key: cord-0007666-zkjnzqax authors: Sato, K.; Inaba, Y.; Miura, Y.; Tokuhisa, S.; Matumoto, M. title: Inducement of cytopathic changes and plaque formation by porcine haemagglutinating encephalomyelitis virus date: 2002-11-13 journal: Vet Microbiol DOI: 10.1016/0378-1135(83)90001-9 sha: e50347e4c7fbc6685835616033f2a3fb2cefdc88 doc_id: 7666 cord_uid: zkjnzqax ESK cells were shown to be a good medium for propagating the 67N strain of porcine haemagglutinating encephalomyelitis virus, although no cytopathic effect was observed. The virus induced a readily recognizable cytopathic effect in ESK cells, when a non-cytotoxic amount of diethylaminoethyl-dextran (DEAE-dextran) was incorporated in the culture medium. Based on this finding, a sensitive, practical assay method for the virus was developed. When DEAE-dextran was incorporated in the agar overlay medium, 67N virus formed plaques in ESK cell monolayers. The cytopathic effect as well as the plaque formation were specifically inhibited by antisera against the virus. Neutralization tests were developed on the basis of these findings. Neutralization and haemagglutination-inhibition tests on swine serum samples indicated a wide dissemation of haemagglutinating encephalomyelitis virus or antigenically-related viruses in Japanese pigs. isolated a haemagglutinating virus (strain 67N) in primary cultures of embryonic pig kidney cells from the nasal cavity of an apparently healthy feeder pig in the United States. This virus was subsequently shown to be the porcine haemagglutinating encephalomyelitis virus (HEV) which had been isolated previously in Canada (Greig et ai., 1962; Greig and Girard, 1963) . The HEV (67N strain) multiplied, but failed to induce readily recognizable cytopathic effect in primary cultures of swine embryonic kidney cells. Therefore, the virus has been assayed in these cultures by a rather cumbersome method in which the presence of virus infection was determined by haemadsorption with chicken erythrocytes or by microscopic examination for syncytia of cultures stained with May--Griinwald--Giemsa stain (Mengeling et al., 1972) . Recently we observed that the 67N strain replicated readily, but induced no cytopathic effect in ESK cells, a continuous cell line from swine embryo kidney (Sugimori et al., 1969) . However, the strain was shown to induce cytopathic effect in ESK cells, when a non-cytotoxic amount of diethylaminoethyl-dextran (DEAE-dextran) was incorporated in the culture medium, thus providing a sensitive, practical assay method. The method was readily adapted to a neutralization test. Furthermore, the strain formed plaques in ESK cells under an agar overlay medium containing DEAE~lextran. This paper describes these observations. ESK cells, a stable cell line derived from swine embryo kidney (Sugimori et al., 1969) , were used. The cell line was kindly supplied by Dr. T. Sugimori, National Institute of Animal Health, Japan. Cells were grown in Eagle's minimum essential medium (MEM) containing 10% inactivated calf serum, 10% tryptose phosphate broth (TPB) (Difco), 100 units ml -t penicillin, 100 gg m1-1 streptomycin and 1 tag ml -~ fungizone. Monolayer cultures were prepared in 11 × 100 mm tubes by seeding with 1.25 X l0 s cells suspended in 0.5 ml growth medium and incubating at 37°C in a stationary rack. After removal of the culture fluid and washing with MEM, cultures were inoculated with 0.1-ml volumes of virus suspension, incubated at 37°C for 60 min for virus adsorption, fed with 0.5 ml of maintenance medium and incubated at 37°C in a roller drum. The maintenance medium was MEM containing 10% TPB, 0.05% yeast extract, 0.5% sodium glutamate, 0.1% glucose and antibiotics. Strain 67N of porcine HEV (Mengeling et al., 1972) was kindly supplied by Dr. K. Hirai, Gifu University, Japan, and passaged twice in ESK cells before being used in the present study. A stock solution of DEAE-dextran (Pharmacia Fine Chemicals, Uppsala, Sweden) containing 1 mg m1-1 was made in distilled water, sterilized by autoclaving and stored at 4°C until use. The following method was established on the basis of the results obtained in the present study. Serial decimal dilutions of the test sample were made in maintenance medium and each dilution was inoculated in 0.1 ml volumes into 3 tube cultures of ESK cells. The inoculated cultures were incubated in a roller drum at 37°C for 7 days. The maintenance medium contained 50 pg ml -~ of DEAE-dextran. At the end of incubation, the cultures were examined for cytopathic effect and the 50% tissue culture infectious dose (TCIDs0) was calculated by the method of K~ber. Monolayer cultures of ESK cells prepared in petri dishes, 55 mm in diameter, were inoculated with 0.2-ml volumes of virus diluted in maintenance medium containing 50 pg m1-1 DEAE-dextran and antibiotics. After virus adsorption at 37°C for 60 min, the cultures were washed 3 times with MEM and covered with 6 ml of agar overlay medium. The overlay medium was 0.8% LGT agar (Miles, U.S.A.) in MEM containing 50 pg ml -~ DEAEdextran and antibiotics. The cultures were incubated in an atmosphere of 5% CO2 in air at 37°C for 4 days and stained by incubating overnight at 37°C under a second overlay medium containing 100 pg ml -~ iodonitrotetrazolium violet (Sigma, U.S.A.). The infectious titre was expressed in plaque-forming units (PFU). Antisera against the 67N strain were prepared in rabbits. Supernatant culture fluid, from ESK cells infected with the 67N strain, was centrifuged at 100 000 g for 2 h and the resulting pellets were dissolved in 0.01 volume of phosphate-buffered saline (PBS) at pH 7.2. The animals were inoculated intravenously with the virus suspension and 3 weeks later intramuscularly and subcutaneously with an equal-volume mixture of the virus suspension and Freund's complete adjuvant. Serum was obtained 4 weeks later. The sera were inactivated at 56°C for 30 min and stored at --20°C until use. Serial twofold dilutions of the serum inactivated at 56°C for 30 rain were made with maintenance medium containing 50/~g ml ~1 DEAE-dextran. In some experiments serial fourfold dilutions were used. Each serum dilution was mixed with an equal volume of maintenance medium containing 200 TCIDs0/0.1 ml of virus and incubated at 37°C for 60 rain. The virus--serum mixtures were then inoculated in 0.1-ml volumes into tube cultures of ESK cells, using 2 tubes for each dilution. After incubation at 37°C for 60 min, the inoculated cultures were fed with 0.5 ml of maintenance medium containing 50 pg m1-1 of DEAE-dextran, incubated in a roller drum at 37°C for 7 days, and examined for any cytopathic effect. The antibody titre was expressed as the reciprocal of the highest serum dilution which showed complete inhibition of cytopathic effect in at least one of the two tubes. Haemagglutinin titres were determined by adding 0.025 ml of a 0.5% suspension of washed chicken erythrocytes in PBS to 0.05 ml of each of twofold dilutions of virus. The mixtures were examined after 60 min of incubation at 22°C. The titre was expressed as the maximum virus dilution that caused HA. This was carried out by the microtitre method (Sato et al., 1977) . The serum was inactivated at 56°C for 30 rain and treated with kaolin to remove non-specific inhibitors and with packed chicken erythrocytes to remove antibodies to the cells. The HI antibody titre was expressed as the reciprocal of the highest serum dilution which showed complete inhibition of HA with 4 units of haemagglutinin. Experiments were done to determine the toxic concentrations of DEAEdextran for ESK cells. Tube cultures of ESK cells were fed with 0.5 ml of maintenance medium containing various amounts of DEAE-dextran, and incubated in a roller drum at 37°C for 7 days. The presence of DEAEdextran in concentrations up to 50 ~g m1-1 was not injurious to ESK cells, whereas 60 ~g m1-1 or higher concentrations induced cytotoxic effects consisting of rounding, granulation and eventual disintegration of the cells. These changes began to appear within 24 h and almost all the cells were damaged at the end of incubation in the presence of 100 pg m1-1 of DEAEdextran, while the changes appeared after 2 and 3 days of incubation and 70 and 40% of the cells were destroyed in 7 days of incubation in cultures containing DEAE