|Abstract: ||非結構性NSs 蛋白為番茄斑萎病毒 (Tospovirus) 屬病毒克服植物防禦基因沉寂機制之重要因子，先前的研究發現，西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV)NSs 蛋白的單株抗體不僅能與WSMoV 血清群的成員產生血清反應，更可偵測到甜瓜黃斑病毒 (Melon yellow spot virus, MYSV)、鳶尾花黃斑病毒 (Iris yellow spot virus, IYSV)和番茄黃輪病毒 (Tomato yellow ring virus, TYRV) 等非WSMoV 血清群成員的NSs 蛋白。經epitope mapping 分析得知，WSMoV NSs 蛋白第98 到120 個胺基酸序列「VRKPGVKNTGCKFTMHNQIFNPN」為單株抗體結合的位置，序列分析亦了解此區域為高保留性序列，存在於這些病毒的NSs 蛋白，使得WSMoV NSs 蛋白的單株抗體成為非常有用的檢測工具。此外，另一高保留性序列「ISVCSNTVNTNGVKHQGHLKVLS」亦被發現存在於番茄斑點萎凋病毒 (Tomato spotted wilt virus, TSWV)、花生輪斑病毒(Groundnut ringspot virus, GRSV) 和鳳仙花壞疽斑點病毒 (Impatiens necrotic spot virus,INSV) NSs 蛋白的第98 到120 個胺基酸位置，這些結果暗示了NSs 蛋白的同源性與tospoviruses 的演化有關，而NSs 蛋白的高保留性序列將成為檢tospoviruses 的重要標的。本研究計畫擬採用表現WSMoV NSs 蛋白的策略，應用矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV) 載體系統快速且大量表現TSWV、INSV、花生黃化扇斑病毒 （Peanut chlorotic fan-spot virus, PCFV） 和花生黃斑病毒 (Peanut yellowspot virus, PYSV) 的NSs 蛋白，用以製備具廣效性血清反應之抗血清及單株抗體，建立完整的tospoviruses 檢測系統，不但可成為植物防檢疫工作之利器，更可供給日後研究NSs 蛋白功能時做為偵測蛋白的工具。
Nonstructural (NSs) protein of tospoviruses plays the role as a gene-silencing suppressor that overcomes natural defense mechanisms of plants. In the previously study, the produced monoclonal antibodies (MAbs) against the NSs protein of Watermelon silver mottle virus (WSMoV) serologically reacted not only with all members of WSMoV serogroup but also with those of non-WSMoV serological related tospoviruses, such as Melon yellow spot virus (MYSV), Iris yellow spot virus (IYSV) and Tomato yellow ring virus (TYRV). Epitope mapping showed that the MAbs recognized the amino acid (aa) 98 to 120 region of the NSs protein of WSMoV, the sequence is “VRKPGVKNTGCKFTMHNQIFNPN”. Sequence alignment of the reported NS proteins revealed that the WSMoV NSs MAb-recognized region is consensus at the same position of those of the forementioned tospoviruses. In addition, another consensus sequence “ISVCSNTVNTNGVKHQGHLKVLS” was found at the same position, the aa 98 to 120, among the NSs proteins of Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV) and Impatiens necrotic spot virus (INSV). These results imply that the homology of NSs proteins is related to the evolution of tospoviruses and these conserved regions of NSs proteins are able to be the important target for detection of tospoviruses. In this project, we will carry out the same approach as expression of the NSs protein of WSMoV that applying the Zucchini yellow mosaic virus (ZYMV) vector to fast express large amounts of NSs proteins of TSWV, INSV, Peanut chlorotic fan-spot virus (PCFV) and Peanut yellow spot virus (PYSV). These expressed NSs proteins will be used as antigens for production of antisera and MAbs which with broad-spectrum serological reactions to establish completely detection system for worldwide tospoviruses. These antibodies are not only able to be used as useful tools for tospovirus detection but also to be used as the detective tools for functional analyses of NSs protein in the future studies.