Identification and Molecular Characterization of a Potyvirus Isolated from Native Larkspur (Delphinium glaucum) in Alaska.

Wild larkspur, Delphinium glaucum S. Watson, grows throughout most of Alaska along roadsides and in forests and is planted as an ornamental. Leaves containing distinct vein-clearing and chlorotic mosaic symptoms were first noticed on several D. glaucum plants during 2000 at the Georgeson Botanical Garden in Fairbanks, AK. Although affected plants continued to produce normal flowers, by 2008, the plants developed overall stunting. Initially, virus presence was determined by a general differential centrifugation extraction and concentration protocol followed by examination of the partially purified virus and leaf sap by electron microscopy. Filamentous particles approximately 725 nm long were observed. Virion protein extractions analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a putative coat protein (CP) of ~35 kDa. Potyvirus identity (family Potyviridae) was confirmed with universal potyvirus antiserum in western blots and ELISA assays (Agdia, Inc., Elkhart, IN). Exotic larkspur plants, D. elatum L., growing next to diseased D. glaucum plants, did not exhibit symptoms nor were they positive for potyvirus when tested serologically as described previously. Total RNA was extracted from potyvirus-infected leaves and used in reverse transcriptase-PCR assays that specifically targeted potyviruses (2,4) to generate genomic segments for identification and sequence analysis. Fragments representing portions of the helper component protease gene, HC-Pro (~700 bp), the cylindrical inclusion gene, CI (~700 bp), and the 3'-end (~1.7 kbp) were purified, cloned, sequenced, and deposited in GenBank (Accession Nos. FJ349329, FJ349328, and FJ349327, respectively). The sequenced 3'-end (1,674 nt) revealed a partial nuclear inclusion protein gene, NIb (1 to 630 nt), a CP gene (631 to 1,443 nt), and a 3'-untranslated region (1,447 to 1,674 nt) attached to a poly (A) tail. Blast searches in GenBank for percent identities of the nucleotide and amino acid comparisons resulted in highest similarities in conserved regions among members in the genus Potyvirus. For example, the highest CI, CP, and HP amino acid identities (0 gaps) were 67% with Potato virus A (Accession No. AF543709), 74% with Araujia mosaic virus (Accession No. EF710625), and 65% with Potato virus A (Accession No. AJ131403), respectively. However, none of the identities were sufficient for inclusion with an existing potyvirus species, whereby the CP amino acid sequence identity must be at least 80% (1). Mechanical transmission of purified virus to Chenopodium amaranticolor, C. quinoa, D. elatum, D. glaucum, and Nicotiana benthamiana seedlings was unsuccessful. We conclude that the isolated virus is a new species in the genus Potyvirus and propose the name Delphinium vein-clearing virus (DeVCV). To our knowledge, this is the first report of a virus isolated from D. glaucum and is representative of the growing number of viruses found in native plants (3). The distribution of DeVCV-infected larkspur is not known in managed or natural ecosystems. Identification of new viruses from native plants is important, in that, the host plant may act as a virus reservoir for transmission to other ornamental and crop plants. References: (1) P. H. Berger et al. Family Potyviridae. Page 819 in: Virus Taxonomy-8th Report of the ICTV. C. M. Fauquet et al., eds. Elsevier Academic Press, San Diego, CA, 2005. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) I. Cooper and A. C. Jones. Adv. Virus Res. 67:1, 2006. (4) C. Ha et al. Arch. Virol. 153:25, 2008.