番茄斑萎病毒屬非結構性NSs蛋白血清學親緣關係之研究

The study of serological relationship in the nonstructural NSs protein of the genus Tospovirus

康雅琪(Ya-Chi Kang)

指導教授 ： 陳宗祺

亞洲大學/健康學院/生物科技學系碩士班/碩士(2008年)

摘要

薊馬傳播之番茄斑萎病毒 (tospoviruses) 屬於布尼亞病毒科 (Bunyaviridae) 之番茄斑萎病毒屬 (Tospovirus)，其病毒核鞘蛋白 (nucleocapsid protein, NP) 序列的同源性，為病毒分類為不同病毒種 (species) 之重要依據。依NP的血清學親緣關係，現行已知的19種病毒，可區分成三大血清群 (serogroups)，分別以西瓜銀斑病毒 (Watermelon silver mottle virus, WSMoV)、番茄斑點萎凋病毒 (Tomato spotted wilt virus, TSWV) 和鳶尾花黃斑病毒 (Iris yellow spot virus, IYSV) 為各血清群的代表型 (type members)。而鳳仙花壞疽斑點病毒 (Impatiens necrotic spot virus, INSV)、花生黃化扇斑病毒 (Peanut chlorotic fan-spot virus, PCFV) 及花生黃斑病毒 (Peanut yellow spot virus, PYSV) 與其他tospoviruses沒有任何的血清學關係，所以稱之為血清型 (serotypes)。在先前的研究報導中，以WSMoV之NSs蛋白做為抗原所製備的單株抗體MAb-WNSs，可測得WSMoV與IYSV二血清群的病毒。抗原決定位的分析得知，MAb-WNSs可與這些tospoviruses的NSs蛋白上之高保留性區域結合。NSs蛋白的序列分析得知，INSV與TSWV血清群病毒具有較高的同源性，而PCFV則是與PYSV最相近。本研究利用細菌表現的方式來生產INSV與PCFV的NSs蛋白，分別製備其抗血清RAs-INSs與RAs-PNSs。結果如預期地，RAs-INSs不但可與INSV反應，更可以廣泛地偵測到TSWV血清群病毒，包括TSWV、番茄黃化斑點病毒 (Tomato chlorotic spot virus, TCSV)、花生輪斑病毒 (Groundnut ringspot virus, GRSV) 和菊花莖部壞疽病毒 (Chrysanthemum stem necrosis virus, CSNV)；而RAs-PNSs則僅可測得PCFV。因本實驗室目前並無PYSV的病毒來源，故尚無法釐清PCFV與PYSV的血清關係。有鑑於此，本研究以人工合成PYSV之N基因並以細菌表現PYSV的NP來製備抗血清RAs-PYSV NP。此血清可偵測到PCFV和細菌表現的PYSV NP。綜合本研究結果，我們建議可將tospoviruses重新分類為WSMoV、TSWV、IYSV和PYSV等四大血清群，且本研究中所製備之抗血清可做為檢測tospoviruses之有利工具。

關鍵字

番茄斑萎病毒屬 ； 血清群 ； 血清型

並列摘要

The homology of nucleocapsid protein (NP) is a key criterion for demarcation of thrips-borne tospoviruses (family Bunyaviridae, genus Tospovirus) at the species level. Based on the serological and phylogenetic relationships of NPs, 19 formal and tentative tospovirus species were clustered into three major serogroups with Watermelon silver mottle virus (WSMoV), Tomato spotted wilt virus (TSWV) and Iris yellow spot virus (IYSV) as the type members. While Impatiens necrotic spot virus (INSV), Peanut yellow spot virus (PYSV) and Peanut chlorotic fan-spot virus (PCFV) were serologically distinct from any other tospoviruses and classified as serotypes. A current study reported that a monoclonal antibody (MAb) prepared to the NSs protein of WSMoV, MAb-WNSs, can broadly react with the members of both WSMoV and IYSV serogroups. Epitope mapping revealed that the MAb-WNSs-recognized region is highly conserved among the NSs proteins of those tospoviruses. Sequence analyses of the reported NSs proteins indicated that INSV shares higher degrees homology with the members of TSWV serogroup while PCFV is close to PYSV. In this investigation, the NSs proteins of INSV and PCFV were individually produced by bacterial pET expression system and used as immungens for preparation of antisera, RAs-INSs and RAs-PNSs, respectively. As expected, RAs-INSs reacted broadly with the homologous INSV antigen and the crude samples from the members of TSWV serogroup, including TSWV, Groundnut ringspot virus, Tomato chlorotic spot virus and Chrysanthemum stem necrosis virus. RAs-PNSs specifically reacted with the crude antigen of PCFV. Due to the lack of PYSV in our laboratory, serological comparison between PYSV and PCFV is unavailable at present. Nucleotide sequence corresponding to the N gene of PYSV was artificially synthesized. Furthermore, the NP of PYSV was expressed for preparation of antiserum RAs-PYSV NP. RAs-PYSV NP reacted positively with the crude sample of PCFV as well as the expressed PYSV NP. Taken together, our results of NSs serological relationships suggest that the current tospovirus species should be reclassified as four major clusters, such as WSMoV, TSWV, IYSV and PYSV serogroups. The antisera produced in this investigation are useful tools for inspection of tospoviruses.

並列關鍵字

Tospovirus ； serogroup ； serotype

參考文獻

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