The Complete Genome Sequence of Citrus Vein Enation Virus (CVEV) Obtained through Deep Sequencing of Small RNAs

Abstract

Citrus vein enation (VE), a graft-transmissible disease naturally spread by several aphid species in a persistent mode, has been reported in many citrus growing areas. It causes vein enations on leaves and woody galls on trunk and branches of sensitive citrus species such as Mexican lime, rough lemon and Citrus volkameriana. The disease is currently diagnosed by biological indexing on sensitive indicator plants, an expensive and time-consuming method. In order to identify its causal agent and develop specific and reliable molecular detection methods, we analyzed small RNAs (sRNAs) from VE-infected Etrog citron plants by deep sequencing using the Illumina Solexa platform. Assembly of VE-associated sRNAs yielded several contigs that showed sequence homology with Pea enation mosaic virus 1 (PEMV-1), the type species of genus Enamovirus, family Luteoviridae. The gaps between adjacent contigs were filled by RT-PCR amplification, cloning and sequencing in order to obtain the complete genome sequence of a new virus, Citrus vein enation virus (CVEV). The CVEV genomic RNA has 5,983 nt organized in five open reading frames, resembling that of PEMV-1. Phylogenetic comparison of amino acid signatures in RNA-dependent RNA polymerases of the family Luteoviridae clearly grouped CVEV with PEMV-1. Therefore, we propose that CVEV should be included in the genus Enamovirus. A rapid and specific detection procedure was developed based on RT-PCR with CVEV-specific primers.