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Arch Virol (2012) 157:383385 DOI 10.1007/s00705-011-1168-8
ANNOTATED SEQUENCE RECORD
Complete sequence of a cryptic virus from hemp (Cannabis sativa)
Angelika Ziegler Jaroslav Matouek
Gerhard Steger Jrg Schubert
Received: 9 August 2011 / Accepted: 29 October 2011 / Published online: 11 November 2011 Springer-Verlag 2011
Abstract Hemp (Cannabis sativa) was found to be a useful propagation host for hop latent virus, a carlavirus. However, when virus preparations were analysed by electron microscopy, along with the expected lamentous particles, spherical particles with a diameter of around 34 nm were found. RNA from virus preparations was puried, and cDNA was prepared and cloned. Sequence information was used to search databases, and the greatest similarity was found with Primula malacoides virus 1, a putative new member of the genus Partitivirus. The full sequences of RNA 1 and RNA 2 of this new hemp cryptic virus were obtained.
During purication of virus particles of hop latent virus (HpLV) from Cannabis sativa (variety Fedora 17) a second, spherical virus was puried along with the expected lamentous virus particles (Fig. 1a). The spherical particles had a diameter of about 34 nm, and were not seen in
preparations of HpLV originating from hop. In order to determine the nature of this virus, cDNA was prepared using RNA from the mixed virion preparations, random primers, and the Universal RiboClone cDNA Synthesis System (Promega). The resulting cDNA was ligated into the pJET1.2/blunt cloning vector (Fermentas), and plasmid DNA was prepared for sequencing. Double-stranded (ds) RNA from hemp leaf tissue was prepared using two cycles of cellulose chromatography [12]. Agarose gel electrophoresis resulted in a single dsRNA band of around2.3 kbp. Cloning, the RACE procedure and sequencing, however, subsequently revealed two different sequences of a similar size. After initial sequencing (Beckman) and comparisons [BLAST; 13] with known sequences in databases, a high similarity to Primula malacoides virus 1(PmV1) [9] was found. The sequence information was used to design specic primers, and 50-and 30RACE (SMARTer RACE cDNA Amplication Kit, Clontech) was employed to determine the full sequence of the two viral RNAs. The sequences were assembled using BIOEDIT [8].
The genome of the...