Immunodiagnostics for Cherry virus A and Cherry necrotic rusty mottle virus

Abstract

Cherry necrotic rusty mottle virus (CNRMV) and Cherry virus A (CVA) are important graft transmitted viruses in the family Betaflexiviridae, infecting cherry. It is believed that CVA causes severe symptoms and disease in combination with other stone fruit viruses in both cherry and non-cherry hosts while CNRMV infected tree show reduced growth, significant yield loss and early death. Detection of these viruses is crucial for their sanitation, indexing and certificate programmes. In this study, a polyclonal antibody was produced against recombinant coat protein (CP) of CVA and CNRMV expressed in Escherichia coli for the development serological based diagnostics. The coat protein gene of CVA and CNRMV was amplified by reverse transcription-polymerase chain reaction (RT-PCR) using specifically designed primer pair with restriction sites at both ends and further cloned and expressed in bacterial expression vector (pET32a). CP coding region of both the viruses was expressed separately and successfully as a fusion protein. The fusion proteins were purified and directly used for rabbit immunizations. Antibodies were purified, conjugated with alkaline phosphatase and used in DAS-ELISA. A total of 74 and 43 samples were checked for CVA and CNRMV, respectively. In this analysis, 40/74 samples were found positive for CVA and 20/43 tested samples were positive for CNRMV by DAS-ELISA, further confirmed by RT-PCR. Antibodies raised against recombinant CNRMV CP also detected the virus consistently in western blot analysis with high sensitivity and specificity. IC-RT-PCR was also developed for the detection of CVA using the produced antibody. To the best of our knowledge, this is the first report of DAS-ELISA and IC-RT-PCR based diagnostics for CVA, and first report of DAS-ELISA based diagnostics for CNRMV from India.