DIFFERENTIATION BETWEEN CUCURBIT YELLOW STUNTING DISORDER VIRUS (CYSDV) AND BEET PSEUDO-YELLOWS VIRUS BY RT-PCR AND ALIGNMENT OF THE CYSDV HSP70 HOMOLOGUE GENE WITH SIMILAR GENES FROM OTHER CLOSTEROVIRUSES
IC LIVIERATOS and RHA COUTTS
Biology Department, Imperial College of Science, Technology and Medicine, Prince Consort Road, London SW7 2BB, UK
Background and objectives
Beet pseudo-yellows virus (BPYV)  and cucurbit yellow stunting disorder virus (CYSDV)  are two partially characterized closteroviruses transmitted by Trialeurodes vaporariorum and Bemisia tabaci whiteflies, respectively. BPYV and CYSDV elicit identical yellowing symptoms in several common cucurbit hosts in the Mediterranean area. In an attempt to differentiate between these viruses, reverse transcriptase polymerase chain reaction (RT-PCR) and Northern hybridisation analysis were developed. In order to obtain the complete CYSDV HSP70 homologue gene, 5' and 3' RACE-PCR protocols were used. Computer-assisted comparison of the deduced amino acid sequence of the complete CYSDV HSP70 homologue with similar genes of other closteroviruses was carried out.
Materials and methods
BPYV-infected Greek cucumbers and CYSDV-infected Spanish melons were used in this study. Freeze-dried sample of BPYV-infected Nicotiana clevelandii plants were kindly supplied by Dr G. Wisler (California, USA). Using previously determined sequence information for fragments of the HSP70 homologue genes of both BPYV  and CYSDV , oligonucleotide primers were designed to be used in RT-PCR assays. RT-PCR amplified products for each virus were subsequently cloned, sequenced and used as probes in Northern analysis of dsRNA species extracted from BPYV-infected cucumbers and CYSDV-infected melons. 5' and 3' RACE-PCR protocols were applied in order to obtain the 5' and 3' termini of the CYSDV HSP70 homologue gene. Sequences were analysed using the University of Wisconsin BLAST and PileUp programs.
Results and conclusions
RT-PCR assays specifically detected the presence of BPYV and CYSDV in extracts of infected plants and specifically hybridised with the homologous dsRNA (ca 9 kbp in size for both viruses) when used as probes in Northern hybridization analysis. Minor bands of approximately 5 kbp for both viruses and one band of 3 kbp for CYSDV were also identified and might represent defective dsRNA species. The products generated by 5' and 3' RACE-PCR were cloned and sequenced, providing sequence information concerning the 5' and 3' termini of the CYSDV HSP70 homologue gene. Computer-assisted analysis of the deduced amino acid sequence of the complete HSP70 homolog of CYSDV with similar homologues of BYV, BYSV, CTV, LCHV and LIYV, and the incomplete HSP70 homologue of BPYV, showed that CYSDV is as closely related to LIYV as to BPYV whitefly-transmitted closteroviruses.
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