A TITRATION PROCEDURE OF THE JUNONIA-CAENIA-DENSOVIRUS AND QUANTITATION OF TRANSFECTION BY ITS CLONED GENOMIC DNA IN 4 LEPIDOPTERAN CELL-LINES

A sensitive and reproducible tissue culture biossay method was develop ed based on indirect immunofluorescence to titrate virus suspensions o f the Junonia cania densovirus (JcDNV) and to quantify transfections b y its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodaptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells deri ved from Spodoptera litura ovaries and hemocytes, respectively, and th e SC-LD 135 from Lymantria dispar were compared for their efficiency t o support viral replication. The viral titres expressed as TCID50/ml a veraged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were a bove 10(7) for SPC-PL 65 cells. Even with this most sensitive cell lin e, the rate of infected cells did not exceed 75% and decreased progres sively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant p lasmid encompassing an infectious sequence of JcDNV genome. SPC-SL 52 cells were found to be the most sensitive, and the lipofection method resulted in about a 5-fold increase compared to the calcium phosphate precipitation protocol. The rescued virions proved to be infectious an d the restriction profiles of their DNA were identical to that of wild type virions.