The NSs gene of GBNV-To (K) was amplified by RT-PCR using partially purified viral RNA as template and specific sense and antisense primers, designed on the basis of the genomic sequence of S RNA of GBNV (accession number U27809, table 1). The PCR product was digested with the restriction enzymes NheI and XhoI (purchased from MBI Fermantas) and ligated into the corresponding sites of plasmid pRSET-C (purchased from Invitrogen). The plasmid was named pRSETC-NSs and propagated in E. coli. The NSs coding sequence was confirmed by DNA sequencing. The recombinant NSs clone was transformed into C43 (DE3) E.coli cells and over-expressed by induction with 0.3 mM IPTG (purchased from Sigma) for 3 hours at 37uC. The cell pellet was resuspended in buffer A (20 mM Tris-HCl pH 8.0 and 300 mM NaCl), sonicated and centrifuged at 12000 rpm for 15 min. The supernatant and pellet fractions were analyzed by 12% SDS-PAGE. The rNSs protein was purified from the soluble fraction.