神經壞死病毒 ( Nervous Necrosis Virus, NNV )會感染許多高經濟性魚種如石斑稚魚，造成高死亡率，而使得漁業經歷重大經濟損失衝擊。病毒本身之B1基因是否參與凋亡調控之機制尚不清楚，因此本論文著重於探討神經壞死病毒B1基因所扮演之角色。
首先我們設計degenerate primer從病毒次基因體RNA3選殖非結構性蛋白基因B1，接著以B1基因做為探針進行北方墨漬法分析發現大小約0.38 kb RNA3在石斑魚背鰭細胞株(GF-1)中，經神經壞死病毒(RGNNV TN1)感染第12小時後開始表現。並利用自製抗B1蛋白之多株抗體偵測到在感染後第24小時B1蛋白質(11 kDa)開始持續表現，同時此新生之B1蛋白質也可被蛋白質合成抑制劑cycloheximide可有效抑制其生合成。另一方面，在功能性分析，首先產生大量表現B1蛋白之石斑魚細胞株，並利用NNV感染，以AnnexinⅤ及粒線體膜電位之消失測定中，表現蛋白質B1能降低細胞凋亡之數量與調節病毒蛋白之表現。反之，反義RNA可抑制病毒感染後之基因表現，並從結果得知細胞死亡時程提早約12小時及病毒蛋白表現量顯著增加。另外，將神經壞死病毒B1基因接合到黃色螢光基因(EYFP)後轉染細胞，可觀察到B1蛋白會累積在細胞核內。
於本論文我們第一個針對B1蛋白基因的功能進行分析,希望藉此研究進一步明瞭神經壞死病毒誘發細胞凋亡之分子機制，能有助於對神經壞死病毒所造成之疾病的預防和治療。

Some fish species are known to be infected by betanodaviruses. Betanodaviruses is belong to one of Nodaviridae member, causes an acute, contagious disease in a number of economically important fish species and resulted in severe mortality and significant economic losses to the aquaculture industry. Despite the severe economic impact on the aquaculture industry, betanodaviruses are not well studied, especially on their molecular pathogenesis.
In the present time, we examine a non-structure protein B1 as a newly expression gene and its biological function in fish cells. We cloned that RGNNV B1 cDNA is a 330 nt in length, encoding for a 111 amino acids (11 kDa). Furthermore, by western blot analysis, B1 protein was expressed at 24 h post-infection (p.i.), and the protein expression of B1 could be blocked by protein synthesis inhibitor cycloheximide (CHX ; 0.33 μg/ml). In functional assay, the B1 protein were significantly blocked either PS exposure or mitochondrial membrane permeability (MMP) loss with RGNNV infection in B1 stabe expression of GF-1 cells. Then, knockdown of B1 expression by B1 antisense RNA promoted cell death. Finally, we found that the nuclear localization of B1 protein by fusing with EYFP reporter gene.
Our results suggest that B1 as an early expression protein may play a crucial role on anti-apoptotic function and suppress the viral proteins expression with RGNNV infection.

羅秉真，南台灣養殖石斑魚病毒性神經壞死症之研究。國立台灣大學動物學研究所碩士論文，台北，1997
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