A unique feature of ascovirus infection is cleavage of host cells into virus containing vesicles. It has been suggested that the virus induces apoptosis, either by expression of a caspase or other means, which is then diverted toward vesicle formation. There is little known about the mechanism of vesicle formation. Recent genome sequences of three ascoviruses indicated the presence of several putative open reading frames coding for proteins that could be involved in lipid metabolism. These proteins may play a role in rearrangement of membranes in infected host cells leading to formation of vesicles. Here, we analyzed a lipase-like gene (ORF19) from Heliothis virescens ascovirus (HvAV-3e) expressed from 8 h after infection and essential for virus replication and cell cleavage. In addition, ORF19 knock down by RNA interference inhibited virus replication indicating that the gene is indispensable for HvAV-3e replication. However, under enzymatic assays tested, we did not detect any lipase or esterase activity from ORF19.