Replication of Chilo iridescent virus (CIV) in Bombyx mori cell culture and transcriptional analysis of dnapol and mcp genes

SUMMARY Replication of Chilo Iridescent Virus (CIV) in Bombyx mori Cell Culture and Transcriptional Analysis of dnapol and mcp Genes Chilo iridescent virus (CIV), the type species of the genus Mdovirus, a member of the Iridoviridae, is highly pathogenic for a variety of insect larvae. This is the first study of the replication of CIV in Bombyx mori (SPC-BM-36) cell culture. Studies were also performed on regulation of CIV genes in this study. For this, DNA polymerase {dnapol) and major kapsid protein (mcp) genes were selected. Temporal classes and untranslated sequences of these genes were determined. Detailed studies were performed on detection of the potential promoter sequences. Replication of CIV in B. mori cells was detected by light microscopy, DNA-DNA hybridization and transcriptionally. At 48 hours post infection of B. mori cells by CIV, virus infected cells showed completely diversity from non infected cells. They were 2-3 times larger than controls and small viroplasmic centres were evident in the cytoplasm of infected cells. Results indicate that CIV-Bm infection is permissive. Infection of B. mori cells in the presence of Ara-C (inhibits DNA replication) or cycloheximide (inhibits protein synthesis), followed by RT-PCR on isolated total RNA using specific primers for dnapol and mcp, showed that dnapol is expressed as a delayed-early gene and confirmed that mcp is a late gene. 5 'RACE analysis on RNA isolated from CIV-infected B. mori cells showed that transcription initiated at position -35 for dnapol and position -15/- 16 for mcp, relative to the translational start sites of these genes. To determine the limits of the putative promoters, upstream sequences of various lengths were cloned in front of a firefly luciferase reporter gene. The resulting plasmid constructs were tested in a transfection assay. The mcp promoter activity was strongly reduced when the length of the sequence upstream of the translational start site was reduced from 67 to 43 nucleotides. For dnapol, the promoter activity was reduced to almost zero when the upstream fragment was reduced from 62 to 41 nucleotides. Changing the G base to C in a TTGTTT motif just upstream of the transcription initiation site of dnapol reduced the promoter activity with 25%. Key Words: Chilo iridescent Virus, Bombyx mori, dnapol, mcp, Gene Regulation vn