Molecular characterization of a closterovirus from carrot and its identification as a German isolate of Carrot yellow leaf virus.

A high-molecular-weight dsRNA (approximately 15 kbp) was isolated from chlorotic leaves of a carrot plant and used for determining the entire nucleotide sequence of a closterovirus. The complete genome of this carrot closterovirus (CCV) was 16.4 kb in length and contained ten open reading frames (ORFs). The genome organization of CCV resembled that of beet yellow stunt virus, but ORF2 and ORF3 were in a reversed order. Based on Hsp70h sequences, CCV is most closely related to carnation necrotic fleck virus and mint virus 1, two viruses of the genus Closterovirus (family Closteroviridae). The major coat protein gene of CCV was expressed in Escherichia coli for raising an antiserum. This permitted routine detection of CYLV by DAS-ELISA and immunoelectron microscopy and was used for demonstrating the bipolar nature of the CCV virion. Moreover, the antiserum gave a Western blot reaction with a reference sample of a Carrot yellow leaf virus (CYLV) isolate from the Netherlands, suggesting that CCV is a German isolate of CYLV.