Nerva L, Ciuffo M, Vallino M, Margaria P, Varese G C, Gnavi G, Turina M
Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy; Mycotheca Universitatis Taurinensis (MUT), Department of Life Sciences and Systems Biology, University of Turin, Viale Mattioli 25, 10125 Torino, Italy.
Istituto per la Protezione Sostenibile delle Piante, CNR, Strada delle Cacce 73, 10135 Torino, Italy.
Virus Res. 2016 Jul 2;219:22-38. doi: 10.1016/j.virusres.2015.10.028. Epub 2015 Nov 9.
The number of reported mycoviruses is increasing exponentially due to the current ability to detect mycoviruses using next-generation sequencing (NGS) approaches, with a large number of viral genomes built in-silico using data from fungal transcriptome projects. We decided to screen a collection of fungi originating from a specific marine environment (associated with the seagrass Posidonia oceanica) for the presence of mycoviruses: our findings reveal a wealth of diversity among these symbionts and this complexity will require further studies to address their specific role in this ecological niche. In specific, we identified twelve new virus species belonging to nine distinct lineages: they are members of megabirnavirus, totivirus, chrysovirus, partitivirus and five still undefined clades. We showed evidence of an endogenized virus ORF, and evidence of accumulation of dsRNA from metaviridae retroviral elements. We applied different techniques for detecting the presence of mycoviruses including (i) dsRNA extraction and cDNA cloning, (ii) small and total RNA sequencing through NGS techniques, (iii) rolling circle amplification (RCA) and total DNA extraction analyses, (iv) virus purifications and electron microscopy. We tried also to critically evaluate the intrinsic value and limitations of each of these techniques. Based on the samples we could compare directly, RNAseq analysis is superior to sRNA for de novo assembly of mycoviruses. To our knowledge this is the first report on the virome of fungi isolated from marine environment. The GenBank/eMBL/DDBJ accession numbers of the sequences reported in this paper are: KT601099-KT601110; KT601114-KT601120; KT592305; KT950836-KT950841.
由于目前使用下一代测序（NGS）方法检测真菌病毒的能力，已报道的真菌病毒数量呈指数级增长，大量病毒基因组通过真菌转录组项目的数据在计算机上构建。我们决定筛选一组源自特定海洋环境（与海草波喜荡草相关）的真菌，以检测其中是否存在真菌病毒：我们的研究结果揭示了这些共生体中丰富的多样性，这种复杂性需要进一步研究以阐明它们在这个生态位中的具体作用。具体而言，我们鉴定出属于九个不同谱系的十二个新病毒物种：它们是巨双分病毒、双链RNA病毒、金黄病毒、多分体病毒以及五个尚未明确的进化枝的成员。我们展示了内源性病毒开放阅读框的证据，以及反转录病毒科逆转录病毒元件双链RNA积累的证据。我们应用了不同技术来检测真菌病毒的存在，包括（i）双链RNA提取和cDNA克隆，（ii）通过NGS技术进行小RNA和总RNA测序，（iii）滚环扩增（RCA）和总DNA提取分析，（iv）病毒纯化和电子显微镜观察。我们还试图严格评估每种技术的内在价值和局限性。基于我们可以直接比较的样本，RNAseq分析在真菌病毒的从头组装方面优于小RNA。据我们所知，这是关于从海洋环境中分离出的真菌病毒组的首次报告。本文报道序列的GenBank/eMBL/DDBJ登录号为：KT601099 - KT601110；KT601114 - KT601120；KT592305；KT950836 - KT950841。
