The complete sequence of the genomic RNA of an isolate of Lily virus X (genus Potexvirus) Arch Virol (Apr 2005) 150: 825–832 J. Chen1, Y.-H. Shi1,2, M.

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1 The complete sequence of the genomic RNA of an isolate of Lily virus X (genus Potexvirus)
Arch Virol (Apr 2005) 150: 825–832 J. Chen1, Y.-H. Shi1,2, M. J. Adams3 ,and J.-P.Chen1 林偉志 D

2 Introduction 百合及其發生之病害 病毒鑑定與病害防治 Lily virus X (LVX)
the genomic RNA of an isolate of Lily virus X (LVX)

3 Introduction 百合及其病害 形態：球莖狀如鱗片、色白 肉質如未開之蓮花。 性味：味甘微苦，性味、無毒。
功效：清涼退熱，主潤肺止咳補中益氣。 大仲馬經典名著「三劍客」﹝又名「俠隱記」﹞，以十七世紀的法國為時代背景。從前法國武士在打仗時，所持的盾牌上都畫有三朵百合花，每每凱旋而歸，於是他們視百合為吉利的象徵，而定為國花。

4 Introduction 百合及其病害 百合：Lilum spp. 英文名： Lily
百合屬百合科百合屬植物。目前栽培的百合大多是經過人為雜交選育出來的品種，主要有三大類：鐵砲雜交型百合(Longiflorum hybrid)、亞洲雜交型百合(Asiatic hybrid)、及東方雜交型百合(Oriental hybrid)。 原生的百合有：台灣百合(Lilium formosanum Wall.)、鐵砲百合(L. longiflorum Thunb.)、豔紅百合(L. speciosum)、細葉卷丹(L. callosum)等4種

5 Introduction 百合及其病害 百合感染單一病毒 主要病徵為葉部系統性斑紋 (mottling) 或嵌紋 (mosaic) ，病株葉片通常不會出現畸形，但若同時感染 多種病毒 時，則會出現較明顯之嵌紋及不同程度之畸形病徵。 除葉部病徵外,感病植株通常較正常者矮小，生長速度也較緩慢。部份較敏感之深色花品種花部會出現退色、條斑或畸形病徵。

6 Introduction 百合及其病害 寄主範圍十分狹小，除百合外，部份virus 分離株尚可感染
煙草 (Nicotiana benthamiana) 藜科植物 (Chenopodium spp.) 番杏 (Tetragonia expensa)

7 Introduction 百合及其病害 根據 Derks 之報告，LVX 在任何百合栽培地區均有發生。記載較多之地區包括美國、荷蘭、德國、義大利、法國、日本及臺灣，近年來荷蘭將百合球莖生產外移至南美及南非等地區，病毒之分佈也會因而擴大。

8 Introduction 百合及其病害 蔓延方式主要經帶病毒種球之鱗片繁殖或組織培養方式，快速增加後形成植株感染。
田間病毒傳染，經由人員農事操作所造成之機械性傷口及蚜蟲以非永續型方式 (non-persistent) 媒介傳播。 秋冬乾燥季節乃蚜蟲蟲口高峰期，此期間正值百合栽培旺季，故病毒傳播難以避免。人工栽培或野生之此類植物均可能成為 傳染源。

9 Introduction病毒鑑定與病害防治
病毒偵測：酵素連結血清檢定法 （ELISA） 墨點轉漬血清檢定法 （DBIA） 核酸探針雜合偵測 反轉錄-聚合酵素連鎖反應

10 Introduction病毒鑑定 利用酵素連結血清檢定法如 ELISA 或 SDS-immunodiffusion test 可將 不同百合病毒加以區別。以直接式 ELISA (direct ELISA) 檢定病毒之專一性較間接式 ELISA (indirect ELISA) 為高，此乃基於利用 indirect ELISA 檢定時，virus血清仍可與部份 potyviruses 如 Bean yellow mosaic virus (BYMV)、Clover yellow vein virus (CYVV)、Turnip  mosaic  virus  (TuMV) 及  Tulip chlorotic blotch virus (TCBV) 反應，而使用direct ELISA 時只能與同源病毒反應。 利用已知之病毒核酸序列設計專一性引子對 (primer pair) 進行反轉錄聚合酵素複製連鎖反應 (RT-PCR)，將特定之核酸片段大量增幅複製，再以電泳法將其檢出，亦可作為診斷 感染之依據。

11 Introduction病害防治 一、栽培帶病毒率低之種球或由信用度佳之種苗商購買種球。 二、避免種植於已發病嚴重田附近。
三、田區附近避免有病毒寄主，若發現則必須加以剷除，以降低感染機會。 四、種球定植發芽後應及早巡視，發現病株應立即拔除，但必須避免拔除時造成傷口傳染。

12 Introduction病害防治 五、避免過度密植，以降低經由根部接觸傳染之機會。 六、避免農事操作時隨意造成植株傷口之機會。
七、自行繁殖開花用種球時，應購買無病毒感染或低感染率之種球，並最好在防蟲網室內或選擇隔離地區種植。並防治蚜蟲以降低傳播率。

13 Introduction病害防治 八、避免栽培環境氣溫偏低，而造成病毒病徵趨於嚴重。

14 Introduction -Lily virus X (LVX)
病毒形態為長絲狀易彎曲顆粒體，長度界於 μm 之間。 Lily virus X (LVX) is an approved species in the genus Potexvirus from the family Flexiviridae. It was first reported in Lilium formosanum from England. Stone OE （1980）Two new potexviruses from monocotyledons. Acta Hort 110:59-63

15 Genomic RNA of an isolate of Lily virus X (LVX)

16 Purpose To identify and classify the Lily virus X (LVX) strains.
Try to determine the complete sequence of the genomic RNA of an isolate of Lily virus X (LVX) for the first time. To define genome organization of Lily virus X (LVX) Compare the nucleotide and amino acid sequences with other genus Potexviruses to describe phylogenetic relation.

17 Method Purify viral particles of LVX and isolate Viral RNA.
Synthesize frst-strand cDNA - （RT－PCR） Design the virus-specific primer （ LVX-1、M4、 LVX-RdRp3t、Potex-RdRp3t、 LVX-12 、 Potex-RdRp5t1、 LVX-5t2、 ZHM1 、 ZHM2 ） Analyze the viral genome.

18 Method The LVX samples from the Netherlands were provided by Ms. Haoyan Li, Zhejiang Entry and Exit Inspection Quarantine Bureau (ZIQ). Freeze-dried infected leaves stored at −80 ◦C. and purified viral particles of LVX. Viral RNA was isolated using the RNeasy Plant Mini Kit(QIAGEN).

19 Method Synthesize frst-strand cDNA - （RT－PCR）
Design the virus-specific primer （ LVX-1、M4、 LVX-RdRp3t、Potex-RdRp3t、 LVX-12 、 Potex-RdRp5t1、 LVX-5t2、 ZHM1 、 ZHM2 ）

20 Method

21 Method Frst-strand cDNA was synthesized using M-MLV-reverse transcriptase (Life Technologies Ltd) according to the manufacturer’s instructions and with M4T (5’-GTT TTC CCA GTC ACG ACA C (T)16-3’ ) as the initial primer. The use of primer M4T, which is complementary to the 3’-terminal polyA tail, ensured that the complete 3’-noncoding region (NCR) was amplified.

22 Method PCR reactions were then carried out using an ExpandTM Long Template PCR system (Roche). The 5’-terminus of the RNA genome was obtained by a modified 5’-RACE method. The virus-specific antisense primer LVX-5t2 (-) was used to initiate the synthesis of first-strand cDNA (1st cDNA). After synthesis, the cDNA/RNA hybrids were purified and denatured by incubating at 100 ◦C for 2 min and quickly cooled on ice.

23 Method The ZHM1 primer was added to the 3’-terminus of the 1st cDNAs by T4 RNA ligase (New England Biolabs), and the cDNA-ZHM1 product was then used as a template with LVX-5t2 (-) and ZHM2 (complementary to ZHM1) to amplify the 3-terminus of the 1st cDNA. Successful amplification of fragments of the expected size was confirmed by electrophoresis through 1% (w/v) agarose gels.

24 Method The fragments were then purified using the QIAGEN Gel Extraction Kit and cloned into the pGEM-T vector (Promega). Clones were auto-sequenced in both directions by the ABI PRISMTM 377 DNA Sequencer. Sequence analysis used programs from the Wisconsin (GCG) package version 10.3 GAP for the pairwise comparisons for amino acid comparisons.

25 Method For phylogenetic analysis, peptide sequences were used as a template to generate the corresponding nucleotide (nt) by program EMBOSS. Phylogenetic trees were constructed by DNADIST.

26 Method

27 Method

28 Result non-specific bands occurred presumably because of primer binding to host plant RNAs 3 kb 2.5 kb 2 kb 1.5 kb 1 kb 750 bp

29 Result The complete sequence of LVX was obtained from the sequences of four overlapping fragments from the purified virus preparation. Four open reading frames (ORF) were identified by computer analysis. The 5-proximal ORF extends from nt 74 to nt 3970, and encodes 1298 aa.

30 Result Excluding the poly(A)tail, the genomic RNA of LVX was 5823 nucleotide(nt) which is the shortest reported potexvirus sequence. The nucleotide sequence of the 5-non-coding region begins with the motif “GGAAAA” which is the same as that of Scallion virus and the isolates of Cym-bidium mosaic virus (CymMV) from Korea and Taiwan. （ strange CyMV Singapore strain )

31 Result Comparison of amino acid similarities varies from comparison of nucleotide identities.

32 Result Phylogenetic trees A Phylogenetic trees B Discussion

33 Discussion Within the genus Potexvirus, 35 complete sequences from 18 distinct species, nearly all of these the 5-NCR begins with the motif 5’-GAAAA-3’. The only other exceptions are Strawberry mild yellow edge virus (SMYEV) Hence this motif has been proposed to be an essential sequence element for RNA replication or protein translation .

34 Discussion The hexamer “ACUUAA” is present in the 3-NCR of all sequenced potexviruses and occurs in the LVX sequence at nts 5786–5791. This motif may be a cis-acting element involved in the synthesis of viral RNAs.

35 Discussion The ORF1 extends from nt 74 to nt 3970, and encodes 1298 aa. This 146 kDa protein contains several well-conserved sequences (underlined) found in the replicase proteins of other potexviruses and carlaviruses. (i) the putative methyltransferase domain at the N- terminus. (ii) the NTPase/helicase domain beginning with the NTP- binding motif IHGAGGSGKS. (iii) the RNA-dependent RNA polymerase (RdRp) domain at the C-terminus, characterized by the motif SNDFTAFDQSQ X39 AIMRMSGEGPTFDANTECAIAY X12 QLYAGDD.

36 Discussion The most variable region was between the putative methyltransferase and NTPase/ helicase domains, and here the sequences of distinct potexviruses showed significant variation in length and very low sequence similarity. Over the whole replicase protein, there was 43.7–53.5% aa identity between LVX and other potexviruses. (Table 2).

37 Discussion There is no normal start codon for a TGBp3. This was confirmed by sequencing this region of 1st cDNA. Computer analysis has revealed that the closely-related allexiviruses also contain a TGBp3-like sequence but without a normal start codon. Whether such a protein is expressed invivois not yet known.

38 Discussion TGB proteins are involved in the cell-to-cell movement of potexviruses, and it has been suggested that the low virus concentration in LVX-infected tissue is due to the absence of such a TGBp3-like protein. If expressed, the percentages of identity of the aa sequences of LVX TGBp3 with those of other potexviruses would range from 19.7% to 38.5%. (Table 2).

39 Discussion ORF2 encodes a 216 aa protein of 23.6 kDa (TGBp1) that also contains an NTPase/helicase domain characterized by an ATP/GTP-binding site motif (VTVVHTVAGSGKTTFIR, aa 22–38) . Percentages of identity between the TGBp1 of LVX and other potexviruses are lower than those observed for the replicase protein, ranging between 29.3% and 40.9% (Table 2). ORF3 encodes a 108 aa protein of 11.8 kDa (TGBp2) containing the conserved potexvirus sequence GDNLHSLPHGGTYCDGTK (aa 38–55) and has 32.4% to 51.0% aa identity to other potexviruses (Table 2).

40 Discussion ORF4 encodes a 201 aa coat protein (CP) of 21.6 kDa. It might be responsible for binding of potexviral RNA to the CP. （ aa,nt table） The percentages of identity of the aa sequences of LVX CP with those of other potexviruses ranged from 26.3% to 41.8% (Table 2).

41 Discussion A phylogenetic analysis of the replicase nucleotide sequence, LVX clearly belonged with the potexviruses and closely related to SMYEV and provided value (74%)(Fig. 2a). A phylogenetic analysis of complete sequence confirmed the relationship of LVX to SMYEV and provided value (88%)(Fig.2b).

42 Discussion Substitution and delection of overlapping region induce the frameshift of TGB gene. The frameshift will not affect the function of TGB gene.

43 Discussion Longer Primer ZHM2 and ZHM1 make PCR amplification well.
Utilize E.mail and MSN well.

44 Further research It is expected that the result of this study can help in the construction of the antivirus transgenic plants for controlling LVX in the future. TGBp3-like sequence exists in the expected region but without an AUG start codon. Whether such a protein is expressed in vivo.

45 Further research Both of Lily virus X and Bamboo mosaic virus are potexvirus and with similar genome. ORF6 exists in BaMV RdRp gene to encode hypothetical 14k protein. Does ORF6 exist in LVX？14k

46 Thank you for attention
輕鬆一下 短片欣賞

47 Discussion CP基因及3‘端未轉譯區之核苷酸序列通常是病毒分類上具有參考意義的區域( Kekarainen et al., 1999；Aleman-Verdaguer et al., 1997；Bousalem et al., 2000；Shukla and Ward, 1988 )。 本文未作探討，僅就RdRp基因比對。

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