| Summary: | 碩士 === 中臺科技大學 === 生命科學研究所 === 97 === Koi herpesvirus (KHV) infection has threatened the worldwide production of Cyprinus carpio (common carp) and C. carpio koi (koi). KHV, with genes similar to their counterparts of Cyprinid herpesvirus-1 and 2, was recently designated Cyprinid herpesvirus 3 (CyHV-3) and classified into a new family Allophersviridae. Very little information is available for KHV virus replication, pathogenesis and latency. Previous proteomic analysis of KHV particles led to the identification of 35 structural protein genes within approximate 200 genes encoded in KHV genome. The goals of this study were to confirm the results of the proteomic analysis and to characterize several structural proteins. Using ORF81 and 92 antisera prepared in this report and ORF83 antiserum prepared previously, results of Western blotting analysis showed that ORF83 and ORF92 expression was first detected at 13 h post infection. However, ORF83 protein was not detected in KHV-infected koi fin cells (KFC) at all time points, suggesting low expression levels of this protein. ORF81, 83 and 92 were all detected in purified KHV particles, confirming the results of the proteomic analysis. Inhibition of KHV genome replication by DNA synthesis inhibitor, phosphonoacetic acid (PAA), was confirmed by dot blotting analysis. Absence of ORF81 and 92 proteins in KHV-infected KFC cells in the presence of PAA suggested that they are late genes. A mild detergent, Triton X-100, in combination with various concentrations of NaCl were used to separate different substructures of KHV particles. Results of Western blotting analysis showed that ORF81 and 83 were partially released from KHV particles only in the presence of higher concentrations of NaCl, suggesting both are tegument proteins. However, ORF92 can not be released from KHV particles even in the presence of high NaCl concentrations, implying ORF92 is a capsid protein. ORF92 was found in the nucleus in KHV-infected KFC cells by IF staining, while faint signals of ORF81 and 83 were detected in the cytoplasm. ORF81 and 83 transiently overexpressed in BHK and ZF4 cells were both found in the cytoplasm. The results of this study confirmed the proteomic analysis results and helped characterize ORFF81, 83 and 92 proteins. ORF81 overexpression resulted in condensed nuclei and cell detachment. The significance and mechanisms of this phenomenom remain to be investigated.
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