ARS | Publication request: Biologically Active Transcripts of Oat Blue Dwarf Virus (OBDV) – the First Infectious Clone of a Marafivirus

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 19, 2009
Publication Date: June 1, 2009
Citation: Edwards, M.C., Weiland, J.J. 2009. Biologically Active Transcripts of Oat Blue Dwarf Virus (OBDV) – the First Infectious Clone of a Marafivirus. Meeting Abstract. Phytopathology 99:S31.

Technical Abstract: Marafiviruses are a small group of phloem-limited, leafhopper-borne viruses closely related to those in the Tymovirus genus. We now report the development of infectious cDNA clones of OBDV – the first such clones of any marafivirus. Prior to clone construction, the reported sequence of the 5’ and 3’ ends was confirmed using 5’ RACE, primer extension, and ligation-anchored PCR. Primers designed to published terminal sequences and internal regions were used to generate 5’ and 3’ amplicons subsequently fused into full-length clones. The 5’ primer incorporated the T7 RNA polymerase promoter sequence to facilitate production of RNA transcripts. The 3’ primer incorporated 28 adenosine residues and an Spe I restriction site to allow clone linearization prior to transcription. Using vascular puncture of maize seeds with capped transcripts, multiple clones were shown to be infectious at an average rate of 24.3% (range 14-36%), as determined by ELISA. Proteins and RNAs consistent in size with those expected in OBDV infection were detected in young leaves via western and northern blotting, respectively. Aster leafhoppers successfully transmitted OBDV to oats and barley after feeding on detached, infected maize leaves. Infectious clones of these viruses will be valuable in further studies of marafivirus functional genomics, the interaction of the virus with its insect and plant hosts, and the relationships between marafiviruses and others within the family Tymoviridae.