Identification of temporal class promoters and evaluation of temporal regulatory expression of foreign gene product in the channel catfish virus vector

Identification of differential temporal promoters of channel catfish virus (CCV) is a prerequisite for understanding the mechanisms involved in gene regulation during lytic infection, and permits and engineering of CCV vectors with temporally controlled foreign gene expression.;In this study, a third CCV immediate early (IE) gene, IE3C, was identified. Its product affects protein expression, suggesting that CCV IE mediated gene regulation may be similar to HSV-1. The full-length transcript of IE3C is 1,412 bp, unspliced, and represents open reading frame (ORF) 12 of terminal repeat portions of the CCV genome.;Transcription analysis using RNase protection assays coupled with DNA and protein synthesis inhibitor identified three differential temporal groups of CCV genes. The results demonstrated that the IE3C is a IE gene, the thymidine kinase (TK) gene is an early gene, and ORF39 and ORF46 are the true-late genes. The transcriptional start sites of IE3C, ORF39 and ORF46 genes were mapped.;The construction of two recombinant viruses was attempted using the promoters of IE3C and ORF39 to control the lacZ gene expression. The CCO cells infected with the recombinants stained blue in the presence of 5-bromo-4-chloro-3-indoyl-;Finally, the kinetic analysis of the lacZ gene expression demonstrated that the lacZ gene of the recombinant CCVlacZ is regulated similarly to the CCV TK gene. The methods used in evaluating the lacZ gene expressions in CCVlacZ would be useful for evaluating temporal gene expression in CCV recombinants.