Identification of a Previously Undescribed Satellite RNA Associated with a Cucumber mosaic virus Subgroup II Strain from Pratia pedunculata in Ohio

Cucumber mosaic virus (CMV) is the type species of the Cucumovirus genus in the Family Bromoviridae. The virus is distributed worldwide, has a very broad host range infecting over 1000 species in more than 85 plant families, and is transmitted by more than 80 aphid species in 30 genera in a non-persistent manner. The viral genome is positive-sense, single-stranded RNA divided among three segments which encode five proteins. RNAs 1 and 2 encode three proteins with methyl transferase/helicase, replicase, and suppression of RNA silencing functions. The movement protein (MP) gene is expressed directly from the 5' half of RNA 3, and the coat protein (CP) gene is expressed from the 3' half via a subgenomic RNA, referred to as RNA 4. CMV is divided into two subgroups based on serological and nucleotide sequence relatedness (3). Some isolates are also reported to harbor small satellite (sat) RNAs ranging in size from 330-405 nucleotides (nt) which are dispensible for CMV replication but rely on CMV for replication and on the CMV-CP for encapsidation. The majority of CMV satRNAs attenuate symptoms induced by the helper virus but pathogenic variants can induce chlorosis or necrosis diseases on tobacco or tomato hosts (4,5,6,7). In the spring of 2012, a landscape sample of Blue Star Creeper (Pratia pedunculata) was submitted to the Ohio Plant Diagnostic Network for a root health issue but it was also showing small, white spots on the leaves. The sample tested positive for CMV and negative for Impatiens necrotic spot virus, Tobacco mosaic virus, and Tomato spotted wilt virus by immunostrips (Agdia Inc., Elkhart, IN). Immunocapture RT using sheep anti-rabbit conjugated magnetic beads incubated with rabbit anti-CMV IgG (Agdia Inc.) followed by PCR with CMV-MP, CP, and satRNA specific primers was done as previously described (2) and all three sets of primers amplified products of expected size (Fig. 1). The amplicons were excised from the gel and cloned into pGEM-T vector as previously described (2). Clones were screened for inserts by PCR using M13 sequencing primers, the plasmid DNA was purified and subsequently sequenced (Plant Microbe Genomics Facility, The Ohio State University), and vector sequences were trimmed from raw sequences (Chromas v. 2.33), assembled, and subjected to pairwise and multiple sequence alignments (Vector NTI Advance 11.5, Invitrogen) as previously described (2).