Evaluation of Factors Related to Growth of Rift Valley Fever Virus in Suspended Cell Cultures

The effect of several controlled variables on the peak titer and fold increase of Rift Valley fever virus grown in suspension culture on two variants of Earle's L cell, L-DR and L-MA clone 1-1, was studied. No significant amount of cell-associated virus was found at 24 hr, indicating a release of virus soon after its formation. Mild sonic treatment of the virus produced in serum-free medium increased the infective titer about 10x. This difference was not observed with virus produced in medium supplemented with serum. Peak titer was not affected by medium used during the infection period, by multiplicity of inoculum (MOI), or by initial cell concentration within the test range of 10(4) to 2 x 10(6) cell/ml. Cell strain employed influenced titer, because the L-DR cell did not produce virus efficiently at low MOI and low initial cell concentration. The time of peak titer and fold replication was dependent on MOI and initial cell concentration. Differences in virus propagation in monolayer and suspension systems are discussed.     

Semliki Forest Virus in HEp-2 Cell Cultures

The growth and development of Semliki Forest virus (SFV), an arbovirus of serological group A, in HEp-2 cells in tissue culture was examined by various techniques at frequent intervals. Infectivity and fluorescent-antibody studies demonstrated the presence of infective virus and viral antigens within the cells at 8 hr after infection. The antigen was particulate and distributed throughout the cytoplasm. Thereafter, there was rapid progression of virus production and cell destruction. By electron microscopy, tubular structures bounded by a fine membrane were observed in cytoplasm at 12 hr. Rows of small (25 mmu) virus particles were often present on the outer surface of these membranes, and at later times they became progressively more encrusted with the small virus particles. These structures subsequently increased rapidly in number, size, and complexity, and the space between the membrane and the tubules increased, thus forming vacuoles which contained tubules and were covered with the small particles. At later times (24 hr and later) larger (42 to 50 mmu) particles were observed, usually inside of the vacuoles. These larger particles (and occasionally the smaller ones) were also seen at the cell periphery and in the extracellular space. The large SFV particles appear to form by three distinct processes: (i) from the smaller particles, (ii) by development on an intravacuolar membrane, and (iii) at the ends of the tubules. The mode of development of SFV is unique among viruses studied to date, but in some characteristics it resembles that of other group A arboviruses. Its development differs from that of most arboviruses of group B and other serological groups.     

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.     

Growth of Nosema algerae in Pig Kidney Cell Cultures*

SYNOPSIS. Nosema algerae, a microsporidan parasite of mosquitoes, can infect pig kidney cell cultures. Spores germinated in the culture medium, infected the cells within 30 min of germination, multiplied, and produced spores. The early developmental stages in the N. algerae life cycle are described.     

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.     

Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Changes of Photosystem Stoichiometry during Cell Growth in Dunaliella salina Cultures

The photosystem stoichiometry in Dunaliella salina thylakoidswas measured during cell growth in a fully contained culture.In dilute cultures, obtained after inoculation of cells intofresh growth medium, the PS II/PS I stoichiometry was about2.2/1.0. This ratio was gradually lowered to about 1.2/1.0 inmature cultures. The decrease of the PS II/PS I ratio is discussedin terms of increasing self-shading in the culture and increasingpH in the growth medium. Changes in the pH occurred from 7.7in young cultures to 8.9 in mature ones and caused a significantdepletion of soluble CO₂ from the growth medium. A correlationof the CO₂/HCO₃^– ratio in the growth medium with the PSII/PS I ratio in the thylakoid membrane is presented. ¹ Permanent address: Department of Physics, Palacky University,tr. Svobody 26, 771 46 Olomouc, Czechoslovakia (Received September 12, 1990; Accepted April 4, 1991)     

Effects of Activated Charcoal on Growth and Morphogenesis in Cell Cultures

The effects of activated charcoal on growth and morphogenesis in plate cultures of different plant cells have been studied. It was shown that medium containing charcoal induced embryogenesis in cultures of Daucus carota in which embryo formation could not be brought about by omitting auxin from the medium. Charcoal-medium also induced abundant root formation in older cultures of Allium cepa, which normally did not produce roots. The growth of cultures of Glycine max and Haplopappus gracilis was totally inhibited by charcoal. It is thought that activated charcoal removes substances from the medium, one of which might be auxin.     

Growth and Plating of Cell Suspension Cultures of Datura innoxia

Suspension cultures of Datura innoxia Mill, were successfully grown on a modified Murashige and Skoog medium with 2,4–D, NAA or BAP as growth substances, provided the micronutrient levels were reduced to 1/10. Normal amounts of micronutrients were toxic. Attempts to identify the toxic elements did not succeed. Cultures grew exponentially on a shaker at 27°C in the light. Their doubling times varied from 1.1 days on 2,4–D (10^–6M) or NAA (10^?5M)+ 1 g/1 casein hydrolysate to 2.7 days on BAP (3 × 10^?7M) and 5.1 days on supraoptimal levels of 2,4-D (10^?5M). Cultures grew on NH₄⁺-N alone (from ammonium malate) or on NO₃^?-N alone. Dry weight yield was proportional to the amount of nitrate-N added (47 mg/mg N). Filtered suspension cultures containing single cells (plating cultures) could be grown in agar in petri dishes when NAA or 2,4-D were used as growth substances. Cells grew at densities above 500 units/ml in the agar. Most colonies grew from cell aggregates but division in single cells was observed. The highest plating efficiency was about 50% on 10^?6 M 2,4-D + 1 g/1 casein hydrolysate.     

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.     

Preparation of La Crosse Virus Hemagglutinating Antigen in BHK-21 Suspension Cell Cultures

Hemagglutinating and complement-fixing antigens of La Crosse virus (California arbovirus group) were produced in serum-free suspension cultures of BHK-21/13S cells. The appearance and production of these antigens were correlated with the titer of infectious virus. No significant differences in antigen titers were produced by varying virus dose 10-fold. Hemagglutinin appeared 6 to 8 hr after inoculation and reached peak titer in 14 to 22 hr. Both beta-propiolactone and Tween 80-ether treatment inactivated infectious virus in the antigens. Unlyophilized antigen was stable at -60, 5 and 24 C for at least 117 days but not for 1 year. Lyophilized antigen was stable for at least a year, however, at -20 and 5 C. Cell culture-produced antigen was more sensitive than brain-produced antigen in detecting hemagglutination inhibition antibody in human sera.     

Growth of Respiratory Syncytial Virus in Suspension Cell Culture

Respiratory syncytial virus, Burnett strain, adsorbed efficiently and grew to high titers in suspension cultures of HEp-2 and MA-160 cells. Our results compared favorably with previous experience with the growth of respiratory syncytial virus in monolayer cell cultures. The use of suspension cell cultures provides a convenient and simple procedure for producing high-titering respiratory syncytial virus pools.     

Growth and Differentiation of Plant Tissue Cultures: II. Synchronous Cell Divisions in Developing Callus Cultures

Explants isolated from Jerusalem Artichoke tubers are stimulatedto divide when placed in contact with a nutrient medium containingsucrose, mineral salts, coconut milk, and 2: 4-dichlorophenoxyaceticacid. The first two or three cell divisions, which only occurin the outer layers of the explant, do not occur uniformly withtime but are, at least, partially synchronous. This synchrony,which decreases with successive divisions, is inherent. DNAsynthesis, which is an essential prerequisite for division inthese cells, is also partially synchronous. These conclusions,while being of some significance in relation to the interpretationof the early development of the callus, are also of some interestin relation to the possible exploitation of this system forthe study of cell division.     

Studies on Insect Growth Regulating Substances with Insect Cell Cultures

The sensitivity of cultured insect cells to a moulting hormone depended on their origin. Cell proliferation of cell-line C6/36, which originated from an Aedes albopictus larva, was suppressed by high concentrations of 20-hydroxyecdysone but was greatly promoted by low concentrations. Similar phenomena were observed with extracts of cedar and pine pollen. By employing the C6/36 cell-line for the screening of insect growth regulating substances, a highly active cell-growth promoting substance was found in a bovine pancreas extract. When 1 ppt of the partially purified substance was added to 2.5% fetal calf serum, the growth of cells was so greatly promoted that it was more than equal to that in the standard medium containing 10% of the serum.     

Complement-Fixing Antigen from BHK-21 Cell Cultures Infected with Lymphocytic Choriomeningitis Virus

Infection of BHK-21 cells with lymphocytic choriomeningitis (LCM) virus resulted in the production of significant titers of complement-fixing (CF) antigen. The antigen was spontaneously released from the cells, but the highest titer of 1:16 was recovered by disruption of the infected cells by freeze-thawing in tryptose phosphate broth. The antigen could be partially separated from infectious virus by centrifugation. Furthermore, it was possible to detect LCM virus infection of cell cultures by the production of the CF antigen, but this method proved less sensitive than titration by intracerebral inoculation of mice. The CF antigen from cell cultures was at least as sensitive and specific as the reference antigen prepared from infected guinea pig spleen.     

Changes in the Character of the Cell Wall in Growth ofBacillus megaterium Cultures

1. (1)
   Исследовалась кинетика образования протопяастов и освобождения C¹⁴ из клеток Bacillus megaterium, предварительно меченых C¹⁴ диаминопимеловой кислотой, под действием лмзоцима в фосфатной среде.