First Report and Characterization of Rhynchosia golden mosaic virus in Honduras.

Abstract

Rhynchosia minima was suspected to be a weed host of Bean golden yellow mosaic virus (BGYMV, previously designated Bean golden mosaic virus type II). Leaf tissue that exhibited yellow mosaic foliar symptoms characteristic of a geminivirus infection was collected in the Comayagua Valley in Honduras in July 1999. Extraction of viral DNA from the symptomatic leaves was accomplished with the DNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA). Subsequent viral DNA amplification was accomplished with degenerative primers for the cp gene (AV494/AC1048) (4). The 570-bp fragment was cloned into the pGEM T-Easy vector (Promega Corp., Madison, WI) producing the recombinant plasmid pRhyb5. The viral insert was sequenced, and from this sequence, specific primers (RHc549 and RHv29) were designed to amplify the remaining part of DNA-A. The 2.1-kb-amplified polymerase chain reaction (PCR) fragment was cloned into the pGEM T-Easy vector producing the recombinant plasmid (pRhya-sp), and the viral insert was sequenced. Nucleotide sequence comparison (GAP program, Wisconsin Package Version 10.0, Genetics Computer Group, Madison, WI) of the complete 2,624-bp DNA-A (GenBank accession no. AF239671) to geminiviruses representing the major phylogenetic clusters (1) showed nucleotide identities ranging from 63 to 82%. Sequence comparisons for the common region and rep, trap, ren, and cp genes with the most closely related geminivirus, Pepper hausteco virus (PHV, X70418), gave 76, 82, 79, 81, and 82% nucleotide identities, respectively. There is a direct repeat (TATCGGT) of 7 nt 5' (viral sense polarity) of the conserved TATA box, and this repeat is most analogous to that in PHV (1). Specific primers were designed in the complementary sense (RGBc2414, BGBc2553) from the common region DNA-A sequence and used with a degenerative viral sense primer for the DNA-B (PBC1v2039) (3) to amplify a 647-bp fragment. Sequence comparison for the common region (134 nt from the rep gene start codon toward the 3' end) from the DNA-B sequence had 88% nt identity to the DNA-A sequence, thus indicating that this geminivirus is bipartite. These sequence analyses indicated that this geminivirus isolated from R. minima is distinct from previously described geminiviruses, and we propose the name Rhynchosia golden mosaic virus (RGMV). From rep gene sequence alignments, RGMV has an apparent genome recombination between Old and New World geminiviruses (Tomato yellow leaf curl virus and Bean dwarf mosaic virus) as previously noted for PHV (2). Our results indicate that RGMV is a distinct geminivirus from BGYMV, and, thus, additional studies are needed to establish the importance of R. minima as a reservoir for vegetable-infecting geminiviruses. This study is the first report of another virus in the PHV phylogenetic cluster and is thus of importance in the understanding of recombinant viruses and their phylogenetic relationship to other characterized geminiviruses.