1.11.20
SEQUENCE ANALYSIS OF BROAD BEAN WILT FABAVIRUSES

Y KOBAYASHI1, M NAKANO2 and Y HONDA1

1National Agriculture Research Centre, Tsukuba, 305-8666, Japan; 2Japan International Research Centre for Agricultural Sciences, Tsukuba, 305-0851, Japan

Background and objectives
Broad bean wilt virus (BBWV), the type member of the genus Fabavirus, has two positive-sense, single-stranded RNAs. BBWV has been isolated from a wide range of plants worldwide, and the isolates are divided into two serotypes I and II [1]. So far, no complete sequence data have been published for BBWV. In this study, we cloned RNA-1 and RNA-2 of one Japanese isolate of BBWV (BBWV-IP), and determined their complete nucleotide sequences. We also sequenced the coat protein gene and 3' non-translated region in RNA-2 of five other isolates of BBWV for comparison.

Materials and methods
Four isolates of BBWV, IP, E, L and 1-2, were obtained from Capsicum annuum, Pisum sativum, Eustoma russellianum and Gentiana scabra, respectively, collected at different sites in Japan. These isolates and two other isolates from ATCC, PV132 and PV176 (both serotype I), were propagated in Chenopodium quinoa, and partially purified. The viral RNAs were extracted using guanidine thiocyanate and purified with oligo(dT)cellulose. cDNA clones covering parts of BBWV-IP RNAs were obtained by cDNA synthesis using oligo(dT) or specific primers and by the 5' RACE method, and sequenced. The sequence data were compared with those of another BBWV isolate PV131 (serotype II) and Comoviruses in the EMBL database. Based on the conserved sequence among these viruses, a degenerate primer was designed and used to amplify the 3' region in RNA-2 of five other BBWV isolates by PCR.

Results and conclusions
The complete nucleotide sequences of RNA-1 (6kb) and RNA-2 (3.5 kb) of BBWV-IP were determined. They share no extensive sequence homology, except for a conserved sequence of 50 nucleotides at most 5'-ends. Both RNA-1 and RNA-2 encode single large polyproteins (210 and 120 kDa, respectively) in which functional proteins are arranged in a manner similar to Comovirus RNAs. The coat protein gene and 3' non-translated region in RNA-2 of BBWV isolates, E, L, 1-2, PV132 and PV176, were sequenced and compared with those of IP and PV131. Four Japanese isolates, IP, E, L and 1-2 have high sequence homology to one another (77-94 and 89-98% at the nucleotide and amino acid levels, respectively). They have higher sequence homology to PV131 (77-87 and 90-96%) than to PV132 and PV176 (61-64 and 62-63%). These results suggest that the four Japanese BBWV isolates all belong to serotype II.

References
1. Uyemoto JK, Provvidenti R, 1974. Phytopathology 64, 1547-1548.