Characterization and antibody preparation of the gene products of grouper iridovirus ORF120L

Abstract

Grouper iridovirus (GIV) is regarded as a prominent viral pathogen of grouper, particularly during the larval and juvenile stages. The aim of this study was to comprehensively characterize the GIV-120L gene during viral infection. The results of sequence analysis suggest that GIV-120L is a 1,470 bp gene encoding a Ranavirus-specific viral protein. Recombinant GIV-120L protein was purified using a nickel-affinity column, and its molecular weight was found to be 59.1 kDa. To obtain antibodies against GIV, mice were immunized with recombinant GIV-120L protein, the spleen was harvested 8 weeks later, and hybridoma testing was performed using Sp2/0 myeloma cells. Polyclonal and monoclonal antibodies against GIV-120L were obtained. To characterize GIV-120L gene expression, grouper kidney (GK) cells were infected with GIV using cycloheximide (CHX) and cytosine arabinoside (AraC). GIV-120L transcripts and protein were found at 12–30 h post infection (hpi) and 18–30 hpi, respectively. In addition, inhibition with CHX and AraC confirmed that GIV-120L was a late gene. Immunofluorescence staining using the antibodies produced in the study confirmed that GIV-120L protein is expressed at viral assembly sites at 24 hpi. The findings of this study provide functional characterization of the GIV-120L viral gene, enhance understanding of GIV assembly, and offer insights for GIV diagnostic applications.