Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: May 18, 2005
Publication Date: October 1, 2005
Citation: Reed, S.E., Tsai, C., Willie, K.J., Redinbaugh, M.G., Hogenhout, S.A. 2005. Shotgun Sequencing of the Negative-Sense RNA Genome of the Rhabdovirus, Maize Mosaic Virus. Journal of Virological Methods. 129(1):91-96. Interpretive Summary: Knowledge of the genome structure and sequence for plant pathogens is critical to understanding pathogenesis and developing advance approaches for disease control. Although high throughput approaches are commonly used for sequencing bacterial and fungal genomes, viral genomes have usually been sequenced using the more traditional approach of sequencing large insert cDNA clones carrying virus sequences. In this study, ~95% of the genomic sequence for Maize mosaic virus was determined using a 'shotgun' sequencing approach in which 94 clones from a viral cDNA library were sequence. The cost for sequencing was about $0.30 per base, representing a significant savings in both time and cost relative to the more traditional approach. The shotgun approach used to determine the genomic sequence of Maize mosaic virus may be used by researchers to determine genomic sequence for plant viruses, saving time and money for publicly and privately funded research programs. It will add to our ability to take a genomic approach to the study of viral genomes. For example, this approach would greatly facilitate projects compare the sequences for all known viruses in a specific virus family. The research has the potential for moderate to high impact in the scientific community.
Technical Abstract: The maize-infecting nucleorhabdovirus, Maize mosaic virus (MMV), was sequenced to near completion using the random shotgun approach. After compiling sequences of 94 randomly primed cDNA clones from a library constructed using viral RNA, a single 12,000 nt contig containing six open reading frames was obtained. The gene junction sequences upstream of the ORFs were conserved and the consensus sequence was similar to that of other plant and animal rhabdoviruses. Nucleotide sequences of open reading frames were compared to protein sequences using the BlastX search in the NCBI website. Significant similarities were found between the deduced protein sequences of cognate MMV genes and the nucleocapsid protein, glycoprotein and polymerase protein sequences of other rhabdoviruses. A gene order of 3'-N-P-ORF3-M-G-L-5' was obtained by comparison with the genomes of sequenced rhabdoviruses. This genome organization is most similar to Sonchus yellow net virus and Lettuce necrotic yellows virus. The costs of MMV shotgun sequencing were calculated to be 30 cents per base pair and it took 90 hours to complete the project, making it an affordable and quick method for sequencing a plant rhabdoviruses.