Serological detection of Grapevine rupestris stem pitting-associated virus (GRSPaV) by a polyclonal antiserum to recombinant virus coat protein

The coat protein gene of Grapevine rupestris stem pitting-associated virus (GRSPaV) was amplified with primers based on the completely sequenced Californian GRSPaV isolate, The protein expressed in Escherichia coli was used to raise an antiserum in rabbit. This antiserum was successfully used to detect virus coat protein in infected grapevine extracts either spotted on polyvinyl difluoride membranes (dot immunobinding) or blotted on membranes after gel separation (Western blot). The antiserum titre was 1:5,000 in Western blot. GRSPaV was detected in leaf petioles and cortical scrapings from dormant canes during the whole vegetative season. Several accessions of Vitis rupestris, currently used as presumptive virus-free indicators of Rupestris stem pitting, were found to be infected by this virus. While the application of the antiserum in ELISA was ineffective, the availability of similarly simple and effective serological tools, such as dot immunobinding, may allow a wide survey for GRSPaV.