| Viruses are widespread by heterotrophic parasitic on nearly all creatures, some of which can cause a lot of harm and even death. Traditional methods of virus identification including filteration, electron microscopy, serology, cell culture and so on that are all culture-dependent or sequence-dependent, which made it’s impossible to identify a novel virus by the traditional methods. Therefore, an emerging improved pathogen detection method that’s named viral metagenomics was produced.The widely used viral metagenomics is a cluture-independent and sequence-independent approach that can be defined as the characterization of virus genetic information directly from samples. Nowadays, there are a lot of methods can be used to identify virus directly from samples based on viral metagenomics, which include the analysis of small RNAs, total RNA/DNA, viral RNA/DNA and so on. In this article, we made an analysis of the small RNAs in the samples of diseased areca palm and tobacco. As a result, two novel closteroviruses were obtained. Besides, we also made an analysis of the feces sample of diarrhea infants based on viral metagenomics.11,246contigs were obtained by assembling of46,613,994reads after sequencing of the small RNA library that constructed from diseased areca palm. And26from all the contigs were found to have distant similarities with viruses, including21contigs that have distant similarities with members of Clostervirade family. So we used little cherry virus1as reference and identified a new virus named APV1through RT-PCR,5’-RACE-PCR、3’-RACE-PCR as well as Sanger sequencing. APV1that’s belonging to genus velarivirus consists of16,080nucleotides and possesses11ORFs. APV1is different with other genus Velarivirus with the reason that ORF10is unique to APV1, and both ORF1a and ORF4have two internal repeats as well as two transmembranes in ORF1a. This identification of APV1will serve as a foundation for further study of the relationship between APV1and the areca palm yellow disease.A novel closterovirus named TV1-AH was identified in tobacco with symptoms of mosaic and yellowing based on viral metagenomics. TV1-AH consists of15,362nucleotides, and possesses a185nts5’-UTR, a330nts3’-UTR as well as9ORFs. These ORFs includes the virus replication associated ORF1, virus assembly and cell-to-cell movement associated ORFs2-6, a RNA silencing repressor ORF7, and an unknown function ORF8. All of these ORFs products share highest amino acid identity with the proteins from mint virus1(49.74%,83.44%,58.73%,65.62%,56.52%,65.74%,49.48%,32.48%,40.88%), and also shares different similarities with other closteroviruses. What’s important, TV1-AH is the first closterovirus that has been identified in tobacco and this study will provide insights into the futher research.In addition,2,806,916reads were obtained after preprocess of the Ion torrent sequencing data from RNA-seq library that constructed from the feces of diarrhea infants. And63,448seqs were produced through clustering and assembly, from which2,911were assigned to the class of virus including29seqs that were classified to rotavirus. Then, we made a blat search through all the2,806,916reads based on the29seqs that were classified to rotavirus. As a result,49seqs were associated with rotavirus after filter, and29of which were mapped to rotavirus A1. So rotavirus A1can be used as a reference to get the new virus genome. All of these will serve as a foundation for further research to study the relationship between the new virus and the infantile diarrhea. What’s more, we have integrated all the analysis process, which will provide guidance for identifying viruses in metagenomics. |
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