Discovery of Jasmin Virus H. Discovery of Jasmin Virus H

Small RNA libraries were constructed from total RNA from Jasminum sambac plants exhibiting virus-like symptoms. After sequencing, small RNAs were assembled into contigs with MetaVelvet and assembled contigs were aligned against the NR database of NCBI using BLASTx. Top hits that reported a virus as subject were considered putative viral sequences. Based on such alignments, the whole genome of a virus, we tentatively name Jasmine Virus H was recovered and cloned. Two more small RNA libraries were made in a confirmatory experiment. One from Jasminum sambac and another one from Nicotiana benthamiana plants infected with the newly-cloned virus. The small RNA libraries were aligned against the full-length sequence of Jasmine Virus H to determine the spacial distribution of virus-derived small RNAs along the virus genome. Overall design: Symptomatic leaves were collected in the field and used for virus discovery and isolation as described in the summary. Collection was repeated once and the small RNAs libraries repeated. In addition an infectious clone was produced and used to infect Nicotiana benhtamina plants, from which total RNA was extracted and viral small RNAs characterized again. All libraries were sequenced in an Illumina HiSeq 2500 instrument.

本研究从表现出类病毒症状的茉莉（Jasminum sambac）植株的总RNA中构建小分子RNA（small RNA, sRNA）文库。测序完成后，利用MetaVelvet软件将小分子RNA组装为重叠群（contig），并通过BLASTx将所得组装重叠群比对至NCBI非冗余（Non-Redundant, NR）数据库。将以病毒为匹配靶标的最高得分比对结果认定为潜在病毒序列。基于此类比对结果，我们成功获取并克隆了一株暂命名为茉莉病毒H（Jasmine Virus H, JVH）的病毒的全基因组。为开展验证实验，本研究额外构建了两个小分子RNA文库：其一取自感染上述新克隆病毒的茉莉植株，其二取自感染该病毒的本氏烟（Nicotiana benthamiana）植株。将这两个小分子RNA文库比对至茉莉病毒H的全长基因组序列，以解析病毒来源小分子RNA在病毒基因组上的空间分布特征。实验整体设计如下：首先于田间采集表现症状的叶片，用于如前文概述的病毒发现与分离实验；该采样流程重复一次，并同步完成小分子RNA文库的构建。此外，本研究构建了该病毒的侵染性克隆，用以接种本氏烟植株，随后提取其总RNA并再次对病毒来源小分子RNA进行鉴定分析。所有小分子RNA文库均在Illumina HiSeq 2500测序平台上完成测序。