利用小 RNA 深度测序和 PCR 技术对巴西橡胶树的病毒组进行分析，揭示了一种潜在的新病毒的存在。
Virome analyses of Hevea brasiliensis using small RNA deep sequencing and PCR techniques reveal the presence of a potential new virus.
机构信息
Department of Microbiology, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG, 31270-901, Brazil.
Department of Chemistry, Centro Federal de Educação Tecnológica de Minas Gerais (CEFET-MG), Belo Horizonte, MG, 30421-169, Brazil.
出版信息
Virol J. 2018 Nov 26;15(1):184. doi: 10.1186/s12985-018-1095-3.
BACKGROUND
Hevea brasiliensis is an important commercial crop due to the high quality of the latex it produces; however, little is known about viral infections in this plant. The only virus described to infect H. brasiliensis until now is a Carlavirus, which was described more than 30 years ago. Virus-derived small interfering RNA (vsiRNAs) are the product of the plant's antiviral defense triggered by dsRNA viral intermediates generated, during the replication cycle. These vsiRNAs are complementar to viral genomes and have been widely used to identify and characterize viruses in plants.
METHODS
In the present study, we investigated the virome of leaf and sapwood samples from native H. brasiliensis trees collected in two geographic areas in the Brazilian Amazon. Small RNA (sRNA) deep sequencing and bioinformatic tools were used to assembly, identify and characterize viral contigs. Subsequently, PCR amplification techniques were performed to experimentally verify the presence of the viral sequences. Finally, the phylogenetic relationship of the putative new virus with related viral genomes was analyzed.
RESULTS
Our strategy allowed the identification of 32 contigs with high similarity to viral reference genomes, from which 23 exhibited homology to viruses of the Tymoviridae family. The reads showed a predominant size distribution at 21 nt derived from both strands, which was consistent with the vsiRNAs profile. The presence and genome position of the viral contigs were experimentally confirmed using droplet digital PCR amplifications. A 1913 aa long fragment was obtained and used to infer the phylogenetic relationship of the putative new virus, which indicated that it is taxonomically related to the Grapevine fleck virus, genus Maculavirus. The putative new virus was named Hevea brasiliensis virus (HBrV) in reference to its host.
CONCLUSION
The methodological strategy applied here proved to be efficient in detecting and confirming the presence of new viral sequences on a 'very difficult to manage' sample. This is the second time that viral sequences, that could be ascribed as a putative novel virus, associated to the rubber tree has been identified.
背景
巴西橡胶树是一种重要的商业作物，因为它生产的乳胶质量很高；然而，人们对这种植物的病毒感染知之甚少。到目前为止，唯一被描述为感染巴西橡胶树的病毒是一种卡尔病毒，它是在 30 多年前被描述的。病毒衍生的小干扰 RNA（vsiRNA）是植物抗病毒防御的产物，这种防御是由 dsRNA 病毒中间体在复制周期中产生的。这些 vsiRNA 与病毒基因组互补，并已被广泛用于鉴定和描述植物中的病毒。
方法
本研究调查了从巴西亚马逊两个地理区域采集的天然巴西橡胶树叶和边材样本的病毒组。使用小 RNA（sRNA）深度测序和生物信息学工具来组装、鉴定和描述病毒序列。随后，采用 PCR 扩增技术实验验证病毒序列的存在。最后，分析了假定新病毒与相关病毒基因组的系统发育关系。
结果
我们的策略能够识别 32 个与病毒参考基因组高度相似的序列，其中 23 个与 Tymoviridae 科的病毒同源。读取结果显示，21nt 大小的分布占主导地位，这与 vsiRNA 图谱一致。通过液滴数字 PCR 扩增实验证实了病毒序列的存在和基因组位置。获得了一个 1913 个氨基酸长的片段，用于推断假定新病毒的系统发育关系，表明它在分类上与葡萄斑点病毒属的 Maculavirus 相关。为了参考其宿主，将假定的新病毒命名为巴西橡胶树病毒（HBrV）。
结论
应用于此处的方法策略被证明在检测和确认 '非常难以管理' 的样本中新病毒序列的存在方面是有效的。这是第二次在橡胶树上发现与病毒相关的序列，可归为假定的新病毒。
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