Characterization, and Expression Analysis of ORF32R, ORF40L, ORF66L and ORF77L of Grouper Iridovirus

• Main Authors: Hiseh Wen Yu, 謝文毓
• Other Authors: Yu-Shen Lai
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/41917536181386128231

碩士 === 國立宜蘭大學 === 生物技術與動物科學系 === 104 === Iridoviruses, belonging to the family Iridoviridae, are large, icosahedral, double-stranded DNA viruses with a viral particle diameter of 120–350 nm. Grouper iridovirus (GIV), which was isolated from Epinephelus spp. (also called groupers) in southern Taiwan, belongs to the Ranavirus genus and has caused significant economic losses in the grouper aquaculture industry. Although the GIV genome sequence is now known, molecular mechanisms underlying iridovirus pathogenicity are not well understood, mainly owing to insufficient viral genetic information. The aim of this study was to investigate the expression and subcellular localization of GIV-32R, GIV-40L, GIV-66L, and GIV-77L during GIV infection in vitro. RT-PCR analyses revealed that during GIV infection in grouper kidney cells, GIV-40L is detected as early as 3 hours post infection (hpi), and GIV-32R and GIV-40L at 9 hpi, GIV-77L was not detected at any time-point post infection. Analyses with cycloheximide (a protein synthesis inhibitor) or cytosine arabinoside (a DNA synthesis inhibitor) revealed that GIV-40L is expressed immediately after infection, whereas GIV-32R and GIV-66L are expressed at a later stage. Western blot analysis using 4 mouse polyclonal antibodies against GIV-32R, GIV-40L, GIV-66L, and GIV-77L proteins in grouper kidney cells during GIV infection detected GIV-32R, GIV-40L, and GIV-66L at 12 hpi, while GIV-77L was not detected at any time-point post infection. The localization of GIV-32R, GIV-40L and GIV-66L in GIV-infected cells was further characterized by immunofluorescence microscopy with the corresponding mouse polyclonal antibodies. In the infected cells, GIV-66L was mainly aggregated in the cytoplasm, while GIV-32R and GIV-40L were distributed in both the nucleus and cytoplasm. The results of this study will enable investigators to better understand the mechanism underlying iridovirus infection.