基于NGS发现滇重楼中新的负链RNA病毒及致病性鉴定

After the genome of -ssRNA virus getting into cells, the complementary RNA is reproduced and used as mRNA, which is an important basis for virus classification. the -ssRNA viruses could cause viral diseases on watermelon and other crops. There are few reports of the viral disease in Paris polyphylla var.yunnanensis, in recent years we found that viral diseases occured in Yunnan, which has an effect on production. NGS technology has become a new way to detect and identify viruses. Pathogenicity identification of newly discovered -ssRNA virus is a prerequisite for Disease control. On the basis of the preliminary data, the -ssRNA virus sequence was found from Paris polyphylla var.yunnanensis by NGS. The existence of the virus was confirmed by RT-PCR. In the core region of RdRp, characteristic sequences SDD and EFNS existed. It shared identities of 32% at amino acid level to the RdRp of all available Bunyaviales viruses. In this study, the full length genome of the virus would be amplified by designing back-to-back primers, and a cDNA infectious vector would be constructed and transformed into differential hosts. The hosts and indicator plants would be inoculated to study the host range of the virus and its pathogenicity,the field distribution also be investigated to identify correlation between the virus and symptoms. It is the first time to report -ssRNA virus on Paris polyphylla var.yunnanensis. It is important to study its classification, host range and pathogenicity, and it can supply the basis for further studying on control and epidemiology of viral disease.

负链RNA病毒基因组进入细胞经复制产生互补RNA才作为mRNA，是病毒分类的重要依据，能引起西瓜等作物发病。滇重楼病毒病报道较少，近年调查发现在云南有发生，对生产造成影响。NGS技术已成为发现鉴定病毒的新途径，对新发现的负链RNA病毒进行致病性鉴定是防控病害前提。在前期基础上，从滇重楼转录组数据中发现一负链RNA病毒序列，经RT-PCR证实存在该病毒，在RdRp核心区域有特征基序SDD和EFNS，与Bunyavirales成员RdRp氨基酸序列同源性仅达32%，分类地位待定。本研究通过设计背向背引物，扩增病毒全长序列并构建侵染性克隆及植物表达载体，用摩擦接种和农杆菌侵染接种寄主和指示植物，确定寄主范围并鉴定致病性。同时调查田间发生分布，确定病毒和田间症状之间的对应关系。首次在滇重楼上发现负链RNA病毒，明确其分类地位、寄主范围及致病性不仅为诊断防控奠定基础，也对研究其流行规律具有重要意义。

滇重楼病毒病近年在云南发生严重，对生产造成较大影响。本项目对滇重楼上新的负链病毒Yunnan paris negative-stranded virus” (YPNSV)进行基因组结构特征分析，发现YPNSV基因组由2条RNA片段组成。RNA1包含一个开放阅读框（ORF），在反义链上编码依赖于RNA的RNA聚合酶（RdRp），RNA2是一条双义链，包含2个ORF，分别编码运动蛋白（MP）和核衣壳蛋白（NP），ORF之间有一段富含A和U碱基长基因间隔区。YPNSV的RdRp、MP、NP蛋白氨基酸序列均与WCLaV-2具有最高的同源性，分别为69.1%、50.4%、60.9%。在以RdRp和NP氨基酸序列构建的系统发育树中，YPNSV与Coguvirus属成员聚为一簇，与WCLaV-2密切相关。结合基因组结构特征和系统进化树分析，确定YPNSV是Bunyavirales目Phenuiviridae科Coguvirus属的一个新种。克隆YPNSV的全长cDNA基因，并将其连接到原核表达载体pBWA(V)HS-35S上，并插入外源基因GFP。将获得的重组质粒转化至大肠杆菌表达菌株DH5α细胞内，经IPTG诱导表达蛋白，以此为抗原分别免疫新西兰大白兔，制备YPNSV全长基因组RdRp、MP和NP的多克隆抗体，采用间接ELISA检测抗体效价。采用电击法将重组质粒转化到农杆菌GV3101和EHA105菌株内，通过浸润注射接种法接种植物鉴定致病性。在滇重楼接种60d后，通过GFP检测和特异性引物RT-PCR检测到YPNSV，在心叶烟和苋色藜接种30d后，通过GFP检测和特异性引物RT-PCR检测到YPNSV。对昆明、曲靖、大理、玉溪、文山和普洱等6个地区110份滇重楼样品进行特异性引物RT-PCR检测和抗血清检测，其中在玉溪样品YPNSV检出率为6.45%、普洱样品YPNSV检出率为20.00%，文山样品YPNSV检出率为33.33%。感染YPNSV的滇重楼表现出下部叶片扭曲皱缩、有的叶脉上有坏死条纹，上部叶片出现大面积褪绿。首次在滇重楼上发现负链RNA病毒YPNSV并构建了侵染性克隆，确定YPNSV能侵染心叶烟苋色藜。研究结果不仅为滇重楼病毒病害的诊断和防控奠定基础，而且也对今后进一步研究该病毒致病机制和发生流行规律具有重要意义。