| Summary: | 碩士 === 國立臺灣海洋大學 === 水產養殖學系 === 97 === The influence of temperature on the Grouper iridovirus of Taiwan (TGIV) multiplication at 18, 25 and 32℃, respectively were investigated in the study. First, PCR results showed TGIV-infected cells at 18 and 25℃ have 1339 bp specific TGIV amplicon but could not be found at 32℃. Then cell-associated virus (CAV) and released virus (RV) at different time points after TGIV inoculation was estimated for determining the growth curve of TGIV. Virus yields of CAV under hyperthermia (32℃) group only 104.25 TCID50/ml, lower than the other two temperature groups of titer 106∼107 TCID50/ml. Furthermore, cell cycles and apoptosis cells were measured by flow cytometric analysis using propidium iodide (PI) and Annexin-Ⅴ. The TGIV-infected cells at 32℃ showed apoptosis in the sub-G1 phase at 5 dpi and reached peak at 9 dpi. And it had 24% apoptosis cells at 9 dpi in Q4 region. The double labeling assay method combines the staining properties of Annexin-Ⅴ and PI was observed by microscopy. The test detected apoptosis-dependent phosphatidylserine translocation from the inner to the outer leaflet of the plasma membrane at 32℃. Western blot assays analyzed heat-shock protein 70 (HSP 70), anti-apoptosis protein bcl-2 and pro-apoptosis protein bak expression of TGIV-infected cells at the above three temperatures. There are obvious expressions of HSP 70 and pro-apoptosis protein bak at 32℃. And anti-apoptosis protein bcl-2 was found expressing significantly at 25℃ only. Finally, fish were injected TGIV and held at 25 and 32℃. After 2 weeks, the cumulative mortality of virus-infected fish was 100% and 37.5% respectively. Besides, the 32℃ group showed more obvious TUNEL positive reaction than 25℃ group. The results revealed that hyperthermia-induced apoptsis might closely related with host defense mechanism against TGIV infection.
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