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Serological characterization and generation of polyclonal and monoclonal antibodies against Phalaenopsis chlorotic necrosis spot virus
|摘要:||蝴蝶蘭（Phalaenopsis spp.)為台灣重要的外銷花卉之一，然而在2002年從呈現黃化壞疽斑點病徵的蝴蝶蘭植株上，分離到一病毒分離株，並發現此病毒與西瓜銀斑病毒（Watermelon silver mottle virus, WSMoV）有血清學的反應，經由電子顯微鏡檢查發現該病毒具球形之病毒顆粒；另外，比對此病毒的L RNA的部分序列，發現此病毒為一新發現感染蝴蝶蘭之番茄斑萎病毒屬（Tospovirus）病毒，並暫定名為蝴蝶蘭黃化壞疽斑點病毒 （Phalaenopsis chlorotic necrosis spot virus, PCNSV）。為了發展有效的血清學工具來檢測PCNSV及釐清此病毒與其他tospovirus之間的血清學關係，以接種PCNSV之奎藜來純化病毒核鞘蛋白（nucleocapsid protein, NP），其分子量大小約為30 kDa並用以製備抗血清，利用紐西蘭白兔生產抗PCNSV核鞘蛋白之多元抗體，其力價經間接酵素聯結免疫吸附法（indirect ELISA） 檢測感病之奎藜或蝴蝶蘭葉片測試，結果為1/32000。而單元抗體之製備是以PCNSV核鞘蛋白免疫BALB/c老鼠，經篩選後得到一個穩定表現單元抗體，並對PCNSV有專一性反應之細胞株331D3E4，再以該細胞注入老鼠腹腔以產生大量腹水抗體，收集腹水後再利用間接免疫酵素聯結吸附法測試其力價為106。此外為了進一步確認蝴蝶蘭葉片上黃化壞疽斑點病徵的確為PCNSV所引起，故調查不同蝴蝶蘭栽培種對PCNSV之感病性，將PCNSV接種於18個蝴蝶蘭栽培種（每品種10棵），其中17個栽培種於接種後1-3個星期，蝴蝶蘭葉片出現黃化壞疽輪斑之病徵；而另一個栽培種A78084（P. Little Steve ‘Brother’）於接種後不僅感病植株較少（4棵），其病勢發展亦較為緩慢及輕微。利用免疫擴散法、間接酵素免疫聯結吸附法以及西方墨點法分析PCNSV與其他5個tospoviruses，包括WSMoV、彩色海芋黃化斑點病毒（Calla lily chlorotic spot virus, CCSV）、番茄斑萎病毒（Tomato spotted wilt virus, TSWV）、花生輪斑病毒（Groundnut ringspot virus, GRSV）及鳳仙花壞疽斑點病毒（Impatiens necrotic spot virus, INSV）之血清學關係，結果顯示PCNSV、WSMoV及CCSV的NP具有相近的血清學關係，但與TSWV、GRSV及INSV則無血清學關係，因此PCNSV、WSMoV及CCSV應該被分類在WSMoV 血清群，然而TSWV及GRSV間具有明顯之血清交互反應，應可將其分類為相同的血清型。綜合以上試驗結果，蝴蝶蘭上黃化壞疽斑點病徵確實為PCNSV所引起，而利用PCNSV核鞘蛋白產生之多元或單元抗體可有效的應用在偵測此一病毒; 由蝴蝶蘭上分離到的PCNSV是一個新發現感染蝴蝶蘭的tospovirus並應歸類為WSMoV血清型。|
In 2002, a virus isolate was obtained from Phalaenopsis moth orchid, one of the important ornamental plants exported from Taiwan. The isolate was found to cause symptoms of chlorotic and necrotic spot on Phalaenopsis moth orchids and reacted serologically with Watermelon silver mottle virus (WSMoV). Electron microscopic examination revealed virus-like spherical particles in the crude sap preparation and the molecular analysis of the partial L RNA of the virus indicated that it is a putative new tospovirus infecting Phalaenopsis moth orchids. The virus was thus tentatively designated as Phalaenopsis chlorotic necrosis spot virus (PCNSV). To develop efficient serological tools for detecting PCNSV and to characterize it's serological relationships to other tospoviruses, the nucleocapsid protein (NP) of PCNSV was purified from PCNSV-infected Chenopodium quinoa and was used to generate polyclonal antibody (PAb) in New Zealand white rabbit and monoclonal antibody (MAb) in BALB/c mouse. The titers of the PAb determined by indirect ELISA using crude sap of PCNSV-infected C. quinoa and Phalaenopsis moth orchid were 1/32000. Hybridoma cell lines were produced by the fusion of mouse myeloma cells and the spleen cells of a BALB/c mouse immunized with the purified PCNSV NP. One stable PCNSV-specific monoclonal antibody (MAb) secreting hybridoma cell line, 331D3E4, was obtained and used for the preparation of ascitic fluids. The titers of ascitic fluid were both 106 as determined by indirect ELISA with crude sap of PCNSV-infected tissue of C. quinoa and Phalaenopsis moth orchid. To further confirm that the chlorotic and necrotic spot symptoms on Phalaenopsis moth orchids are indeed caused by PCNSV and to survey the susceptibility of moth orchid cultivars, 18 cultivars of moth orchids were inoculated with PCNSV. The symptoms of chlorotic, necrotic to ringspot were developed on the leaves of the 17 out of 18 inoculated cultivars 1-3 weeks post inoculation. One cultivar A78084 (P. Little Steve ‘Brother') showed not only less plant infection (four out of ten) but also slow and mild symptom development after inoculation with PCNSV. In addition, immunodiffusion, indirect ELISA and western blotting assays were used to analyze the serological relationships of PCNSV with other tospoviruses including WSMoV, Calla lily chlorotic spot virus (CCSV), Tomato spotted wilt virus (TSWV), Groundnut ringspot virus (GRSV) and Impatiens necrotic spot virus (INSV). The results indicated that the NPs of PCNSV, WSMoV and CCSV have close serological relationships, distinct from those of TSWV, GRSV, and INSV. Thus the viruses, PCNSV, WSMoV and CCSV should be classified into the WSMoV serogroup. Whereas strong cross reaction between TSWV and GRSV indicated that they should be reclassified in the same serogroup. We conclude that the chrolotic and necrotic spot symptoms found on moth orchids are indeed caused by PCNSV and the antisera produced against purified PCNSV NP can be successfully used for PCNSV detection. The tospovirus, PCNSV, isolated from moth orchids is a new reported virus infecting moth orchids and should be classified into WSMoV serogroup.
|Appears in Collections:||植物病理學系|
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