptor density or affinity. This system contrasts with that of picornaviruses, where tissue tropisms appear to be determined predominantly by the presence or absence of viral receptors [6, 7]. The rate of viral penetration into cultivated cells, discerned by quantitation of the decline of trypsinreleasable mumps virus after adsorption, was similar for heart, lung, and liver. Both the efficiency of virus production in relation to viral adsorption and the ratio of virogenic to hemadsorbing cells suggest that defects in later stages of viral replication, assembly, or release may contribute to lower levels of virus production in lung and liver cells than in heart cells. Experiments with puromycin revealed that infected chicken embryonic liver cells could be induced to increase their production of mumps virus a short time after protein synthesis was inhibited. Increased production of virus by puromycin-treated liver cells was not dependent upon an increase in the number of hemadsorbing cells, an observation suggesting that newly pro-
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were 99% inhibition with 20 JLg/ mI). Tests of the long-term effect of actinomycin D on mumps virus infection showed a stimulation of production of virus (up to 14-fold) and percentages of hemadsorbing cells (up to fourfold) in heart and liver cell cultures between one and three days after treatment with 0.1 or 1.0 JLg/ml of the antibiotic. Less stimulation by actinomycin D was seen in lung cultures. Results from two experiments in which virusproducing heart, lung, and liver cells were treated with puromycin showed a concentration-dependent increase in production of mumps virus by liver cells within 4-5 hr but no stimulation of virus production by heart or lung
Experimental Avian Myocarditis
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duced virus evolved from cells already capable of hemadsorbtion. Indeed, the viral precursor proteins were probably synthesized before treatment with puromycin to permit production of virus during the time at which amino acid incorporation was >98% inhibited. Neither lung nor heart cells showed an increase in production of mumps virus after puromycin treatment, but the fact that they continued to produce virus for 4-5 hr after protein synthesis had been almost completely inhibited suggests that adequate stores of viral structural proteins were available within the cells. The lability to freeze-thawing of virus produced by all cultures in the presence of higher concentrations of puromycin may reflect a deficiency of certain structural components of the virus or faulty assembly under these conditions. Enhancement of production of virus in liver cells by puromycin may provide a key to control mechanisms that limit mumps virus production in these cells. Northrop [8] showed a rapid increase in production of mumps virus in a persistently infected human conjunctival cell line after treatment with puromycin and actinomycin D. However, several differences between Northrop's system and that described herein are apparent. Firstly, in the persistently infected human conjunctival cells, increased production of virus after treatment with puromycin was accompanied by a dramatic increase in the percentage
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of hemadsorbing cells; this fact suggested that production of virus was blocked at a stage before incorporation of hemagglutinin into the cell membrane. In mumps virus-infected chicken embryonic liver cells, on the other hand, the increased amount of virus was derived from cells that were already producing viral hemagglutinin and were capable of hemadsorbtion. Secondly, persistently infected human conjunctival cells could be stimulated to produce more mumps virus than controls within a short time after treatment with actinomycin D, whereas any stimulation of virus production by actinomycin D in chicken embryonic heart, lung, or liver cells was delayed for 24-72 hr. The rather general stimulation of production of virus and percentages of hemadsorbing cells in chicken embryonic heart, lung, and liver cells after treatment with low doses of actinomycin D may have resulted from reduced competition for ribosomes with cellular messenger RNA. An effect via inhibition of interferon induction or action is an unlikely explanation si-nce interferon has not been detected in culture fluids overlaying these cells, nor were the cultures resistant to challenge by vesicular stomatitis virus. Puromycin may affect the balance among transcription, translation, and replication of mumps virus, as do other antibiotics for related viruses [9-11]. Variations in the efficiency of late stages of assembly or release in different types
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Figure 3. Effect of puromycin on incorporation of [14C]amino acids and production of mumps virus. Cultures -of heart, lung, and liver cells from 12-day-old chicken .embryos were infected with mumps virus; three days later 1 ml of maintenance medium (see text) containing a specific puromycin concentration was added to replicate tubes. After 1 hr 0.1· ml of maintenance medium containing 0.51L Ci of [I4C]amino acids was added to each tube. After a further 4 hr the medium was recovered for immediate virus titration, and cells from replicate tubes were processed for determination of percentages of hemadsorbing cells and incorporation of [ 14 C]amino acids into protein.
131
132
References
I. S1. Geme, J. W., Jr., Peralta, H., Farias, E., Davis, C. W. C. Noren. G. R. Experimental gestational mumps virus infection and endocardial fibroe1astosis. Pediatrics 48:821-826, 1971. 2. St. Geme, J. W., Jr., Davis, C. W. C, Peralta, H. J., Farias. N. E., Yamauchi. T., Cooper, M. D. The biologic perturbations of persistent embryonic mumps virus infection. Pediatr. Res. 7:541-552, 1973. 3. Davis, C W. C, S1. Geme, J. W., Jr. Differential sus-
4.
5.
6.
7.
8.
9.
10.
I I.
12.
13.
14.
15.
ceptibility of chick embryo cells to mumps virus. Fed. Proc. 28:697, 1969. Davis, C W. C, S1. Geme, J. W., Jr. A rapid hemadsorption plaque assay for mumps virus. Proc. Soc. Exp. BioI. Med. 136: 1319-1322, 1971. Durand, D. P., Borland, R. Effect of input multiplicity on infection of cells with myxoviruses as studied by hemadsorption. Proc. Soc. Exp. BioI. Med. 130:4447. 1969. McLaren. L. C, Holland. J. J., Syverton, J. T. The mammalian cell-virus relationship. I. Attachment of poliovirus to cultivated cells of primate and nonprimate origin. J. Exp. Med. 109:475-485, 1959. Holland. J. J. Receptor affinities as major determinants of enterovirus tissue tropisms in humans. Virology 15:312-326, 1961. Northrop, R. L. Effect of puromycin and actinomycin D on a persistent mumps virus infection in vitro. J. Viro!. 4: 133-140, 1969. Bratt,' M. A. RNA synthesis in N DV-infected chick embryo cells treated with different concentrations of actinomycin D. Virology 39: 141-145, 1969. East, J. L., Kingsbury, D. W. Mumps virus replication in chick embryo lung cells: properties of ribonucleic acids in virions and infected cells. J. Virol. 8:161-173, 1971. Scholtissek, C., Rott, R. Synthesis in vivo of influenza virus plus and minus strand RNA and its preferential inhibition by antibiotics. Virology 40:989-996, 1970. Holmes, K. V., Choppin, P. W. On the role of the response of the cell membrane in determining virus virulence. Contrasting effects of the parainfluenza virus SV5 in two cell types. J. Exp. Med. 124:501-520, 1966. Compans, R. W., Holmes, K. V., Dales, S., Choppin, P. W. An electron microscopic study of moderate and virulent virus-cell interactions of the parainfluenza virus SV5. Virology 30:411-426, 1966. Darlington, R. W., Portner, A~, Kingsbury, D. W. Sendai virus replication: an ultrastructural comparison of productive and abortive infections in avian cells. J. Gen. Viro!. 9: 169-177, 1970. Knight, P., Duff, R., Rapp, F. Latency of human measles virus in hamster cells. J. Virol. 10:995-1001, 1972.
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of cell have been documented for other paramyxoviruses [12-15]. By analogy with these studies, we are presently investigating the possible role that the lipid composition of plasma membranes or the formation of incomplete virus may play in favoring production of mumps virus in chicken embryonic heart cells. Formation of more incomplete mumps virus by lung and liver cells than by heart cells would certainly explain the smaller virogenic-to-hemadsorbing cell ratios in lung and liver cell cultures [5]. It may seem surprising that primary chicken embryonic cell cultures, which contain several different types of cells of which the relative proportions may vary between experiments, have yielded such con~istent results in terms of organ-related differences in production of mumps virus, and that these differences correspond to those seen in ovo. In spite of the low overall level of mumps virus production in these cultures, it should be possible to elucidate further the mechanisms controlling production of mumps virus, especially when more is known about the synthesis of viral intermediate and structural components in the different types of chicken embryonic cell.
Davis et al.
