柑橘破葉病毒感染性選殖株之構築及偵測方法之改良

Abstract

柑橘破葉病毒 (Citrus tatter leaf virus，CTLV)，分類上屬於 Betaflexiviridae 科，Capillovirus 屬，基因體為 (+)ssRNA，被視為柑橘重要病毒病害之一，唯相關研究至今仍相當有限。本論文針對台灣椪柑 CTLV 分離株 (CTLV-Pk) 進行全長 cDNA 感染性選殖株 (infectious clones) 之構築，定序結果顯示其基因體全長為 6496 核苷酸，比大多數已知的CTLV 序列長 (6495核苷酸)，其基因體組成與其他 Capillovirus 屬病毒相似，具有兩個轉譯框 (open reading frames，ORFs)，ORF1 (37-6354 nt) 可產生一聚合蛋白 (polyprotein) p242，包含了複製相關區域 (replication associated region) 以及外鞘蛋白 (coat protein，CP)，ORF1 尚有兩處變異較大區域存在，分別為變異區一 (variable region I) 位於胺基酸 532 到 570 位置以及變異區二 (variable region II) 位於胺基酸 1583 到 1868位置；ORF2 (4788-5750 nt) 與 ORF1為不同轉譯框，推測可產生移動蛋白 (movement protein，MP)。CTLV-Pk 與其他 CTLV 分離株及同屬於 Capillovirus 的 Apple stem grooving virus (ASGV) 相比，在基因體全長序列相似度達 79.4-94%，胺基酸序列相似度方面 ORF1 約 85.3-95.8%、CP 約92-95.8%、MP約93.4-99.1%。進而針對基因體全長序列及 ORF1 胺基酸序列做親緣分析，結果顯示，Capillovirus 屬病毒親緣關係為同一地區相似度較高。CTLV-Pk 感染性選殖株 (pCTLV-Pk) 可利用胞外轉錄 (in vitro transcription) 方式合成病毒基因體全長 RNA，於 22°C 下進行白藜感染力試驗，篩選出其中的一選殖株 (pCTLV-Pk-8) 轉錄之 RNA 可成功感染白藜，感染成功率約為 73.3%，且引發的病徵與原自然分離株相似，但病徵出現的時間稍微延遲，且可繼續感染白藜。同時觀察到 pCTLV-Pk-8轉錄 RNA 感染白藜所引起的病徵分佈並不均勻，且頂端葉病毒含量較多。本研究亦嘗試將 CTLV-Pk 外鞘蛋白轉譯起始碼 (5642 nt) 進行突變，會使 pCTLV-Pk-8 轉錄 RNA不能對白藜造成系統性病徵。另外本論文也改良了 CTLV 傳統 RT-PCR 偵測以及開發Real-time RT-PCR定量檢測技術。新設計的CTLV-501引子對可專一性的檢測 CTLV，其 RT-PCR 偵測敏感度可達到 1 pg RNA。另外，利用設計的TaqMan primers / probes 套組 (命名為CTLV-RT-TP) 進行 Real-time RT-PCR 定量分析，其敏感度可達到 10 fg RNA，可提供往後學術研究與檢疫應用之所需。

Citrus tatter leaf virus (CTLV) belongs to Capillovirus, Betaflexiviridae with the genome of (+) ssRNA packaged by a filamentous virion. CTLV has been considered to be one of iportant citrus virus diseases, but the scientific data associated with CTLV are still rare so far. This thesis was dedicated to construct the infectious clones of full-length cDNA of CTLV to obtain more molecular and pathological information about CTLV. A CTLV isolate named CTLV-Pk collected from the diseased Ponkan mandarin was used for the cloning. The sequencing data reveals that the genome of CTLV-Pk consists of 6496 nucleotides (nt). The size is larger than most other genome sequences of CTLV isolates (6495 nt). The genome organization is similar to other capilloviruses, with two overlapping open reading frames (ORFs). ORF1 encodes a polyprotein p242 containing replication-associated domains and coat protein (CP). There are two variable regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868); ORF2 encodes a putative movement protein (MP). The nucleotide sequences of full-length genome of CTLV-Pk are similar (79.4-94%) to those of the other CTLV and Apple stem grooving virus (ASGV) isolates. The similarity of amino acid sequences is 85.3-95.8% for ORF1, 92-95.8% for CP and 93.4-99.1% for MP among those isolates. The phylogenetic analyses showed that the geographic separation might lead to the molecular differences among various capilloviruses. The complete genomic RNA of CTLV can be synthesized from infectious clones through in vitro transcription. In the inoculation tests, the selected infectious clone (pCTLV-Pk-8) could successfully infect Chenopodium quinoa at 22°C and incite the symptoms similar to the original CTLV-Pk isolate. The symptoms induced by pCTLV-Pk-8 were slightly later than those by CTLV-Pk. Approximately 73.7% of tested plants were positive for CTLV-infection in the inoculation tests, and they were able to maintain stability through serial passages on C. quinoa. Meanwhile, the results also indicated that the symptoms caused by in vitro transcripts from pCTLV-Pk-8 were not evenly distributed on C. quinoa. The trial using point mutation at translational start codon of CTLV-Pk coat protein (5642 nt) demonstrated that the mutant did not cause systemic symptoms on C. quinoa. For more sensitive detection of CTLV, this study also attempted to improve the RT-PCR and develop Real-time RT-PCR assays. The RT-PCR detection of CTLV with the newly devised primer pair CTLV-501 could obtain more specific and sensitive results even using only 1 pg of RNA template. The Real-time RT-PCR with the devised “CTLV-RT-TP TaqMan primers / probes” could better results even using only 10 fg of RNA template. The results presented in this thesis were expected to provide important references for the academic research and quarantine application in the future.