一種與構樹嵌紋病相關的carlavirus之特性研究

Characterization of a Carlavirus Associated with Paper Mulberry Mosaic Disease in Taiwan

構樹（Broussonetia papyrifera(L.) L´H’erit. ex Vent.）為桑科（Moraceae）落葉性喬木或灌木，2005年於屏東地區田間發現構樹葉片呈現嚴重黃綠嵌紋病徵。田間病葉經由奎藜（Chenopodium quinoa）進行單斑分離，可分離出一種絲狀病毒，病毒粒子大小約為600-650 nm，暫稱為W1。此病毒的熱不活化溫度為70℃，耐稀釋度為10-4，室溫（24℃）下活性可維持4天，冷凍（-80℃）則活性可保存10個月。電子顯微鏡觀察奎藜單斑及黃化嵌紋構樹病組織之切片，可於細胞質中見到病毒粒子成束狀之聚集及細胞胞器之病變。機械接種16科64種供試植物，僅藜科的奎藜、紅藜（C. maranticolor）、綠藜（C. murale）及豆科之豇豆（Vigna unguiculata）等4種植物會被感染，接種葉呈現黃色局部斑點病徵。經電泳分析估算病毒鞘蛋白分子量約為36 kDa。白兔經免疫注射，可製備出力價1024之抗血清；瓊脂擴散反應可與單斑奎藜病葉、純化病毒及田間構樹嵌紋病葉呈現同源正反應，不會與Carlavirus屬之Lily symptomless virus （LSV）及Lycoris virus T （LVT）發生沉澱反應。以針對Carlavirus屬病毒聚合酶基因之廣效性引子對進行RT-PCR，構樹嵌紋病葉及所接種之奎藜病葉皆可增幅出一286 bp核酸產物，經解序及與GenBank中已知25種59支分離株之carlaviruses序列比對後，發現其與其他carlaviruses之核苷酸序列相同度（identity）為67-77%，胺基酸序列相同度為77-88%。綜合以上結果顯示，本研究之構樹病毒分離株W1為一種carlavirus，建議暫時先將其命名為構樹嵌紋病毒（Paper mulberry mosaic virus）。

"Broussonetia papeyrifera (L.) L´H’erit. ex Vent. commonly named as paper mulberry in Moraceae family is a kind of deciduous arbor or shrub which leaves can be used as feeds for pigs, cattle, sheep and deer, and barks can be used for paper-making. It is a pioneer plant growing in plains or regions with lower altitude. At present, the commercial cultivation has never been implemented in Taiwan, only pioneer plants on roadsides or wastelands, so they are distributed widely but sporadically in Taiwan. In 2005, a mosaic symptom on paper mulberry was found in Pingtung. Symptoms fluctuated seasonally. A filamentous virus of 600-650 nm in particle size, denoted W1, was successfully isolated by mechanical inoculation from a symptomatic paper mulberry leaf. The thermal inactivation point of W1 was 70℃, dilution end point was 10-4 and the longevity in vitro was 4 days at 24℃ and more than 10 month at -80℃. W1 has a very narrow host range restricted to three chenopodiaceous plant species and one leguminosae plant species among 64 tested plant species belonging to 16 families. Numerous bundles of virus particles scattered through the cytoplasm were found in local lesion of Chenopodium quinoa and mosaic leaf of paper mulberry. The disease incidence was about 15.2% by seed transmission. A purified virus preparation was obtain with a yield of 3.6 mg from 100 g diseased leaves. The coat protein (CP) with molecular weight of 36 kDa was detected from purified virus particles by sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS-PAGE) and by western blotting using the produced rabbit antiserum against W1 virions. In SDS-agar gel double diffusion test, the W1 antiserum reacted strongly with its homologous antigens, but not to antigens of two other carlaviruses Lily symptomless virus (LSV) and Lycoris virus T (LVT). A DNA fragment of 286 nucleotides (nts) amplified from the polymerase gene of W1 was sequenced and analyzed to reveal that the amplicon sequence of W1 shares 67-77% nt identity and 77-88% amino acid (aa) identity with those of 59 strains of 25 carlavirus species. Based on the results, the virus W1 associated with the mosaic symptom on leaves of paper mulberry is identified as a carlavirus and is proposed as Paper mulberry mosaic virus tentatively."