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Hemagglutination with Nebraska Calf Diarrhea Virus

Nebraska calf diarrhea (NCD) virus or reovirus-like agent of calf diarrhea has been shown to be a primary etiological agent of neonatal calf diarrhea by Mebus and his associates (1, 4, 5). Recently Spence et al (6) prepared hemagglutinin from African green-monkey kidney cells infected with NCD virus. This antigen agglutinated human group O cells. We also examined hemagglutination (HA) with NCD virus grown in calf kidney (BK) cells and found that the virus agglutinated erythrocytes of human and some other animals. This paper describes our observations on the HA with this virus and its inhibition (HI) by specific antiserum. Lincoln strain of NCD virus (4) maintained by serial passage in BK cells, was supplied by Dr. C.A. Mebus, University of Nebraska, Lincoln, Nebraska, and used in the present study after several passages in BK cells in our laboratory. BK cell cultures were prepared as described previously (2). The growth medium used was LE medium (Earle's solution containing 0.5% lactalbumin hydrolysate) supplemented with 10% calf serum. Primary BK cell monolayers prepared in 500-ml bottles were inoculated with 0.1 TCID50 per cell of virus and incubated at 37 C for 2 days with LE medium containing 0.1 % yeast extract. As described later, in some preliminary experiments, culture fluid harvested from infected cells was used as HA antigen after centrifugation to remove coarse debris. In subsequent experiments, however, HA antigens concentrated from infected culture fluid were used. HA and HI tests were carried out by the microtiter method. The diluent used was VBS (veronal-buffered saline, pH 7.0, containing 0.1 % bovine serum albumin and 0.001% gelatin), unless otherwise stated. Blood was obtained in Alsever's solution and stored at 4 C. Erythrocytes from horse, cattle, sheep and goat were used as 0.3% suspensions, and those from other species as 0.5% suspensions. In HA tests, serial 2-fold dilutions of HA antigen were prepared in 0.05-ml amounts, and mixed with 0.025 ml of an erythrocyte suspension. The mixtures were incubated at 37 C for 1 hr before the results were read, unless otherwise stated. The HA titer was expressed as the reciprocal of the highest antigen dilution showing complete HA. For HI tests, 0.2 ml of the serum was inactivated by heating at 56 C for 30 min, diluted 5-fold, mixed with 0.2 ml of packed erythrocytes and incubated at 37 C for 1 hr. After removal of the cells by centrifugation, the supernatant fluid was mixed with 0.5 ml of 25% kaolin, incubated at room temperature for 1 hr, and centrifuged to remove kaolin. The resulting supernatant fluid was taken as the 10-fold dilution