In Vitro expression of NSs protein of Melon yellow spot virus infecting melon in Thailand and serological activity of NSs antibody in virus diagnosis

Melon yellow spot virus (MYSV) is one of the most devastating plant viruses in the genus Tospovirus and adversely affects productivity, and quality of cucumber and melon. The locally made polyclonal antibody against nucleocapsid (N) protein of Thai isolate of MYSV has been previously used for the diagnosis of tospovirus species in serogroup IV as well as MYSV, therefore, causing problems for specific MYSV diagnosis. In this research, polyclonal antibody against MYSV- non structural (NSs) protein was produced and evaluated for use in MYSV diagnosis. The N and NSs genes were directly amplified by RT-PCR from MYSV-infecting melon and cloned. Analysis of N gene indicated MYSV infection in diseased melon (designated as isolate MYSV-SUT). The cloned NSs gene of MYSV-SUT is composed of 1,410 nucleotides which encoded for 470 amino acid residues. In vitro protein expression of NSs gene inserted in pQE80L expression vector yielded a recombinant 6xHis-NSs polypeptide. The rabbit polyclonal antibodies against the expressed 6xHis-NSs protein (PAb-MYSV-NSs) had titer up to 1:12,800 when tested with 3 mu g/ml of the 6xHis-NSs protein by DAC-ELISA. This antibody reacted positively to crude sap from MYSV-infected melon and cucumber leaf tissue by nitrocellulose ELISA (NCM-ELISA), and not to crude sap of symptomatic plants infected with Watermelon silver mottle virus (WSMoV), Capsicum chlorosis virus (CaCV) and Tomato necrotic ring spot virus (TNRV) isolates from Thailand. However, crude sap of MYSV-infected melon from fields did not react with PAb-MYSV-NSs in the DAC-ELISA conditions used in this study.