(65(2):194-206)Serological and Molecular Identification of Nerine latent virus Infecting Blood Lily (Haemanthus multiflorus Martyn)

由未呈現病徵之火球花 (Haemanthus multiflorus Martyn) 植株葉片，經機械接種於奎藜 (Chenopodium quinoa Willd.) 葉片後出現局部斑點病徵，以3 次連續單一斑點分離獲得1 個分離株 (HM4)。將感染HM4 之火球花及其接種後之奎藜單斑組織於間接式- 酵素連結免疫分析法 (indirect enzyme-linked immunesorbent assay; indirect ELISA) 及西方墨點法中，均可與已知之Nerine latent virus (NeLV) 金花石蒜分離株 (NeLV-LY56) 之抗血清產生正反應。於反轉錄-聚合酶連鎖反應 (reverse transcription-polymerase chain reaction; RT-PCR) 中應用NeLV-up/Oligo-d(T)14 引子對，可增幅出HM4 基因體核酸3’ 端約1.5 kbp 之片段，經解序後證實此片段涵蓋NeLV 之全長度鞘蛋白 (coat protein; CP) 及富含半胱氨酸蛋白 (cysteine-rich protein; CRP) 之轉譯架構(open reading frame)。將HM4 及其他6 個選殖自火球花之Carlavirus 屬病毒分離株核酸序列，與GenBank 上已登錄之NeLV (Acc. no. JQ395044) 進行分析比對，結果顯示此等病毒序列於鞘蛋白核苷酸及胺基酸相同度(identity) 均可達96–99%，確認HM4 及其他6 個選殖序列均為NeLV。將此7 個火球花NeLV 選殖株與來自金花石蒜 (Lycoris aurea)、孤挺花 (Hippeastrum hybridum Hort.) 及水仙 (Narcissus spp.) 之NeLV 分離株之CP 序列進行類緣分析 (phylogenetic analyses)，發現此等序列均同屬NeLV 群組，而火球花分離株則自成1 個類緣分支。由上述血清學及核酸分子鑑定、類緣分析結果顯示，自火球花上所分離之HM4 及其他選殖株均為NeLV 之成員。本試驗亦證實，利用對應NeLV 金花石蒜分離株之抗血清於indirect ELISA 及西方墨點法等免疫檢測中，可順利檢測火球花、金花石蒜、孤挺花及水仙等不同寄主受NeLV 感染之樣品。且其結果與利用HM4 核酸序列所設計之引子對 (NeLV552u/NeLV552d) 進行RT-PCR 檢測之結論相符。本研究於2006–2014 年期間以indirect ELISA 法調查台灣地區火球花等4 種石蒜科花卉之NeLV 感染率 [(檢出數/樣品數) × 100%]，結果分別為火球花18.4%、金花石蒜32%、孤挺花79% 及水仙13.3%，且印證受NeLV 單獨感染者均未顯現病徵。經血清學與分子生物學鑑定結果，本研究亦首次證實火球花為NeLV 之天然寄主。 A virus isolate (HM4), obtained from a symptomless blood lily (Haemanthus multiflorus Martyn) plant, was established on Chenopodium quinoa Willd. by three successive single lesion reisolation and inoculation. The original blood lily plant and inoculated C. quinoa leaf tissues of HM4 were reacted strongly with the antibody against the LY56 isolate of Narine latent virus (NeLV) from golden spider lily in indirect ELISA and western blotting. A 1.5-kbp DNA fragment was amplified by RT-PCR from HM4-infected blood lily and C. quinoa tissues using a primer pair specific to the 3'- end of genomic sequences of NeLV. Sequence analyses revealed that the amplicon comprises 1,560 nucleotides spanning the entire open reading frame of coat protein (CP) and the complete cysteinerich protein region of NeLV. Comparison of nucleotide and amino acid sequences indicated that the CP of HM4 shares 96–99% identities with those of the other six blood lily isolates and the known NeLV isolates documented in the GenBank. Results indicated that HM4 and the other six blood lily isolates are the isolates of NeLV. In phylogenetic analysis, these blood lily isolates and those NeLVs from golden spider lily (Lycoris aurea), amaryllis (Hippeastrum hybridum Hort.) and narcissus (Narcissus spp.) were grouped in the same clade evidently differing from that of Hippeastrum latent virus (HLV) and Narcissus common latent virus (NCLV). Phylogenetic analysis indicated that the blood lily isolates are grouped in the same sub-clade. Our studies also confirmed that all the NeLV isolates from blood lily, golden spider lily, amaryllis and narcissus can be detected by the same antiserum against NeLV-LY56 in indirect ELISA and western blotting. Moreover, a 552-bp DNA fragment can be amplified from all these NeLV isolates in RT-PCR using the primers designed from the genomic sequence of HM4. Field surveys were conducted during 2006–2014 by indirect ELISA and showed 18.4, 32, 79, and 13.3% of the incidences of NeLV in blood lilies, golden spider lilies, amaryllis and narcissus, respectively, in Taiwan. Based on the serological and molecular evidences, NeLV naturally infecting blood lilies without showing discernible symptoms is first reported