1.11.81
THE GENOME STRUCTURE OF ORCHID FLECK VIRUS SHOWS TO BE A UNIQUE BIPARTITE GENOME VIRUS THAT RESEMBLES RHABDOVIRUSES

H KONDO1, T MAEDA1, T TAMADA1 and Y SHIRAKO2

1Research Institute for Bioresources, Okayama University, Kurashiki 710-0046, Japan; 2Asian Center for Bioresources and Environmental Sciences, University of Tokyo, 113-0032 Japan

Background and objectives
Orchid fleck virus (OFV) is an economically important virus of orchids. It has been reported in Japan [1], and also occurs in Australia, Brazil, Germany, Korea and USA. It is sap-transmissible to several species of orchids and a few dicotyledonous plants. Partially purified virions are non-enveloped and bacilliform, 150 nm long and 40 nm in diameter. In cells of infected orchids, virus particles are visible in both nuclei and cytoplasm; enveloped virions are occasionally seen in the cytoplasm. OFV was classified tentatively in the family Rhabdoviridae in the 6th ICTV Report, although no details of its genome were available. We have determined the complete nucleotide sequence of the OFV genome and compared it with those of other plant and vertebrate rhabdoviruses.

Materials and methods
OFV (isolate So) was originally obtained from Cymbidium sp. [2] and was propagated in Tetragonia expansa leaves by mechanical inoculation. RNA was extracted from purified virus particles and used as a template for CDNA synthesis using random priming. The CDNA was cloned into pGEM3Z. The 3' and 5' terminal regions were cloned by RACE. Nucleotide sequences were determined by dideoxynucleotide chain termination. Total RNA extracted from OFV-infected leaves was used for the analysis of transcribed MRNA by RT-PCR.

Results and conclusions
OFV genome consists of two single-stranded RNA molecules (RNA 1 and RNA 2). The 3'and 5' terminal sequences of the two RNAs are conserved and complementary. RNA 1 has 6413 nucleotides and includes five open reading frames (ORFs) which encode 49-, 26-, 42-, 20- and 62-kDa proteins. RNA 2 has 6001 nucleotides and includes a single long ORF which encodes a 212-kDa protein. Amino acid sequence comparisons indicate that the 212-kDa protein has similarities with the L (polymerase) proteins of sonchus yellow net virus (SYNV; genus Nucleorhabdvirus) and of some rhabdoviruses of vertebrates. The 49- and 60-kDa proteins coded by RNA 1 have slight sequence similarities to the nucleoprotein (N) and glycoprotein (G) of SYNV, respectively. The 3' end sequences of the six MRNA species corresponding to each ORF were detected in total RNA extracted from infected leaves. The potential transcription termination and polyadenylation sites of each MRNA have highly conserved consensus sequences. These results indicate that OFV is similar to rhabdoviruses but that it is unique in having a bipartite genome.

References
1. Doi Y, 1977. CMI/AAB Descriptions of Plant Viruses: No. 183.
2. Kondo H, Matsumoto J. Maeda T, Inouye N, 1995. Bulletin of the Research Institute for Bioresources, Okayama University 1, 21-34.