BHK-21 Cell Cultures Research Articles

Herpes simplex virus replication in the presence of DNA polymerase alpha inhibitors

2-( p- n-butylanilino)deoxyadenosine (BuAdA), and N-2-( p- n-butylphenyl)deoxyguanosine (BuPdG), selective inhibitors of mammalian DNA polymerase alpha, were added to BHK-21(C13) cell cultures infected with herpes simplex virus type 1 (HSV-1) strain 17 syn +. Infectious virus production decreased significantly in the presence of the inhibitor at concentrations varying from 1 nM to 100 μM. BuPdG was more effective than BuAdA at all concentrations tested, while it inhibited virus yield as much as BuAdA when CVG2, a thymidine kinase deficient (TK-) HSV-1, was employed. HSV DNA synthesis, determined by quantitation of CsCl separated DNA peaks, was inhibited by each compound. BuPdG inhibited viral DNA replication more than BuAdA, while the effect on cell DNA synthesis was the same as that of BuAdA. CVG2 DNA replication was inhibited to the same level by BuAdA as by BuPdG. These results indicate that HSV DNA replication is partially dependent on cell DNA polymerase alpha activity, and that the greater effect of BuPdG on viral replication may be ascribed to its action on HSV thymidine kinase.

Identification of two species of actin depolymerizing factor in cultures of BHK cells.

High-speed supernatant obtained from the lysate of cultured BHK cells has been chromatographed on Sepharose-4B, DEAE-cellulose and hydroxyapatite columns, and a fraction has been identified with characteristics similar to an actin depolymerizing factor (ADF), a small protein previously isolated from embryonic chick brain. Using a rabbit antibody against the chick brain protein, two immunoreactive forms were identified: a 19 kDa form co-migrating in SDS-polyacrylamide gels with embryonic chick brain ADF, and a 20 kDa form. The two species could be separated on a hydroxyapatite or green A dye matrix columns and only the 20 kDa protein was active when assayed for effects on pyrene-G-actin assembly. It enhanced the rate of F-actin assembly, but only after an initial lag phase, and decreased the final proportion of actin in filamentous form. These effects were calcium-independent. Actin depolymerizing factor constituted at least 0.5% of the total protein in the cytoplasmic fraction. A Triton extract of plasma membrane-enriched fraction from BHK cells was fractionated on a Sepharose-4B column and again, a fraction was found which had an ADF-like activity and also contained the two immuno-cross-reactive forms, 19 kDa and 20 kDa. These results suggest a novel regulation of the microfilament system in eukaryotic cells via the control of the ADF activity.

Isolation of bluetongue virus from Culicoides sp. in India

Summary: Over 200 non-engorged midges (Culicoides) were caught in the vicinity of a flock of sheep infected with bluetongue virus (BTV) serotype 1. An isolate of virus, recovered from the midges by inoculating cultures of BHK-21 cells, was confirmed as BTV by using the double immunodiffusion test.

A preliminary study for development of an inactivated Chuzan virus vaccine.

A preliminaly study for development of an inactivated vaccine for Chuzan virus was performed. The best propagation of the virus was observed in BHK21 cell cultures. The culture fluid after treatment with formalin had good immunogenicity in cattle and guinea pigs. Immunized calves did not develop leukopenia and viremia, which were observed in a control calf, after challenge inoculation.

Growth of bluetongue and epizootic hemorrhagic disease of deer viruses in poikilothermic cell systems

Continuous cell lines from the ticks Dermacentor variabilis, D. parumapertus, D. nitens, Rhipicephalus sanguineus and R. appendiculatus, the mosquitoes Aedes albopictus and Culex quinquefasciatus and the African toad Xenopus laevis were tested for their ability to replicate bluetongue (BT) and epizootic hemorrhagic disease of deer (EHD) viruses, and for their sensitivity as potential isolation systems. BT serotype 17 grew to peak titers of 10 4.5−10 7.5 TCID 50 ml −1 in all except one of the tick cell lines, EHD 2 virus attained titers similar to that of BT 17 in the mosquito and toad cells, but failed to replicate in tick cells. Only Aedes albopictus and Xenopus laevis cells were as sensitive to infection with low-passage BT 11 and EHD 2 viruses as control cultures of Vero and BHK cells. At 27°C, persistent infection of Xenopus laevis cells occurred, producing low yields of BT 17 and EHD 2. When shifted to 32°C, these cultures expressed virus in exponential increments. No cytopathic effect (CPE) was seen in any of the tick-virus systems, but infected mosquito and toad cells detached from the monolayer within 3–6 days after inoculation with either virus. In the toad cells, this CPE was presaged by the development of plaques within 48 h after infection. Potential applications of poikilotherm systems in orbivirus research are discussed.

Evidence that the mature form of the flavivirus nonstructural protein NS1 is a dimer

Evidence is presented which indicates that the dengue-2 virus nonstructural protein NS1 (soluble complement fixing antigen) exists in infected BHK and mosquito cell cultures as part of a stable oligomer. Identification of the dissociation products of the isolated oligomer and comparison of the number of N-linked glycans in native and denatured NS1 is consistent with the idea that the high-molecular-weight form of NS1 is a homodimer. By analyzing lysates of BHK cells infected with St. Louis encephalitis virus or Powassan virus and proteins from dengue-2 virus-infected mouse brain we have demonstrated that the appearance of the high-molecular-weight form of NS1 is a general feature of flavivirus infection. It is formed between 20 and 40 min after NS1 is synthesized and before the protein passes the Golgi apparatus. Both soluble and pelletable extracellular NS1 are also found as the high-molecular-weight form.

Phosphatidylserine functions as the major precursor of phosphatidylethanolamine in cultured BHK-21 cells.

Pulse-chase experiments with [3H]serine provide evidence that significant amounts of phosphatidylserine turn over to form phosphatidylethanolamine in mammalian cells in tissue culture. Phospholipase C hydrolysis of [3H]phosphatidylethanolamine synthesized from [3H]serine by baby hamster kidney (BHK-21) cells demonstrates that nearly all of the radiolabel remains in the ethanolamine moiety. Uniform labeling experiments with [3H]serine further demonstrate that the distribution of radiolabel in phosphatidylserine and phosphatidylethanolamine is nearly identical to the mass ratio of these lipids. Physiological concentrations of ethanolamine (20 microM) have only a marginal effect upon the ability of cells in culture to incorporate radiolabeled serine into either phosphatidylserine or phosphatidylethanolamine. These data provide compelling evidence that phosphatidylethanolamine synthesis via phosphatidylserine and phosphatidylserine decarboxylase contributes significantly to membrane biogenesis in mammalian cells.

4-Deoxy-4-fluoro-D-mannose inhibits the glycosylation of the G protein of vesicular stomatitis virus.

The effect of 4-deoxy-4-fluoro-D-mannose (4F-Man), a synthetic analog of D-mannose, on the synthesis of the glycoprotein (G) of vesicular stomatitis virus was examined. Nearly confluent monolayers of cultured BHK21 cells infected with vesicular stomatitis virus were incubated for 2 h with 4F-Man (0-10 mM) or for 1 h with tunicamycin (2 micrograms/ml) and then pulse-labeled with [35S]methionine or [3H]glucosamine. After a 90-min chase period, the cells were lysed and the viral proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. The 35S-labeled G protein from cells exposed to greater than or equal to 1 mM 4F-Man migrated more rapidly than G protein isolated from control cells and with the same electrophoretic mobility as the glycoprotein produced by cells treated with tunicamycin. When infected cells were labeled with [3H]glucosamine, little or no radioactivity was associated with G protein synthesized in the presence of greater than or equal to 1 mM 4F-Man. The conclusion that 4F-Man blocks the glycosylation of the G protein was supported by experiments which demonstrated that the fluorosugar inhibits the synthesis of lipid-linked oligosaccharides.

Utilizacao da cultura de celulas de rim de hamster para o isolamento do virus da caxumba (parotidite epidemica).

Continuous-line cultures of hamster kidney cells (BHK-21) was found to be a more rapid and sensitive procedure for isolation of mumps virus as compared with the embryonated chicken egg procedure. BHK-21 cells can be easily maintained in the virus labotory, thus contrasting with similarly sensitive primary cells of monkey kidney wh ich are difficult to obtain and cannot be maintained. Use of BHK-21 cell cultures for the isolation of mumps virus seems to be a useful alterna tive procedure.

Characterization of aphtovirus subtype Asia 1/2 recovered in India and development and potency testing of a highly concentrated purified vaccine.

Summary : The aphthovirus subtype Asia 1/2 (strain India 21/80) recovered in India was further characterized by microneutrali zation, guinea pig protection and challenge tests in cattle. These tests confirmed it to differ from subtype Asia 1/1 and corroborated the results of microcompleme nt fixation tests. It multiplied to high titre in BHK21 cell culture. A PEG-concentrat ed vaccine was prepared using this strain, which when tested in cattle proved to be highly immunogenic and protective in a 1 ml dose. The studies also demonstrated that it is possible to readily adapt an indigenous strain and manufacture an effective vaccine in a short time to meet the need of an effective control of FMD in a given area.

In vitro toxicity and transformation potency of nickel compounds.

An in vitro bioassay utilizing BHK-21 cells in culture is used to determine the relative transformation potential of a number of nickel compounds including, as relatively insoluble particulates a known carcinogen (Ni3S2) and several oxides either of commercial interest or found in the working environment in the metal industry (e.g., NiO), and a soluble salt [Ni(CH3COO)2]. Although a wide range of transformation potency is found as a function of the dose of Ni per area of culture, all substances produce the same number of transformed colonies at the same degree of toxicity (e.g., 50% survival). If toxicity is a direct measure of intracellular concentration, then apparently nickel per se is the ultimate transforming agent independent of source or uptake mechanism.

In vitro Toxicity and Transformation Potency of Nickel Compounds

An in vitro bioassay utilizing BHK-21 cells in culture is used to determine the relative transformation potential of a number of nickel compounds including, as relatively insoluble particulates a known carcinogen (Ni3S2) and several oxides either of commercial interest or found in the working environment in the metal industry (e.g., NiO), and a soluble salt [Ni(CH3COO)2]. Although a wide range of transformation potency is found as a function of the dose of Ni per area of culture, all substances produce the same number of transformed colonies at the same degree of toxicity (e.g., 50% survival). If toxicity is a direct measure of intracellular concentration, then apparently nickel per se is the ultimate transforming agent independent of source or uptake mechanism.

Serum rapidly mobilizes calcium from an intracellular pool in quiescent fibroblastic cells

Addition of dialysed fetal bovine serum to quiescent cultures of Swiss 3T3 cells loaded with 45Ca 2+ causes a very rapid increase in the rate of 45Ca 2+ efflux from an intracellular pool. Exposure to serum for 2 min leads to a fall of 0.59 nmol Ca 2+/mg protein in the intracellular Ca 2+ content of the cells. Inhibitors of mitochondrial function prevent the stimulation of 45Ca 2+ efflux by serum. The stimulation of 45Ca 2+ efflux by serum is also observed in quiescent cultures of Rat-1, Swiss 3T6 and BHK cells and in secondary cultures of whole mouse embryo fibroblasts.

Isolation of cell-surface glycopeptides from bovine cerebral cortex that inhibit cell growth and protein synthesis in normal but not in transformed cells.

Glycopeptides were isolated from the cell surfaces of bovine cerebral cortex that could inhibit increase in cell numbers in tissue culture and cellular protein synthesis. This cell growth inhibition apparently affected all cells exposed, could completely block cell division in a reversible manner and synchronized BHK-21 cell cultures. Polyoma-virus-transformed BHK-21 cells were completely insensitive to the inhibitor. Fractionation of the inhibitor on a Bio-Gel P-100 column revealed two peaks of biologically active material eluting at apparent molecular weights of 45 000 and 10 000 with A 1cm,280 1% 11.0. Affinity purification of the inhibitory fractions on a Ulex europaeus agglutination I lectin column resulted in retention of the inhibitory activity, suggesting the inhibitor material was a glycopeptide. Subsequent elution with 0.10 M-fucose resulted in a 244-fold increase in the specific biological activity over the starting material. Although purified from bovine brain, the material could inhibit baby-hamster kidney cell protein synthesis by 50% at a concentration of 5 x 10(6) molecules per target cell. Analysis by competitive radioimmunoassay or immunoadsorption indicated that the bovine inhibitor was structurally related to, although not necessarily identical with, a similar inhibitory glycopeptide preparation that we had previously isolated from mouse brain.

Epizootic Hemorrhagic Disease Virus Infection in Salivary Glands ofCulicoides Variipennis

Epizootic hemorrhagic disease virus (EHDV) is transmitted to ruminants by the biting gnatculicoides variipennis. In cultured cells, EHDV replicated in cytoplasmic matrixes; filaments and macrotubules were observed in the cytoplasm and in association with viroplasms. This report describes EHDV infection ofC. variipennissalivary gland cells.Normal colony female flies were infected either by intrathoracic inoculation of EHDV propagated in BHK-21 cell cultures (CC) or orally on EHDV-infected CC fluids mixed 1:9 with normal sheep blood. EHDV-exposed flies and noninfected controls were maintained alternately on water and a 10% sugar solution until salivary glands were removed at 9 (inoculated flies) and 19 (orally exposed flies) days for processing and viewing in thin section.Viroplasms were observed in the cytoplasm often in intimate association with progeny virions and macrotubules (Fig. 1). Macrotubules and progeny virions also were found free in the cytoplasm away from viroplasms (Fig. 2). Macrotubules were found in bundles in ordered array whether in association with viroplasms or free in the cytoplasm.

The association of calf serum with the contamination of BHK21 clone 13 suspension cells by a parvovirus serologically related to the minute virus of mice (MVM).

An investigation of persistent cell deaths of BHK21 suspension cells during subculturing resulted in the isolation of a viral agent. The agent was isolated from samples of dead cells, cell growth media and 2 batches of calf serum. It was established that the agent was associated with the use of certain batches of calf serum. The isolated virus was found to replicate effectively only in rapidly growing BHK cell cultures. In monolayers it caused the formation of large intranuclear inclusion bodies. The isolate was a strong haemagglutinin; it was stable to ether, chloroform, pH 3 and heating at 56 degrees C. It was shown to be a DNA-virus and by electron-microscopy it was evident as unenveloped spherical, small particles (21 nm in diameter). In sucrose density gradients the virus sedimented as 2 peaks at approximately 114S and 85-92S, and by caesium chloride equilibrium centrifugation, two main peaks at densities of 1.39 and 1.31 g/ml were evident with a minor peak at 1.35. The capsid of the complete virion consisted of 3 polypeptides. The agent has therefore been provisionally designated a member of the Parvoviridae family, genus parvovirus. Serologically it was found to be related to MVM. The isolated parvovirus was inactivated by 0.05 per cent peracetic acid and 0.05 per cent acetylethyleneimine.

Connecticut Virus: A New Sawgrass Group Virus from Ixodes Dentatus (Acari: Ixodidae)1

A new member of the Sawgrass serogroup, Connecticut virus, was isolated from a pool of 21 nymphal Ixodes dentatus removed from 2 nonviremic Eastern Cottontails ( Sylvilagus floridanus ) trapped on 22 August 1978 in Lyme (New London Co.), Connecticut, USA. This strain (Ar-1152-78) killed suckling but not weanling mice by intracerebral inoculation; neither suckling nor weanling mice were killed by intraperitoneal or subcutaneous inoculation. The only evidence of infection in adult female laboratory rabbits and guinea pigs inoculated subcutaneously was neutralizing antibody in the guinea pigs. Plaques were first observed in Vero and BHK-21 cell cultures on 6 and 8 days, respectively, after inoculation. Neutralizing antibody was detected in 3 of 14 (21.4%) Eastern Cottontails from south-central Connecticut, suggesting an I. dentatus –rabbit maintenance cycle.

Vesicular stomatitis virus and sindbis virus glycoprotein transport to the cell surface is inhibited by ionophores

The effect of two cationic ionophores, monensin and A23187, on the replication of Sindbis and vesicular stomatitis virus in cultures of BHK and chicken embryo fibroblast cells has been studied. Treating these cells with the ionophores at 10 −6, M 2 hr prior to infection at high multiplicities of infection did not affect viral protein synthesis but severely inhibited release of viral particles into the media. Glycoproteins of these viruses were synthesized in normal amounts but they did not appear on the surface of infected cells. Proteolytic cleavage of Sindbis virus glycoprotein PE2 to E2 was inhibited in ionophore-treated cells, although fatty acid attachment to PE2 proceeded normally. Fatty acid attachment to VSV G protein and the posttranslational removal of mannose residues from G oligosaccharides occurred in the drug-treated cells. These data indicate that these ionophores block the movement of viral glycoproteins from the Golgi apparatus to the cell surface membrane where budding and release of these two viruses occur.

Short-lived minus-strand polymerase for Semliki Forest virus.

Semliki Forest virus (SFV)-infected BHK-21, Vero, and HeLa cells incorporated [3H]uridine into 42S and 26S plus-strand RNA and into viral minus-strand RNA (complementary to the 42S virion RNA) early in the infectious cycle. Between 3 and 4 h postinfection, the synthesis of minus-strand RNA ceased in these cultures, although the synthesis of plus-strand RNA continued at a maximal rate. At the time of cessation of minus-strand RNA synthesis, two changes in the pattern of viral protein synthesis were detected: a decrease in the translation of nonstructural proteins and an increase in the translation of the viral structural proteins. Addition of cycloheximide and puromycin to cultures of SFV-infected BHK cells actively synthesizing both viral plus- and minus-strand RNA resulted within 15 to 30 min in the selective shutoff of minus-strand RNA synthesis. Removal of the cycloheximide-containing medium led to the resumption of minus-strand synthesis and to an increased rate of viral RNA synthesis. We conclude that the minus-strand polymerase regulates the rate of SFV plus-strand RNA synthesis by determining the number of minus-strand templates and that the synthesis of the minus-strand templates is regulated at the level of translation by a mechanism which utilizes one or more short-lived polymerase proteins.

The effects of egta and trypsin on the serum requirements for cell attachment to collagen

Cells growing on plastic or glass surfaces in vitro may be brought into suspension by proteases (e.g. trypsin) or chelating agents (e.g. EGTA). Trypsin and EGTA remove different quantities and types of molecules from cell surfaces. Previous studies have revealed that when confluent cultures of either BHK or PyBHK cells are brought into suspension by exposure to trypsin, foetal calf serum (or fibronectin) is required for cell attachment to films of denature type I collagen, but not to 3-dimensional gels of native collagen fibres. In this communication the serum requirements for the attachment of BHK and PyBHK cells to collagen substrata have been examined as a function of (a) the method used to prepare the cell suspension (EGTA or trypsin), and (b) cell density. Data are presented consistent with the view that cell surface-associated fibronectin is able to mediate cell attachment directly to films of denatured collagen.