Replicative kinetics of classical swine fever virus in PK-15 cells

Abstract:

In order to understand the replication kinetics of classical swine fever virus(CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37℃ in 5% CO₂ environment when growing to 80% confluence，the cells were infected with CSFV strain Shimen at 100 TCID₅₀ per well. At various time post infection(p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay(IFA)，RNA replication using reverse transcription real-time PCR and viral production using titration of TCID₅₀. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining.the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However，the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID₅₀ demonstrated that the production of live virions increased at 8h and peaked between 48-72 hrs p.i. without significant lose of titer.