分離自大花曼陀羅的哥倫比亞曼陀羅病毒（Colombian datura virus）特性分析

Characterization of a Colombian datura virus (CDV) isolated from Angel’s trumpet (Brugmansia suaveolens)

2022年12月於南投鹿谷溪頭（Xi-Tou）發現大花曼陀羅葉面密布褪綠黃點或輪斑和嵌紋狀等疑似病毒感染的病徵。病組織粗汁液中可觀察到大小約750-800 x 13 nm的長絲狀病毒顆粒，罹病細胞內有典型的卷軸狀、層狀和風車狀的內含體。使用馬鈴薯Y病毒屬（Potyvirus）簡併式引子對（PNIbF1: 5'-GGBAAYAATAGTGGNCAACC-3'、PCPR1: 5'-GGGGAGGT-GCCGTTCTCDATRCACC-3'）於RT-PCR，可增幅出預期的1.0-1.2 kb、涵蓋potyvirus NIb 3'-後半和CP 5'-前半的cDNA片段。該片段（1022 nt）之序列與哥倫比亞曼陀羅病毒（Colombian datura virus, CDV）有98%的相似度，故單離後之供試病毒暫以哥倫比亞曼陀羅病毒溪頭分離株（CDV-XT）稱之。CDV-XT接種於數種供試植物進行初步之寄主範圍測試。結果顯示奎藜（Chenopodium quinoa）為單斑寄主；茄科之番茄（Solanum lycopersicum）、茄子（S. melongena）、菸草（Nicotiana benthamiana, N. tabacum cv. Xanthi, N. rustica）等則為系統性寄主。CDV-XT感染番茄引起嵌紋、葉柄捲曲下垂、葉片變小、全株矮化；感染茄子引起葉片嵌紋；感染菸草於N. benthamiana引起葉片嵌紋、上位葉萎凋枯死；於N. tabacum Xanthi之接種葉和系統葉引起壞疽性斑點與葉脈壞死等症狀；於N. rustica則引起系統性嵌紋病徵。解析CDV-XT基因，全長具9621個核苷酸（不含3'-端poly-A尾部），包含5'-端未轉譯區（5'-untranslated region, 5'-UTR）[nt 1 - 154]、大蛋白轉譯框（polyprotein open reading frame）[nt 155 - 9382]、3'-端未轉譯區（3'-UTR）[nt 9383 - 9621]，為potyvirus典型基因結構。大蛋白轉譯框可轉譯產生一個包含3076個胺基酸之複合蛋白。CDV-XT全長度核苷酸序列與已發表之CDV全長度核苷酸序列比對，其相似度介於98.9%（CDV-AT-Kr和CDV）與98.4%（CDV-TA-Br2）之間，但與其他potyviruses的全長度核苷酸序列相似度則低於66.6%。CDV-XT之鞘蛋白基因具有822個核苷酸序列，與其它已發表之CDV鞘蛋白基因核苷酸序列相似度在96.9-98.1%之間；胺基酸序列相同度則在98.5-99.3%之間。本文為CDV發生於台灣的首次報導。

Angel's trumpet (Brugmansia suaveolens) plants with symptoms of systemic chlorotic spots or ringspots and mosaic on leaves were found at Xi-Tou, Nantou in December, 2022. A quick diagnostic examination with transmission electron microscope (TEM) revealed filamentous virus-like particles of about 750-800 x 13 nm and typical inclusions of potyvirus in infected cells. Degenerate primers (PNIbF1: 5'-GGBAAYAATAGTGGNCAACC-3' and PCPR1: 5'-GGGGAGGT-GCCGTTCTCDATRCACC-3') for the genus Potyvirus were applied in RT-PCR to amplified an expecting cDNA fragment of about 1.0-1.2 kb. The amplified cDNA of 1022 nt consisted the 3'-half of potyvirus NIb and 5'-half of CP genes and shared more than 98% similarity with that of Colombian datura virus (CDV). The isolated virus thus designated as a Xi-Tou isolate of CDV (CDV-XT). CDV-XT induced local lesions on inoculated leaves of Chenopodium quinoa. It systemically infected economically important solanaceous crops including tomato (Solanum lycopersicum), eggplant (S. melongena) and tobacco (Nicotiana benthamiana, N. tabacum cv. Xanthi, and N. rustica) but excluding pepper (Capsicum annuum). The viral genome of CDV-XT (LC771070) consists of 9621 nuclotides (nt) including 5'-UTR (154 nt), polyprotein ORF (9228 nt / 3075 a.a.), and 3'-UTR (239 nt) excluding poly-A tail. The closest potyvirus being Colombian datura virus isolates at more than 98% nt sequence identity. Based on biological, morphological and molecular characters, the virus was J. Plant Med.determined to be an isolate of CDV. This is the first report of CDV in Taiwan.