- 期刊
Molecular Characterization and Serological Diagnosis of the New Emerging Cucurbit chlorotic yellows virus in Taiwan
台灣新興瓜類褪綠黃化病毒分子特性與血清學診斷之研究
摘要
Crinivirus為Closteroviridae科之一病毒屬，為長絲狀病毒，具有由兩條正極性單股RNA所組成之基因體，分別命名為RNA1及RNA2，且其病毒顆粒較短，介於650－900nm×12nm之間，是由粉蝨以半永續性的方式傳播。Crinivirus危害全球許多重要的經濟作物，造成植株的葉片呈現黃色斑點、褪綠黃化、甚至白化的病徵，導致收成量降低而造成嚴重的經濟損失。在2009年四月間，於台灣雲林縣崙背鄉的洋香瓜田中，發現洋香瓜上出現典型的Crinivirus病徵。採集洋香瓜的罹病組織並以針對Closterovirus及Crinivirus所設計之簡併性引子對進行巢式反轉錄－聚合酶鏈鎖反應（nested reverse transcription-polymerase chain reaction，nested RT-PCR），可增幅出0.5kb的DNA片段，經定序及比對分析得知與日本所發現的新興瓜類褪綠黃化病毒（Cucurbit chlorotic yellows virus，CCYV）有100%的相同度。本研究以粉蝨傳毒方式將新興病毒感染於菸草（Nicotiana benthamiana）中，以獲得一crinivirus分離株，命名為Crinivirus-TW。將Crinivirus-TW的RNA2完全解序，可得其全長度有8,041個核苷酸，可對應產生八個蛋白，分別為P5、Hsp70h、P6、P60、P9、CP、CPm和P26。RNA2的核苷酸序列與日本原始的CCYV分離株序列具有99%的相同度，因此Crinivirus-TW應為CCYV的分離株，並更名為CCYV-TW。將所有已知Crinivirus病毒種的Hsp70h、P60、CP、CPm及P26等基因進行親緣關係比對，結果得知CCYV-TW與Lettuce chlorosis virus（LVC）、Bean yellow disorder virus（BYDV）及Cucurbit yellow stunting disorder virus（CYSDV）最為親近。利用pET細菌表現系統表現CCYV-TW的CP用以生產單株抗體（monoclonal antibody），所得之單株抗體可用於西方墨點法（western blotting）以進行田間之病毒檢測，並可應用於組織點漬免疫分析法（tissue blot immunoassay，TBIA）以檢視病毒於韌皮部細胞累積之情形。此外，設計自Hsp70h序列的引子對Crini-hsp70h-f/Crini-hsp70-r與設計自植物粒線體中的NADH dehydrogenase（nad5）基因之引子對mt-F2/mtR1共同使用所開發的multiplex one-step RT-PCR，為相當有效率的檢測方法，可應用於田間CCYV之檢測。以此方法進行田間調查，2010年2－6月間，自台灣中部地區共採集253個罹病洋香瓜樣本，檢測結果發現，病毒之檢出率從3.8%上升至100%。本調查結果顯示CCYV已成為台灣瓜類作物生產上的一大重要限制因子，值得密切注意。
並列摘要
Crinivirus, a genus of the family Closteroviridae, has large positive-sense single-stranded RNA genomes encapsidated in long flexuous virions and is transmitted by whitefly. The virus bipartite particles of 650-900 × 12 nm comprise two genome segments, denoted RNA1 and RNA2. Criniviruses infect several important crops worldwide, causing symptoms of interveinal chlorosis, yellowing and brittleness of leaves accompanied by severe yield losses. In April 2009, typical crinivirus-induced symptoms were observed on melon (Cucumis melo L.) plants in Yunlin County, Taiwan. The degenerate primers designed for Closterovirus and Crinivirus were used in nested reverse transcription-polymerase chain reaction (RT-PCR) to amplify a 0.5 kb DNA fragment from the symptomatic melon tissues. Subsequently, the amplified DNA fragment was determined to share 100% identity with the reported nucleotide (nt) sequence of Cucurbit chlorotic yellows virus (CCYV), a new Crinivirus species found in Japan. In this study, an isolate of crinivirus denoted as Crinivirus-TW was obtained from a diseased melon plant and maintained in Nicotiana benthamiana by whitefly transmission. Furthermore, the complete nt sequence of Crinivirus-TW RNA2 was determined as 8,041 nt in length containing eight open reading frames (ORFs), including P5, Hsp70h, P6, P60, P9, CP, CPm and P26. Sequence analyses revealed that the RNA2 sequence of Crinivirus-TW shares 99% nt identity with that of the original Japan isolate of CCYV. Thus, Crinivirus-TW was identified as an isolate of CCYV, renamed CCYV-TW. Phylogenetic analyses of the Hsp70h, P60, CP, CPm and P26 among Crinivirus species revealed that CCYV-TW is closely related to Lettuce chlorosis virus (LCV), Bean yellow disorder virus (BYDV) and Cucurbit yellow stunting disorder virus (CYSDV). The recombinant CP of CCYV-TW expressed by the bacterial pET expression system was used as an immunogen for production of monoclonal antibodies (MAbs). The MAbs prepared were successfully used to detect CCYV in diseased plants by western blotting and to examine lie ace mulation of virus in phloem cells by tissue blot immunoassay (TBIA). Moreover, primer pair Crini-hsp70h-f/Crini-hsp70-r, designed from the Hsp70h gene, coupled with primer pair mt-F2/mtR1, designed from the plant mitochondrial NADH dehydrogenase (nad5) gene, used in multiplex one-step RT-PCR was developed as an effective detection method and applied in field survey. A number of 253 diseased melon samples collected from central Taiwan during February to June in 2010 were tested by multiplex one-step RT-PCR. An increased incidence of 3.8% to 100% was noticed. Our results indicated that CCYV has become an important threat for the production of cucurbits in Taiwan, and it should be majorly concerned.