Development of a polyclonal antibody against the coat protein of Prunus necrotic ring spot virus

The Prunus necrotic ring spot virus (PNRSV) poses a significant threat to the global stone fruit industry. In this study, samples suspected of PNRSV infection were gathered from Iran’s primary stone fruit-growing areas. The coat protein (CP) gene of the isolates was amplified, and the nucleic acid and amino acid sequences of the PNRSVs were determined. Phylogenetic analysis revealed that these isolates belonged to the PV-96-II, a prominent group of PNRSVs found worldwide. An isolate from this clade was chosen to express the CP gene in Escherichia coli. The CP gene of this isolate was cloned into the pET28 vector for expression, and the resulting coat protein was purified as a native protein using Ni-NTA agarose. The purified protein served as a recombinant antigen to generate anti-PNRSV-CP antiserum in rabbits. Purified anti-PNRSV-CP IgG and conjugated IgG showed specificity and sensitivity, successfully detecting expressed CP and PNRSV isolates in infected stone fruit trees through various serological and sero-molecular techniques such as plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), Double Antibody Sandwich-ELISA (DAS-ELISA), and western blotting. These antibodies can be valuable in virus and plant screening studies, as well as serological and sero-molecular tests. This study represents the first documentation of a polyclonal antibody preparation against the CP of an Iranian PNRSV isolate, offering potential assistance in controlling and preventing this economically significant virus in the future.