Microarray and RT-PCR screening for white spot syndrome virus immediate-early genes in cycloheximide-treated shrimp

• Published in: Virology (New York, N.Y.) Vol. 334; no. 2; pp. 327 - 341
• Main Authors: Liu, Wang-Jing, Chang, Yun-Shiang, Wang, Chung-Hsiung, Kou, Guang-Hsiung, Lo, Chu-Fang
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 10.04.2005
• Subjects: 60 APPLIED LIFE SCIENCES; Amino Acid Sequence; amino acid sequences; Animals; Base Sequence; Cells, Cultured; CYCLOHEXIMIDE; Cycloheximide - pharmacology; Cycloheximide-insensitive gene; dsDNA viruses; FLUORESCENCE; Gene Expression Profiling; gene expression regulation; Gene Expression Regulation, Viral; GENES; Genes, Immediate-Early; GLOBAL ANALYSIS; Immediate-early gene; immediate-early genes; Immediate-Early Proteins - genetics; Immediate-Early Proteins - metabolism; INSECTS; Microarray; microarray technology; Molecular Sequence Data; Nimaviridae; Nuclear polyhedrosis virus; nucleotide sequences; Oligonucleotide Array Sequence Analysis - methods; Orgyia pseudotsugata; Penaeidae - drug effects; Penaeidae - virology; Penaeus monodon; POLYMERASE CHAIN REACTION; promoter regions; Promoter Regions, Genetic; Protein Synthesis Inhibitors - pharmacology; PROTEINS; Reverse Transcriptase Polymerase Chain Reaction; Shellfish - virology; SHRIMP; Spodoptera; transcription (genetics); viral proteins; VIRUSES; White spot syndrom virus; white spot syndrome; White spot syndrome virus; White spot syndrome virus 1 - drug effects; White spot syndrome virus 1 - metabolism; WSSV; Taiwan; Penaeus monodon; Microarray; Immediate-early gene; WSSV; Cycloheximide-insensitive gene
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/j.virol.2005.01.047

Here, we report for the first time the successful use of cycloheximide (CHX) as an inhibitor to block de novo viral protein synthesis during WSSV (white spot syndrome virus) infection. Sixty candidate IE (immediate-early) genes were identified using a global analysis microarray technique. RT-PCR showed that the genes corresponding to ORF126, ORF242 and ORF418 in the Taiwan isolate were consistently CHX-insensitive, and these genes were designated ie1, ie2 and ie3, respectively . The sequences for these IE genes also appear in the two other WSSV isolates that have been sequenced. Three corresponding ORFs were identified in the China WSSV isolate, but only an ORF corresponding to ie1 was predicted in the Thailand isolate. In a promoter activity assay in Sf9 insect cells using EGFP (enhanced green fluorescence protein) as a reporter, ie1 showed very strong promoter activity, producing higher EGFP signals than the insect Orgyia pseudotsugata multicapsid nuclear polyhedrosis virus (OpMNPV) ie2 promoter.