Susceptibility of Autographa californica multiple nucleopolyhedrovirus to inhibitors of DNA replication.
The objectives of this study were to develop methods to evaluate the susceptibility of the type baculovirus AcMNPV to various antiviral compounds and to select potential inhibitors for investigating baculovirus DNA replication. In concert with the classical cytopathic effects (CPE) and cytotoxicity inhibition assays, two approaches, which could be amenable for high throughput application for evaluating several classes of known antiviral compounds were developed. (i) An indirect approach based on spectrofluorimetric analysis of EGFP expression in Sf21 cells infected with a recombinant AcMNPV (AcEGFP) and (ii) a direct DNA quantitative assay based on quantitative real time PCR (qPCR). Initial CPE results suggested that of 21 compounds tested, aphidicolin, abacavir, camptothecin, (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU), l-mimosine, hydroxyurea and phosphonoacetic acid (PAA) were selective inhibitors of AcMNPV replication. Consistent with the CPE results, the EGFP fluorescence and the qPCR of viral DNA accumulation exhibited a dose dependent depression of EGFP expression and DNA accumulation, respectively, in infected cells exposed to them. The inhibitory effects of aphidicolin, abacavir, l-mimosine and hydroxyurea on AcMNPV DNA replication were reversible. Taken together, both spectrofluorimetric and qPCR assays are suitable and rapid quantitative approaches to investigate inhibitors of baculovirus DNA replication in infected cells.
Thumbi
David K
DK
Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ont. N1G 2W1, Canada.
Arif
Basil M
BM
Krell
Peter J
PJ
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D003588:Cytopathogenic Effect, Viral; D004261:DNA Replication / Q000187:drug effects; D004279:DNA, Viral / Q000378:metabolism; D008826:Microbial Sensitivity Tests / Q000379:methods*; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D017924:Nucleopolyhedroviruses / Q000187:drug effects* / Q000502:physiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D018411:Spodoptera / Q000821:virology*; D019562:Viral Load / Q000379:methods*; D014779:Virus Replication / Q000187:drug effects
D000998:Antiviral Agents; D004279:DNA, Viral; D019384:Nucleic Acid Synthesis Inhibitors