ARS | Publication request: Quantification of Maize Fine Streak Virus Genomic and Positive-sense RNAs in Infected Maize Reveals High Level Accumulation of ORF 3 and 4 MFSV Transcripts

Submitted to: American Society for Virology Meeting
Publication Type: Abstract Only
Publication Acceptance Date: February 2, 2010
Publication Date: July 17, 2010
Citation: Willis, D.K., German, T.L. 2010. Quantification of Maize Fine Streak Virus Genomic and Positive-sense RNAs in Infected Maize Reveals High Level Accumulation of ORF 3 and 4 MFSV Transcripts [abstract]. American Society for Virology Meeting.

Technical Abstract: Quantification of Maize fine streak virus genomic and positive-sense RNAs in infected maize reveals high level accumulation of ORF 3 and 4 MFSV transcripts. We improved methods to analyze RNA produced by Maize fine streak virus (MVSF) within infected maize tissue using real-time RT-qPCR. We designed and tested efficient primers corresponding to each of the seven MFSV ORFs. Tagged strand-specific primers and increased cDNA reaction temperature allowed the synthesis of specific cDNA products corresponding to the MFSV virion RNA (vRNA) or virion-complementary RNA (vcRNA) species. These conditions significantly reduced auto-priming and non-specific priming during the synthesis of strand-specific cDNA. Using this methodology, we established that MFSV vRNA is approximately 50-fold more abundant than the replicative vcRNA in symptomatic maize leaf tissue. The abundance of the N, P, M, G and L gene transcripts of MFSV decreased with distance from the 3´ promoter suggesting polar, discontinuous transcription in infected maize similar to transcription in the well studied rhabdovirus Vesticular stomatitis virus. However, the plant specific ORF3 and ORF4 transcripts accumulated to higher (ORF3) or equivalent (ORF4) levels to that of the N mRNA suggesting that these transcripts are produced by an alternate transcription process or exhibit increased stability relative the remaining MSFV transcripts.