Discovery and molecular characterization of a new cryptovirus dsRNA genome from Japanese persimmon through conventional cloning and high-throughput sequencing

• DOI: 10.1007/s11262-014-1127-z
• Publication Date: 2014-10-14T14:07:30Z
• Authors: M. Morelli; M. Chiumenti; A. De Stradis; P. La Notte; A. Minafra
• Keywords: Molecular Sequence Data; Sequence Homology; dsRNA; Genome, Viral; Plant Viruses; 03 medical and health sciences; Japan; Cryptovirus, Partitiviridae, Next generation sequencing, RNA-seq, Persimmon; Cluster Analysis; RNA Viruses; Cloning, Molecular; Cryptovirus; Phylogeny; RNA, Double-Stranded; 0303 health sciences; High-Throughput Nucleotide Sequencing; Sequence Analysis, DNA; Diospyros; Cryptovirus, Partitiviridae, Next generation sequencing, RNA-seq, Persimmon; RNA-Dependent RNA Polymerase; 3. Good health; persimmon; Plant Leaves; NGS; RNA, Viral; Capsid Proteins

Through the application of next generation sequencing, in synergy with conventional cloning of DOP-PCR fragments, two double-stranded RNA (dsRNA) molecules of about 1.5 kbp in size were isolated from leaf tissue of a Japanese persimmon (accession SSPI) from Apulia (southern Italy) showing veinlets necrosis. High-throughput sequencing allowed whole genome sequence assembly, yielding a 1,577 and a 1,491 bp contigs identified as dsRNA-1 and dsRNA-2 of a previously undescribed virus, provisionally named as Persimmon cryptic virus (PeCV). In silico analysis showed that both dsRNA fragments were monocistronic and comprised the RNA-dependent RNA polymerase (RdRp) and the capsid protein (CP) genes, respectively. Phylogenetic reconstruction revealed a close relationship of these dsRNAs with those of cryptoviruses described in woody and herbaceous hosts, recently gathered in genus Deltapartitivirus. Virus-specific primers for RT-PCR, designed in the CP cistron, detected viral RNAs also in symptomless persimmon trees sampled from the same geographical area of SSPI, thus proving that PeCV infection may be fairly common and presumably latent.