| Summary: | 碩士 === 中臺科技大學 === 醫學生物科技研究所 === 96 === The koi herpesvirus (KHV) disease leads to mass mortality in common carp (Cyprinus carpio carpio) and koi (Cyprinus carpio koi). KHV was officially named Cyprinid herpesvirus-3 on the basis that it shares morphological and genetic features with members of the Herpesviridae family. Structural proteins play critical roles in virus replication cycle and pathogenesis. The envelope proteins are potential serological markers and vaccine targets for enveloped viruses such as KHV. The aim of this study was to identify the genes for KHV virion proteins by taking the proteomics approach. The titers of wild-type KHV-T, a KHV isolate collected in Taiwan, were low (103-4 TCID50/mL) for virus purification. After 30 passages in KFC cells, a large-plaque KHV variant, namely KHV-Tp30.1, was isolated by plaque purification. One-step growth curve analysis of KHV-Tp30.1 replication in KFC showed that the eclipse phase was between zero to eight hours post infection and the log phase between eight to forty-eight hours. And its titers reached around 1 x 108 TCID50/ml. Purification of KHV virion was achieved by a combination of 20/60% sucrose step gradient and 30-50% sucrose continues gradient centrifugation. The purity of virus preparation was confirmed by transmission electron microscopy (TEM) analysis of negatively-stained samples. TEM analysis also confirmed that the envelope was separated from the capsid by Triton X-100/0.5 M NaCl treatment. Whole virion and the Triton X-100/0.5 M NaCl-soluable fraction were subjected to trypsin digestion. The resulted peptides were sequenced by liquid chromatography-mass spectrometry/mass spectrometry. Mascot analysis of the peptide sequences using the DNA sequence database at the National Center for Biotechnology Information led to the identification of genes for thirty five KHV proteins. Among these, pORF81 and pORF83 which were predicted to contain trasmembrane domains and the putative major capsid protein pORF92 were further dissected by immunoblotting analysis of KHV virions treated with various combinations of trypsin and Triton X-100. The results suggested that ORF81 and ORF83 encode KHV envelope proteins and ORF92 indeed encodes the major capsid protein. Further characterization of the structural protein genes identified herein shall facilitate the identification of major KHV immunogens and basic virology studies in the future.
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