Genetic variability and evolutionary dynamics of atypical Papaya ringspot virus infecting Papaya

Papaya ringspot virus biotype-P is a detrimental pathogen of economically important papaya and cucurbits worldwide. The mutation prone feature of this virus perhaps accounts for its geographical dissemination. In this study, investigations of the atypical PRSV-P strain was conducted based on phylogenetic, recombination and genetic differentiation analyses considering of it’s likely spread across India and Bangladesh. Full length genomic sequences of 38 PRSV isolates and 35 CP gene sequences were subjected to recombination analysis. A total of 61 recombination events were detected in aligned complete PRSV genome sequences. 3 events were detected in complete genome of PRSV strain PK whereas one was in its CP gene sequence. The PRSV-PK appeared to be recombinant of a major parent from Bangladesh. However, the genetic differentiation based on full length genomic sequences revealed less frequent gene flow between virus PRSV-PK and the population from America, India, Colombia, other Asian Countries and Australia. Whereas, frequent gene flow exists between Pakistan and Bangladesh virus populations. These results provided evidence correlating geographical position and genetic distances. We speculate that the genetic variations and evolutionary dynamics of this virus may challenge the resistance developed in papaya against PRSV and give rise to virus lineage because of its atypical emergence where geographic spread is already occurring

Taxonomy of the order Bunyavirales : second update 2018

In October 2018, the order Bunyavirales was amended by inclusion of the family Arenaviridae, abolishment of three families, creation of three new families, 19 new genera, and 14 new species, and renaming of three genera and 22 species. This article presents the updated taxonomy of the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV).Non peer reviewe

Taxonomy of the family Arenaviridae and the order Bunyavirales : update 2018

In 2018, the family Arenaviridae was expanded by inclusion of 1 new genus and 5 novel species. At the same time, the recently established order Bunyavirales was expanded by 3 species. This article presents the updated taxonomy of the family Arenaviridae and the order Bunyavirales as now accepted by the International Committee on Taxonomy of Viruses (ICTV) and summarizes additional taxonomic proposals that may affect the order in the near future.Peer reviewe

Construction and evaluation of transgenic tobacco plants expressing the coat protein gene of papaya ringspot virus with different translation leaders

Papaya ringspot virus (PRSV) YK isolate used in this study is a local mosaic strain isolated from Yung-Kang, Tainan, and its genome has been cloned and completely sequenced. A NcoI site before the coat protein (CP) reading frame of PRSV YK was generated by oligonucleotide-directed mutagenesis, and then the CP reading frame with the 3' noncoding region of PRSV YK was ligated with the gus leader sequence from the pGEM vector to create the construct pGGCP. To express the CP with a homologous viral translation sequence, the gus leader was replaced by the cDNA sequence corresponding to the 5' region (nt 1-347) of PRSV genome to generate a protein containing 9 kDa polypeptide of PRSV P1 protein fused with the CP, and the construct was designated as pG5'CP. In vitro translation from the transcripts derived from pGGCP and pG5'CP generated protein products of 36 kDa and 45 kDa, respectively. Both proteins reacted with the antiserum to PRSV CP, and the level of 36 kDa protein was higher than that of 45 kDa protein. The CP reading frame with the gus or PRSV 5' leaders was individually subcloned into a Ti binary vector. Transgenic tobacco plants (Nicotiana tabacum L. Havana 423) expressing the PRSV CP gene with the gus leader (GCP lines) or with the viral leader (5'CP lines) were obtained by Agrobacterium-mediated transformation. When the transgenic lines were analyzed by western blotting, the protein products of 36 kDa and 45 kDa reacting to PRSV CP antiserum were detected in the GCP lines and 5'CP lines, respectively. The presence of the CP gene in the transgenic tobacco was also confirmed by polymerase chain reaction (PCR) using primers specific to the CP gene. Analysis of segregation ratios in the R1 plants of four GCP lines and four 5'CP lines indicated that the CP gene in all of them was nuclearly inherited as a single dominant trait. R0 and R1 plants of the four GCP lines and four 5'CP lines were inoculated with tobacco etch virus (TEV), potato virus Y (PVY), or pepper mottle virus (PepMoV). The transgenic lines showed significant delay in symptom development and the severity of symptoms was attenuated. The GCP lines expressing the PRSV CP gene by the gus leader accumulated higher levels of CP and showed higher degrees of resistance than the 5'CP lines with the PRSV 5' leader. Our results indicate that the homologous viral leader does not enhance CP expression either in vitro or in vivo, nor does it provide better resistance in transgenic tobacco

利用番茄斑萎病毒屬(Tospovirus)之RNA複製高保留性區域做轉基因至植物體中而具有抗番茄斑萎病毒屬病毒之方法與其應用

本發明係關於一種利用番茄斑萎病毒屬(Tospovirus)之RNA複製高保留性區域做轉基因至植物體中而具有抗番茄斑萎病毒屬病毒之方法與其應用,利用西瓜銀斑病毒的複製高保留性區域,育成並選殖具廣泛抗性之轉基因植物,其所提供的轉基因植物能夠有效地同時對抗至少五種不同且親緣關係甚遠的番茄斑萎病毒屬病毒

提供瓜類作物雙重病毒抗性之基因轉殖載體

一種提供瓜類作物雙重病毒抗性的載體，其係包含矮南瓜黃化嵌紋病毒(ZYMV)之部分鞘蛋白編碼區片段，及木瓜輪點病毒西瓜系統(PRSV W)之部分鞘蛋白編碼區片段。本發明另相關於一種含有前述載體的細菌。本發明另相關於利用前述載體轉殖在一植物中以達抗病毒的方法。本發明藉由構築一轉殖載體，該載體同時攜帶ZYMV及PRSV W的鞘蛋白基因片段，並將該載體成功轉殖至一植物上，藉以誘導基因沉寂現象，使該植物可同時對此二病毒的單一及複合感染產生高度的抗性。 【創作特點】 有鑑於既有抗病方法的限制，本發明主要係提出一種新穎瓜類抗病毒的方法，以提高瓜類的產量與品質，避免瓜類作物生長及生產的損失，進而提高我國農產品之價值與產量。 為達成前述目的，本發明主要係以一種提供瓜類作物雙重病毒抗性的載體，其係包含ZYMV之部分鞘蛋白編碼區片段，及PRSV W之部分鞘蛋白編碼區片段。 較佳的是，該ZYMV之部分鞘蛋白編碼區片段長度係為544 bp；更較佳的是，該ZYMV之部分鞘蛋白編碼區片段係利用SEQ ID NO：1及SEQ ID NO：2單離。 較佳的是，該PRSV W之部分鞘蛋白編碼區片段長度係為445 bp；更較佳的是，該PRSV W之部分鞘蛋白編碼區片段係利用SEQ ID NO：3及SEQ ID NO：4單離。 較佳的是，經單離的ZYMV之部分鞘蛋白編碼區片段與PRSV W之部分鞘蛋白編碼區片段係接合在雙偶型載體pBI121內；更較佳的是，該ZYMV之部分鞘蛋白編碼區片段與PRSV W之部分鞘蛋白編碼區片段係位於雙偶型載體pBI121的CaMV 35S啟動子及Nos終止密碼子間；最佳的是，含有ZYMV之部分鞘蛋白編碼區片段與PRSV W之部分鞘蛋白編碼區片段的雙偶型載體pBI121，係不具有 GUS閱讀框。 本發明另相關於一種含有前述載體的細菌。 較佳的是，該細菌係為桿菌；更較佳的是，該細菌係為農桿菌卸甲株。 本發明另相關於一種提供植物抗雙重病毒的方法，其係包含：將前述載體轉殖於一植物內，使該植物物表現該載體上所載之ZYMV之部分鞘蛋白編碼區片段與PRSV W之部分鞘蛋白編碼區片段。 較佳的是，該植物係為甜瓜。 較佳的是，轉殖方式係利用帶有該載體的農桿菌接種植物。 藉由前述之技術手段，本發明所達成之主要功效在於：藉由植物表現雙病毒(ZYMV及PRSV W)的部分鞘蛋白編碼區，以使轉基因植物表現後轉錄基因靜默作用，而達到抗病毒的效果

利用番茄斑萎病毒屬（Tospovirus）之RNA複製酶高保留性區域做轉基因至植物體中而具有抗番茄斑萎病毒屬病毒之方法與其應用

本發明係關於一種利用番茄斑萎病毒屬(Tospovirus)之RNA複製酶高保留性區域做轉基因至植物體中而具有抗番茄斑萎病毒屬病毒之方法與其應用，利用西瓜銀斑病毒的複製酶高保留性區域，育成並選殖具廣泛抗性之轉基因植物，其所提供的轉基因植物能夠有效地同時對抗至少五種不同且親緣關係甚遠的番茄斑萎病毒屬病毒

genue)之西瓜銀斑病毒血清群病毒(WSMoV-serogroup tospoviruses)之單株抗體及其製備方法與應用

本發明係一種鑑定番茄斑點萎凋病毒屬(Tospovirus genue)之西瓜銀斑病毒血清群病毒(WSMoV-serogroup tospoviruses)之單株抗體及其製備方法與應用

Resistance Evaluation of the Transgenic Papaya Lines Expressing the Coat Protein Gene of Papaya Ringspot Virus

為了利用植物遺傳工程方式將木瓜輪點病毒(PRV)鞘蛋白(coat protein, CP)基因轉移至木瓜以產生抗病性狀，木瓜轉型作用之進行係以金剛砂製造傷口，經由Agrobacterium tumcfaciens媒介，將GUS或PRV 5’前導序列表現的PRV 鞘蛋白基因導入木瓜幼胚分化組織中，並再生成轉基因植株。經由PCR 及西方漬染法分析，確定CP 基因存在轉型木瓜株系中，並可表現產生PRV的鞘蛋白，計獲得包含GUS 前導序列的10個株系及PRV 5’前導序列的3 個株系。先以組培養技術繁殖其R0植株，經由發根、馴化，使其在溫室生長兩個月後，進行挑戰接種，以測定其抗病性狀。實驗中以來自臺灣、夏威夷及泰國三個不同來源的PRV YK、HA 及TH 系統供試“接種後在溫室中觀察其結果，不表現病徵之植株再以酵素連結免疫呈色法（E LlsA ）及生物分析偵測是否有病毒增殖。抗病篩選得到四個抗同源PRV YK的株系：GCP 16-0、GCP 17-0、GCP 17- 1及GCP 17-3，在此四株系中，GCP 17-1及GCP 17-3對異源的PRV HA 及TH 系統仍有高度抗性，GCP 16-0則對異源病毒只有中度抗性，而GCP 17-0的抗病能力為系統專一性的抗病現象，只對同源病毒系統有抗病力。目前此四抗病毒株系仍繼續於隔離溫室中培養，其生長情形與一般木瓜一致。但此四株系均為雌株，以日昇之花粉授粉，果實生長情形與外觀形狀也都正常。目前GCP 17-1之R1 種子已發芽，並於溫室中栽培，再次進行抗病檢定中。由於GCP 17-1及GCP 17-3對不同來源的三個病毒系統都有極高的抗病力，其未來可望不受地區限制而能廣泛應用以防治木瓜輪點病毒。 The coat protein (CP) of a local mosaic strain of papaya ringspot vrius (PRV YK) was previously constructed in the Ti-vector for generation of transgenic papaya resistant to PRV infection. The CP gene with a GUS or the PRV leader sequence was transferred to embryogenic tissues derived from immature embryos of papaya via Agrobacterium -mediated transformation assisted by carborundumwounding treatment. The presence and the expression of the transgene in the putative transgenic lines regenerated were confirmed by PCR detection and Southern blotting. R0 plants of ten CP-transgenic lines with the GUS leader and three lines with the virus leader were established by micropropagatio n The plants were challenged with three different strains of PRV originated from Taiwan (YK), Hawaii (HA), and Thailand (TH). The infection of PRV was determined by symptom observation, ELISA and bioassay. Four lines of GCP 16-0, 17-0, 17-1, and 17-3 were found to be resistant to the homologous YK strain. Among them, GCP 17-1 and 17- 3 were highly resistant to the heterologous HA and TH strains, GCP 16-0 were moderately resistant to the heterologous strain HA and GCP 17-0 was specifically resistant only to the homologous YK strain. All four lines were female and their horticultural properties were similar to the untransformed papaya. The R1 plants of GCP 17-1 crossed with Sunrise papaya have been obtained and their resistance to PRV infection are currently evaluated under greenhouse conditions. The broad resistance in GCP 17-1 and 17-3 indicated that they have a great potential to be applied in different areas for control of the notorious PRV