1.11.43
IDENTIFICATION AND ANALYSIS OF FOUR FURTHER COMPONENTS OF THE FABA BEAN NECROTIC YELLOWS VIRUS GENOME

L KATUL1, T TIMCHENKO2, B GRONENBORN2 and HJ VETTEN1

1Biologische Bundesanstalt fur Land- und Forstwirtschaft, lnstitut fur Biochemie und Pflanzenvirologie, Messeweg 11-12, 38104 Braunschweig, Germany; 2lnstitut des Sciences Vegetales, CNRS, 91 198 Gif-sur-Yvette, France

Background and objectives
Faba bean necrotic yellows virus (FBNYV) is associated with virus diseases affecting several food and fodder legumes. Available information suggests that its geographical distribution is confined to west Asian and north African countries, in some of which it occurs at a high incidence and causes destructive diseases. FBNYV is persistently transmitted by various aphid species of which Aphis craceivora appears to be the most significant natural vector. It has unusually small isometric particles (18 nm in diameter) containing a capsid protein of about 20 kDa and a genome consisting of several circular ssDNA components each of about 1 kb. In vector transmission and particle properties as well as in genome composition and organisation, FBNYV resembles banana bunchy top virus (BBTV), milk vetch dwarf virus (MDV) and subterranean clover stunt virus (SCSV) all of which, thus, form a new taxonomic group of ssDNA viruses of plants [1]. A recent comparison of six components of the FBNYV genome with the genome components of its relatives suggested that at least two components of the FBNYV genome have not yet been determined [1]. This prompted us to identify further genome components of FBNYV in the original cDNA library.

Materials and methods
Partially sequenced DNA fragments distinct from any of the six previously described components (Cl - C6) were identified in the original cDNA library of a Syrian isolate of FBNYV and served for the design of PCR primers. Missing segments of the circular components were amplified by PCR using ssDNA of the Syrian isolate as template, cloned into an EcoRV-linearised and T-tailed pBluescript SK- vector, and used for transformation of Escherichia coli NM522 and for subsequent sequencing reactions. Sequences were assembled and analysed using the GCG programme package.

Results and conclusions
In addition to the six genome components identified earlier [1], four further components (C7-C10) have now been determined and analysed. All 10 components are about 1 kb in size, contain one major ORF in the virion sense, and have a non-coding region containing a highly conserved sequence possibly forming a stem-loop structure. Similar to Cl and C2, C7 and C9 each potentially code for replicase-associated proteins (Rep) whose molecular masses were estimated to be 33.2 and 32.7 kDa, respectively. The significance of the occurrence of four Rep components in the FBNYV genome is not yet understood. The presence of a common stem-loop region shared only by the Rep component C2 and all six non-Rep components and preliminary results from replication assays suggest that C2 codes for a Rep indispensable for FBNYV replication. The other two components, C8 and C10, are clearly distinct from any of the previously identified components. They encode proteins of 17.4 and 19.7 kDa, respectively, and, based on amino acid sequence homology, have counterparts in the BBTV and SCSV genomes. The genome of the Syrian isolate of FBNYV, thus, comprises four Rep and six non-Rep components. Of the six non-Rep components, C3 is not homologous to any of the BBTV components and C6 has no counterpart in the BBTV and SCSV genomes. Amino acid sequence comparisons of the major proteins encoded by all components of FBNYV, MDV, SCSV, and BBTV are presented. These confirm earlier observations on the degree of serological relatedness among these four viruses [2], namely that FBNYV is closely related to MDV, distantly related to SCSV, and least related to BBTV.

References
1. Katul L, Maiss E, Morozov SY, Vetten HJ, 1997. Virology 233, 247-259.
2. Katul L, Vetten HJ, Maiss E, Makkouk KM, Lesemann D-E, Casper R, 1993. Annals of Applied Biology 123, 629-647.