There are no stable permissive cell lines available for in vitro replication of PlxyGV. In this study, several PlxyGV bacmids containing egfp and plasmids expressing the luciferase gene (luc) were constructed and used to transfect insect cell lines from Plutella xylostella, Trichoplusia ni (Hi5), and Spodoptera frugiperda. Fluorescence was observed only in the cells transfected with a bacmid with egfp driven by a PlxyGV ie1 promoter, but not by a PlxyGV vp39 or granulin promoter. In transient assays, various levels of LUC activity were detected in the cells transfected with individual reporter plasmids containing the luc driven by the promoters of PlxyGV early genes ie1, exon0, dnapol, lef1, lef9, and orf105, s... More
There are no stable permissive cell lines available for in vitro replication of PlxyGV. In this study, several PlxyGV bacmids containing egfp and plasmids expressing the luciferase gene (luc) were constructed and used to transfect insect cell lines from Plutella xylostella, Trichoplusia ni (Hi5), and Spodoptera frugiperda. Fluorescence was observed only in the cells transfected with a bacmid with egfp driven by a PlxyGV ie1 promoter, but not by a PlxyGV vp39 or granulin promoter. In transient assays, various levels of LUC activity were detected in the cells transfected with individual reporter plasmids containing the luc driven by the promoters of PlxyGV early genes ie1, exon0, dnapol, lef1, lef9, and orf105, suggesting that the PlxyGV early genes could be activated in the cells independent of virus infection. The addition of a PlxyGV bacmid in the transfections activated luc expression from the promoters of PlxyGV late genes vp39 and granulin only at minimum levels, and caused significant reduction in luc expression from the early promoters, may be due to apoptosis triggered by the PlxyGV bacmid. PlxyGV reporter bacmids containing Autographa californica multiple nucleopolyhedrovirus (AcMNPV) genes p35 or p35 and ie1 or p35, ie1 and gp64 expressed LUC from a PlxyGV vp39 promoter at levels of 2.6, 8.3, and 23 times higher than those produced by the basic PlxyGV reporter bacmid, respectively, in transfected Hi5 cells. Green fluorescence was present in the cultures of all three cell lines transfected by a PlxyGV bacmid containing egfp with a vp39 promoter and AcMNPV ie1, p35, and gp64 with their native promoters. The fluorescence was also observed in the culture of Hi5 cells inoculated with the supernatant from the transfection. These results suggest that AcMNPV p35 could rescue late gene expression, and the ie1, p35, and gp64 may cooperatively rescue replication of PlxyGV in the cells.
