Abstract
Techniques are described for the culture of bovine embryonic lung (BEL) cells by the primary explant technique and mild enzymatic disaggregation of lung tissue. Preparations of bovine herpesvirus-1 (BHV-1) isolates with titers of 109 plaque-forming units/ml are purified by sucrose and cesium chloride gradient centrifugation after replication in primary cultures of BEL cells. BHV-1 isolated by velocity sedimentation bands in 35% sucrose and sediments at a density of 1.23 g/cc in cesium chloride density gradients. The DNAs of all virus isolates have a velocity sedimentation coefficient of approximately 54S. Virus DNAs from all isolates sediment to equilibrium in cesium chloride at 1.730 g/cc. Differences between virus isolates can be demonstrated by digestion of virus DNA with the restriction endonucleaseKpnI and by polyacrylamide gel electrophoresis of virion polypeptides. These techniques are useful for production of purified high titer virus free of host cell contaminants.
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