Trypsin-enhanced replication of neonatal calf diarrhea coronavirus in bovine embryonic lung cells

Bovine embryonic lung (BEL) cells, grown in 8-chamber cell-culture slides, were inoculated with 2.5, 25, and 250 TCID50 of neonatal calf diarrhea coronavirus (NCDCV)/chamber. The NCDCV inocula were prepared in serum-free, trypsin-free regular medium (RM), and in serum-free medium containing 1 microgram (TM1), 5 microgram (TM5), and 10 microgram (TM10) of trypsin/ml; the the cells were maintained under identical media. Cytopathic effect (CPE), hemadsorption of rat RBC, formation of syncytia, appearance of immunofluorescing cells, and release of infectious virus was followed from postinoculation hours 4 to 100. Maintaining the BEL cells in medium containing trypsin up to 10 microgram/ml did not affect them adversely. The BEL cells appeared resistant to infection with NCDCV in RM by CPE, syncytia, immunofluorescing cells, or infectious virus-release parameters. In TM1, TM5, and TM10, all parameters were positive for NCDCV replication. They manifested and developed in positive correlation with increasing trypsin concentration from 1 microgram to 10 microgram/ml. Release of infectious virions preceded all other parameters of virus replication. Replication of NCDCV was demonstrated earlier by hemadsorption and immunofluorescing cells than by syncytia or CPE. The TM5 and TM10 increased the susceptibility of BEL cells to NCDCV by more than a millionfold, as measured by the titer of released virus in these media (greater than 1-(6) TCID50/ml) as compared with those in RM (infectious virus not demonstrated).