Molecular and immunohistochemical characterization of granulin gene encoded in Pieris rapae granulovirus genome

Abstract

Pieris rapae granulovirus (PiraGV) is highly pathogenic to the cabbage butterfly (P. rapae), an important pest of cultivated cabbages and mustard crops. It therefore holds significant promise towards exploitation as a potent bio-control agent in the field controlling the pest population. Whole-genome elucidation of the Korean isolate of the granulovirus (PiraGV-K), reported the presence of a granulin gene corresponding to ORF 1 in its genome. Comprehensive studies towards functional characterization of the gene, established that it is composed of 744 nucleotides and encodes a peptide of 247 amino acid residues. It possessed significant homology with AoGV and ClanGV with 87% identity at amino acid level. Multiple alignment data suggests that the C-terminus region of the gene had three different conserved regions. Time-course studies conducted in PiraGV-K infected P. rapae larvae revealed a significant upsurge of the transcript (134-fold) at 4 days post infection followed by a significant decline at the most advanced stages of infection. Anti-PiraGV-K granulin antibody was produced and western blot conducted with the infected larvae further confirmed the induction pattern with a protein of 30 kDa. Immunofluorescent staining showed a granulin-specific signal in fat body and integument of the infected larvae. Granulin-specific signals were noticed 2 days post infection with the eventual systemic spread of infection to the associated tracheal matrix witnessed at 4 days post infection. Immunogold labeling and electron microscopic studies further proved the cytopathological effects as the presence of numerous membrane-bound vesicles with nucleocapsids and abruption of intercellular junctions in fat body and hypertrophied cells in the integument.