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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Molecular Plant Pathology Laboratory » Research » Publications at this Location » Publication #314411
Title: Complete genome sequence of the alfalfa latent virus

Author
item Nemchinov, Lev
item Shao, Jonathan
item POSTNIKOVA, OLGA - Russian Academy Of Sciences

Submitted to: Genome Announcements
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/4/2015
Publication Date: 4/16/2015
Citation: Nemchinov, L.G., Shao, J.Y., Postnikova, O.A. 2015. Complete genome sequence of the alfalfa latent virus. Genome Announcements. 3(2):e00250-15.

Interpretive Summary: Complete sequencing of a pathogen’s genome provides important information on the biology of the pathogen, its interaction with the host plant and measures that could be taken to protect crops from the pathogen. When sequences of the pathogen are available, they can be used for the pathogen’s detection, accurate identification and development of novel control strategies. Alfalfa latent virus (ALV) is a strain of Pea streak virus that affects pea, bean and many other plants and is known for more than 60 years. The virus is spread in the USA, Canada and in the Eurasian region. As of today, no complete genomic sequence of PSV or ALV is available. In this work, researchers obtained for the first time a complete nucleotide sequence of the ALV and determined genome structure of the virus. Results of this study will be of interest to the researchers in academia and government organizations working in plant virology field, as well as the scientists working alfalfa.

Technical Abstract: Alfalfa latent virus (ALV) is a member of the carlavirus group and occurs symptomlessly in alfalfa (Medicago sativa). In the US it is prevalent in Nebraska and Wisconsin. The virus is recognized as a strain of Pea streak virus (PeSV) So far, no complete genomic sequence of PSV or ALV is available. ALV-infected tissues were obtained from ATCC (PV-264 isolate from Lancaster County, NE). Total RNA was extracted with the TRIzol protocol (Life Technologies) and used in primer walking employing SuperScript RT-PCR for long templates (Life Technologies). The 5’ end of the genome was amplified with the RACE system (Life Technologies). PCR fragments were sequenced, assembled into contigs and the complete genome consensus sequence was generated. Independently, total RNA was purified with the RNAeasy Mini kit (Qiagen) and used as a template for Illumina RNA sequencing. Paired-end libraries (470 million reads) were cleaned by removing host plant DNA. Using Bowtie2, the reads were mapped to the Medicago truncatula genome (Mt4.0v1), mitochondrial DNA and plastid DNA. The 200 million reads that did not map were assembled with Trinity into 73,574 contigs, which were queried against known plant viruses using BLASTn. A single contig was selected that had a negative E-value with other plant viruses. It was 99.8% identical to the PCR-derived ALV sequence. The 8,041 nt-long consensus sequence was produced and used for further analysis. The ALV genome contains five tentative ORFs encoding a viral replicase, triple gene block proteins and capsid protein. The ALV does not appear to encode a clear 3’ proximal nucleotide acid-binding protein (NABP) typical for carlaviruses. We propose that the sequenced ALV isolate from Nebraska differs substantially from the Czech ALV isolate and PeSV isolate from Wisconsin. A lack of clear NABP indicates that ALV is divergent from other members of the Genus Carlavirus. The sequence has been deposited in Genbank as accession no. KP784454.