Targeting highly conserved 3′-untranslated region of pecluviruses for sensitive broad-spectrum detection and quantitation by RT-PCR and assessment of phylogenetic relationships

Abstract

The 3'-end region of many virus isolates has been shown to possess conserved sequences in addition to the presence of numerous genomic and subgenomic RNAs. Utilizing these sequences, a broad-spectrum reverse transcription-polymerase chain reaction protocol has been developed to detect all the known Indian peanut clump virus and Peanut clump virus isolates, that cause peanut clump diseases in West Africa and India. The primers were targeted at the highly conserved 3'-untranslated regions of the PCV RNA-1 and RNA-2. The conservation was confirmed by sequencing these untranslated regions of RNA-1 for six isolates and RNA-2 for one isolate. The conserved structure of the RNA-1 and RNA-2 was observed and the importance of this region for the virus survival was confirmed. The primers were also designed for virus quantitation using a Taqman(®)-based real-time RT-PCR. The use of RT-PCR and real-time quantitative RT-PCR improved the sensitivity of PCV detection compared to ELISA. RT-PCR also led to the detection of IPCV and PCV on two new natural hosts: Oldenlandia aspera and Vigna subterranea. Real-time RT-PCR is considered to be an ideal tool for identifying resistant sources to both IPCV and PCV.

Unusual Features of Pomoviral RNA Movement

“…Similar to PMTV and other viruses of the Virgaviridae (Goodwin and Dreher, 1998; Dreher, 2009; Dieryck et al, 2010), the 3′ extremities of BSBV and BVQ RNAs fold into tRNA-like structures that are highly conserved with only seven nucleotide variations in the terminal 46 nt. On the other hand, poor sequence similarity is observed inter- and even intra-species for the 5′ UTRs (Koenig et al, 1998).…”

Tropical Food Legumes

“…C. amaranticolor is a good diagnostic host for PCV and IPCV. ELISA and nucleic acid hybridization-based tests as well as RT-PCR have been used for the detection of these viruses (Dieryck, Delfosse, Reddy, & Bragard, 2010;Huguenot et al, 1989;Manohar et al, 1995;Reddy et al, 2008). Both PCV and IPCV are soil-borne and transmitted by the plasmodiophorid obligate biotrophic parasite, Polymyxa graminis .…”