Transcriptome analysis of Frog virus 3, the type species of the genus Ranavirus, family Iridoviridae. Virology 2009

Frog virus 3 is the best characterized species within the genus Ranavirus, family Iridoviridae. FV3's large (∼105 kbp) dsDNA genome encodes 98 putative open reading frames (ORFs) that are expressed in a coordinated fashion leading to the sequential appearance of immediate early (IE), delayed early (DE) and late (L) viral transcripts. As a step toward elucidating molecular events in FV3 replication, we sought to identify the temporal class of viral messages. To accomplish this objective an oligonucleotide microarray containing 70-mer probes corresponding to each of the 98 FV3 ORFs was designed and used to examine viral gene expression. Viral transcription was initially monitored during the course of a productive replication cycle at 2, 4 and 9 hours after infection. To confirm results of the time course assay, viral gene expression was also monitored in the presence of cycloheximide (CHX), which limits expression to only IE genes, and following infection with a temperature sensitive (ts) mutant which at non-permissive temperatures is defective in viral DNA synthesis and blocked in late gene expression. Subsequently, microarray analyses were validated by RT-PCR and qRT-PCR. Using these approaches we identified 33 IE genes, 22 DE genes and 36 L viral genes. The temporal class of the 7 remaining genes could not be determined. Comparison o