番茄斑點萎凋病毒（＂Tomato spotted wilt virus＂）核鞘蛋白抗血清製備及其血清學親緣關係之探討

Production of Antiserum Against the Nucleocapsid Protein of ＂Tomato Spotted Wilt Virus＂ and Investigation of Its Serological Relationship with Other Tospoviruses

番茄斑萎病毒屬（Tospovirus）隸屬於布尼亞病毒科（Bunyaviridae），病毒顆粒呈球型具外套膜（envelope），其內含三條單股RNA片段，依大小稱之為L、M及S RNA，由薊馬類（thrips）昆蟲以永續性方式傳播，核鞘蛋白（nucleocapsid protein，NP）序列的同源性與血清學親緣關係，為該類病毒分類之重要依據。番茄斑點萎凋病毒（Tomato spotted wilt virus，TSWV）是Tospovirus屬的代表種（type species），寄主範圍廣，且在全世界造成嚴重的危害，在台灣亦有危害甜椒及彩色海芋等作物的記錄。為了落實防檢疫工作，本研究以細菌表現系統生產TSWV之NP做為抗原，用以製備抗血清，命名為RAs-TSWV-NP。經間接酵素連結免疫吸附分析法（indirect enzyme-linked immunosorbent assay，indirect ELISA）與西方墨點轉漬法（western blotting）分析RAs-TSWV-NP和16種tospoviruses之反應，可得知RAs-TSWV-NP除了與TSWV反應外，亦與花生輪斑病毒（Groundnut ringspot virus，GRSV）、番茄黃化斑點病毒（Tomato chlorotic spot virus，TCSV）、水仙百合壞疽條斑病毒（Alstroemeria necrotic streak virus，ANSV）、菊花莖部壞疽病毒（Chrysanthemum stem necrosis virus，CSNV）、以及鳳仙花壞疽斑點病毒（Impatiens necrotic spot virus，INSV）產生血清反應。親緣演化分析顯示TSWV、GRSV、TCSV、ANSV、CSNV及INSV屬於同一演化分支（clade）。關於TSWV、GRSV、TCSV和ANSV之間的血清學關係雖早已於先前的文獻中報導，然而本研究結果更進一步指出TSWV與CSNV及INSV之間亦可能存在著血清學親緣關係，此結果與親緣演化分析符合。親緣關係的釐清將有助於Tospovirus屬病毒的診斷與鑑定。

"Viruses belonging to the genus Tospovirus of the family Bunyaviridae have enveloped quasi-spherical particles containing three segmented single-stranded (ss) RNA molecules, named L, M and S RNA based on their molecular sizes. Tospoviruses are transmitted by thrips in a persistent manner. The homology and serological relationships of nucleocapsid protein (NP) are the important criteria for demarcation of a tospovirus species. Tomato spotted wilt virus (TSWV) is the type species of the genus Tospovirus and has a wide host range to cause serious damages worldwide. In Taiwan, TSWV was reported to infest pepper and calla lily. For the assignment of quarantine and inspection, the NP of TSWV was expressed by bacteria for production of antiserum RAs-TSWV-NP. Indirect enzyme-linked immunosorbent assay (ELISA) and western blotting were conducted for investigating the serological reactions of RAs-TSWV-NP with 16 different tospovirus species. Our results showed that RAs-TSWV-NP reacted with not only TSWV but also Groundnut ringspot virus (GRSV), Tomato chlorotic spot virus (TCSV), Alstroemeria necrotic streak virus, ANSV), Chrysanthemum stem necrosis virus (CSNV) and Impatiens necrotic spot virus (INSV). Phylogenetic analysis revealed that TSWV, GRSV, TCSV, ANSV, CSNV and INSV belong to the same clade. The serological relationship of TSWV, GRSV, TCSV and ANSV was reported in the previous studies. CSNV and INSV serologically related with TSWV and that is accordance with their phylogenetic relationship. Verification of phylogenetic and serological relationships is helpful for diagnosis and identification of tospoviruses."