A titration procedure of the Junonia coenia densovirus and quantitation of transfection by its cloned genomic DNA in four lepidopteran cell lines.
A sensitive and reproducible tissue culture biossay method was developed based on indirect immunofluorescence to titrate virus suspensions of the Junonia coenia densovirus (JcDNV) and to quantify transfections by its cloned genomic DNA. Four lepidopteran cell lines, the SPC-SL 52 from Spodoptera littoralis, the SPC-PL 40 and the SPC-PL 65 cells derived from Spodoptera litura ovaries and hemocytes, respectively, and the SC-LD 135 from Lymantria dispar were compared for their efficiency to support viral replication. The viral titres expressed as TCID50/ml averaged 10(5) for SPC-SL 52, SPC-PL 40 and SC-LD 135 cells, but were above 10(7) for SPC-PL 65 cells. Even with this most sensitive cell line, the rate of infected cells did not exceed 75% and decreased progressively by serial subcultures. Two transfection protocols were used to compare the sensitivity of the same four cell lines to a recombinant plasmid 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 and the restriction profiles of their DNA were identical to that of wild type virions.
Li
Y
Y
Unité de Virologie Moléculaire, Station de Recherches de Pathologie Comparée, INRA-UA CNRS 1184, Saint Christol-lez-Alès France.
Jousset
F X
FX
Giraud
C
C
Rolling
F
F
Quiot
J M
JM
Bergoin
M
M
D000818:Animals; D002130:Calcium Phosphates / Q000737:chemistry; D002460:Cell Line; D011232:Chemical Precipitation; D003001:Cloning, Molecular; D004279:DNA, Viral; D017990:Densovirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D016679:Genome, Viral; D009036:Moths / Q000166:cytology; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000166:cytology; D014162:Transfection; D014771:Virion
D002130:Calcium Phosphates; D004279:DNA, Viral; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; C485829:calcium phosphate, dibasic, anhydrous