Chemically Characterized Media for Study of Foot-and-Mouth Disease Virus in Baby Hamster Kidney Cells

Foot-and-mouth disease virus can be grown in baby hamster kidney cells with a chemically characterized medium containing only tris(hydroxymethyl)-amino-methane (Tris) buffer, glucose, glutamine, and salts. Virus infectivity was only 0.5 log unit less than in a complex cell growth medium containing serum, tryptose phosphate, and lactalbumin hydrolysate. At high multiplicity of infection, production was maximal in 5 hr, with the virus remaining largely intracellular. Glucose and glutamine appeared to act independently of each other although both were required at about the same time during the virus production cycle. Glutamine had the greater effect and could not be replaced by amino acids, purines, and pyrimidines. Glutamine also stimulated cellular oxygen uptake in both normal and infected cells. Serum and other organic components added singly to the defined medium did not increase the virus yield. Studies on uninfected cells over a 5-hr incubation period showed that the defined medium maintained protein and ribonucleic acid synthesis at rates similar to the complex cell growth medium. These rates were much lower in media containing only inorganic salts and Tris buffer. Glucose, however, was more important to uninfected cellular metabolism than was glutamine. Defined medium containing dialyzed calf serum produced the highest rate of protein synthesis.     

Growth of Chikungunya Virus in Baby Hamster Kidney Cell (BHK-21-Clone 13) Suspension Cultures

This report describes the methods used to obtain high titers of chikungunya virus with suspension cultures of BHK-21-clone 13 cells. The cells were grown at 37 C to a cell concentration of 10(6) to 2 x 10(6) per ml. After maximum cell growth, the cells were inoculated with chikungunya virus at a multiplicity of 1 to 2 50% suckling mouse intracerebral lethal doses (SMICLD(50)) per cell in the spent Eagle's minimum essential medium for suspension cultures (MEMS), or the cell cultures were centrifuged at 200 x g and resuspended in either fresh MEMS or medium 199 prior to inoculation. The medium used had no effect on virus titer. The inoculated cultures were incubated at 34 C until the cell viability dropped to 30%, which usually occurred 28 to 30 hr postinoculation. After these procedures, chikungunya virus titers of log(10) 10.3 to 11.8 SMICLD(50) per ml were obtained.     

Foot-and-Mouth Disease Virus-Induced Alterations of Baby Hamster Kidney Cell Macromolecular Biosynthesis: Inhibition of Ribonucleic Acid Methylation and Stimulation of Ribonucleic Acid Synthesis

The kinetics of ribonucleic acid (RNA) and protein synthesis and RNA methylation were examined after foot-and-mouth disease virus (FMDV) infection of baby hamster kidney cells. The synthesis of RNA extracted from the whole cells was stimulated two- to threefold above the control level of synthesis. This increased rate was attributed to viral RNA synthesis. The inhibition of host RNA methylation was concomitant with but more pronounced than protein synthesis inhibition. The methylation of transfer RNA was initially inhibited by virus infection, but rose to within 70 to 80% of the control level just prior to the production of maximal amounts of virus-specific RNA polymerase. Cycloheximide studies showed that rapid cessation of protein synthesis did not result in the immediate cessation of RNA methylation. A comparison between the kinetics of inhibition of these processes by cycloheximide and FMDV infection suggests that FMDV selectively inhibits RNA methylation.     

Effect of Actinomycin D on Virus-induced Ribonucleic Acid Polymerase Formation in Foot-and-Mouth Disease Virus-Infected Baby Hamster Kidney Cells

Actinomycin D, at a concentration that inhibits cellular ribonucleic acid (RNA) synthesis, inhibited the production of foot-and-mouth disease virus-induced RNA polymerase in baby hamster kidney cells. Inhibition was proportional to exposure time and reached 85% when actinomycin D was added 90 min before infection. Polymerase production was inhibited to the same extent in growth and minimal media, and the kinetics of its appearance were slightly different than in untreated cells. Enzyme preparations from actinomycin-treated cells having one-third to one-tenth the activity of untreated samples gave products with RNA profiles similar to those of controls. The 37S viral peak, 20S ribonuclease-resistant peak, and 26 to 28S peaks were present in all cases. Actinomycin D did not consistently inhibit virus production in either medium. Insulin did not prevent the actinomycin induced inhibition of polymerase and virus production from occurring.     

Glycolipid Content of Vesicular Stomatitis Virus Grown in Baby Hamster Kidney Cells

Vesicular stomatitis virions grown in baby hamster kidney (BHK-21-F) cells were found to contain hematoside (neuraminosyl-galactosyl-glucosyl-ceramide). This ganglioside, which was the only detectable glycolipid in the virion, is also the only glycolipid found in significant amount in BHK-21-F cells. Approximately 87% of the total neuraminic acid in the virion was found to be linked to protein and 13% to lipid.     

Unrestricted Diffusion of Exogenous and Endogenous PIP2 in Baby Hamster Kidney and Chinese Hamster Ovary Cell Plasmalemma

We used two approaches to characterize the lateral mobility of phosphatidylinositol 4,5-bisphosphate (PIP₂) in the plasmalemma of baby hamster kidney and Chinese hamster ovary fibroblasts. First, nitrobenzoxadiazole-labeled C6-phosphatidylcholine and C16-PIP₂ were incorporated into plasma membrane “lawns” (∼20 × 30 μm) from these cells and into the outer monolayer of intact cells. Diffusion coefficients determined by fluorescence recovery after photobleaching were similar for the two lipids and were higher in lawns, ∼0.3 μm²/s, than on the cell surface, ∼0.1 μm²/s. For membrane lawns, the fractional recoveries (75–90%) were close to those expected from the fraction of total membrane bleached, and labeling by the probes was several times greater than for intact cells. Second, we analyzed cells expressing M1 muscarinic receptors and green fluorescent protein fused with PIP₂-binding pleckstrin-homology domains, Tubby domains or diacylglycerol (DAG)-binding C1 domains. On-cell gigaseal patches were formed with pipette tips >5 μm in diameter. When the agonist carbachol (0.3 mm) was applied either within or outside of the pipette, lipid signals crossed the pipette barrier rapidly in both directions and membrane blebbing occurred on both membrane sides. Accurate simulations of lipid gradients required diffusion coefficients >1 μm²/s. Exogenous DAG also crossed the pipette barrier rapidly. In summary, we found no evidence for restricted diffusion of signaling lipids in these cells. The lower mobility and incorporation of phospholipid at the extracellular leaflet may reflect a more ordered and condensed extracellular monolayer, as expected from previous studies. An erratum to this article can be found at     

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Production and Purification of Large Amounts of Rous Sarcoma Virus

Procedures are described for production and purification of large amounts of Rous sarcoma virus. The virus was produced by Rous sarcoma virus-transformed chicken embryo fibroblasts in roller culture which produced up to 6 mg of virus per day per liter of supernatant fluid. Various methods of concentrating virus were evaluated; pelleting yielded the best results in terms of recovery of infectious virus. Purification was achieved by means of successive velocity and equilibrium density centrifugation by using sucrose solutions made in low-salt buffer. A rapid method for the optical density measurement of virus concentration was also developed.     

Effect of Serum on the Yield of Lymphocytic Choriomeningitis Virus in Baby Hamster Kidney Cells

The yields of the Armstrong and WE strains of lymphocytic choriomeningitis virus in baby hamster kidney (BHK) cells cultivated in either bovine, calf, fetal bovine, or horse serum were investigated. Lines of BHK cells were established in these sera. When the infected cell lines were observed by immunofluorescence, the per cent fluorescing cells for a given virus strain did not vary. However, for both strains, the extracellular virus yields per cell were significantly greater in the fetal bovine-cell line than in the other serum-cell lines.     

Growth of the IB-RS-2 Pig Kidney Cell Line in Suspension Culture and Its Susceptibility to Foot-and-Mouth Disease Virus

The adaptation of the pig kidney cell line IB-RS-2, clone 60, to growth in suspension culture is described. When fully adapted, an approximate threefold increase in viable cells was obtained within 72 hr from initial cell concentrations of 5 x 10(5) per ml in culture volumes up to 1,500 ml. The monolayer cells (99th passage level) used to initiate the suspension cultures and the fully adapted suspension cells were shown to have an aneuploid chromosome karyotype, whereas earlier monolayer cultures (32nd passage level) had a pseudodiploid karyotype. Replicate virus titrations in monolayers prepared from suspension-adapted cells, IB-RS-2 monolayer cells, BHK monolayer cells, and in suckling mice showed that the suspension cells had retained sensitivity to foot-and-mouth disease virus. The geometric mean peak infectivity of seven strains of foot-and-mouth disease virus grown in IB-RS-2 suspension cells was 10(8.2) plaque-forming units per ml, with a mean complement-fixing activity of approximately 135 complement-fixing units per ml. These preliminary results indicate that submerged cultures of these cells on an industrial scale may be useful for commercial foot-and-mouth disease vaccine production.     

Growth of Foot-and-Mouth Disease Virus in the Different Layers of Bovine Omasum in Suspended Cultures

When suspended cultures of bovine omasum were cultured without agitation, the epithelium soon degenerated and foot-and-mouth disease virus multiplied mainly in the corium cells. Five days of preincubation were needed to reach a population of corium cells that could yield virus at a titer of 10(6.70) to 10(6.95) mean tissue culture infective doses per ml. The virus was freely released from the cells into the medium only when the degenerated epithelium was removed from the subepithelial tissue prior to virus inoculation. In agitated cultures, the viability of the epithelium was retained, the virus multiplied in all the layers of the epithelium and was freely released into the medium, and a virus titer of 10(6.95) mean tissue culture infective doses per ml was obtained without preincubation. The omasal laminae could be separated along the line of apposition of the two mucous membranes of the organ. The virus yield from these thin separated membranes was 0.5 to 1.0 log higher than that obtained from nonseparated laminae.     

Inhibition of Host Cell Ribosomal Ribonucleic Acid Methylation by Foot-and-Mouth Disease Virus

A study of protein and ribonucleic acid (RNA) synthesis in cells infected by foot-and-mouth disease virus has indicated possible mechanisms of viral control over host cell metabolism. Foot-and-mouth disease virus infection of baby hamster kidney cells resulted in 50% inhibition of host cell protein synthesis at 180 min postinfection. A viral-induced interference with host cell RNA methylation was observed to be more rapidly inhibited than protein synthesis. To determine the nature of methylation inhibition, the kinetics of several host cell methylated RNA species were examined subsequent to virus infection. Data from sucrose zonal centrifugation and methylated albumin kieselguhr chromatography showed that methylation of nuclear RNA was inhibited 50% at 60 min postinfection. Inhibition of nuclear ribosomal RNA precursors and formation of nascent ribosomes correlated with inhibition kinetics of nuclear RNA methylation. It is suggested that the viral interference with the host nuclear RNA methylation is directly responsible for the observed loss of nascent ribosome formation. Moreover, early in the infectious cycle, methylation inhibition of host cell RNA could, in part, account for the cessation of host protein synthesis.     

Electron Microscopy of Monkey Kidney Cell Cultures Infected with Rubella Virus

Two rubella virus strains isolated in this laboratory were investigated in terms of their growth in LLC-MK(2) cell cultures and their effect on cell morphology. Rubella virus grew readily in LLC-MK(2) cells, but cytopathic effects of the virus were not observed in infected cultures. Such infected cultures can be subcultured indefinitely and continue to shed virus. Examination of rubella-infected cell cultures by electron microscopy showed the presence of annulate lamellae in the cytoplasm of 15% of the cells. No changes were evident in the nuclei. These membranous inclusions varied in complexity from parallel arrays of annulate lamellae to large lamellar structures of complex morphology. An occasional cell contained a crystal lattice structure in association with the lamellae. Larger inclusions, consisting of disorganized arrays of "unit" membranes, were also found. Uninfected cells were devoid of annulate lamellae, crystals, and complex membranous inclusions. No viruslike particles were observed in any part of the cells from infected cultures. The significance of the structures observed has not been determined.     

Immunogenicity of Nanogram to Milligram Quantities of Inactivated Foot-and-Mouth Disease Virus. I. Relative Virus-neutralizing Potency of Guinea Pig Sera

Quantitative antigen dose-neutralizing antibody response curves were established in guinea pigs for purified foot-and-mouth disease virus (FMDV), type A, strain 119, inactivated for 48 hr with N-acetylethyleneimine (AEI). Inactivation of FMDV by 0.05% AEI at 25 C occurred without virus degradation and followed first-order kinetics over a 10(8)-fold decrease in plaque-forming units (PFU) extrapolating to 10(-5) PFU/ml at 48 hr. The AEI-treated virus was administered in doses ranging from 10 ng to 2.62 mg, alone or emulsified in oil adjuvant. Sigmoidal dose-response curves were obtained with 160 ng as the minimum effective dose. The maximum effective dose was 163 mug and 2.62 mg or more at 6 and 28 through 84 days postinoculation, respectively. Oil adjuvant had little effect at 6 days postinoculation, but its use markedly increased the amount of neutralizing antibody obtained at the later testing periods.     

Plaque Assay for Polyoma Virus on Primary Mouse Kidney Cell Cultures

A plaque assay for polyoma virus using primary baby mouse kidney cells is reported.     

Repeated-batch cultures of Baby Hamster Kidney cell aggregates in stirred vessels

Natural aggregates of Baby Hamster Kidney cells were grown in stirred vessels operated as repeated-batch cultures during more than 600 hours. Different protocols were applied to passaging different fractions of the initial culture: single cells, large size distributed aggregates and large aggregates. When single cells or aggregates with the same size distribution found in culture are used as inoculum, it is possible to maintain semi-continuous cultures during more than 600 hours while keeping cell growth and viability. These results suggest that aggregate culture in large scale might be feasible, since a small scale culture can easily be used as inoculum for larger vessels without noticeable modification of the aggregate chacteristics. However, when only the large aggregates are used as inoculum, it was shown that much lower cell concentrations are obtained, cell viability in aggregates dropping to less than 60%. Under this selection procedure, aggregates maintain a constant size, larger than under batch experiments, up to approximately 400 hours; after this time, aggregate size increases to almost twice the size expected from batch cultures.     

Ultrastructure of Measles Virus in Cultures of Hamster Cerebellum

Replication of Edmonston strain of measles virus in cultures of hamster central nervous system tissue was studied by electron microscopy of ultrathin sections. Infected cultures were fixed from 3 hr to 39 days postinoculation (PI). Measles nucleocapsid was first seen within the cytoplasm of giant cells, the latter appearing 5 to 6 days PI. Measles virus particles were most abundant at 10 days PI and appeared to bud off from areas of the cell membrane along which nucleocapsid was aligned. Intranuclear nucleocapsid was more abundant at later stages, and by 39 days PI entire nuclei were seen to be occupied. By this time, the cytoplasmic formations, which had been sequestered by membranes, appeared to lose their regular structure. Budding viral particles at 39 days PI were of a much simplified structure and did not involve the alignment of nucleocapsid about their periphery.     

Inactivation of Foot-and-Mouth Disease Virus with Ethylenimine

Foot-and-mouth disease virus (FMDV) was inactivated by ethylenimine (EI) at three concentrations and two temperatures. Comparison of inactivation kinetics and the antigenic and immunogenic potency of EI and N-acetylethylenimine (AEI)-inactivated FMDV indicates that EI has nearly optimal characteristics as an inactivant for FMDV vaccine preparation. Although AEI-inactivated FMDV has proved to be a potent specific immunogen, an equivalent percentage of EI inactivated FMDV at substantially faster rates and produced an equally potent immunogen. In addition, EI inactivated FMDV at rates that were essentially linear throughout the loss of nearly all measurable infectivity.