Monoclonal antibodies to the recombinant nucleocapsid protein of a groundnut bud necrosis virus infecting tomato in Karnataka and their use in profiling the epitopes of Indian tospovirus isolates

Abstract

A tospovirus infecting tomato in the fields of Karnataka, India, was propagated in greenhouse-grown Nicotiana benthamiana plants by mechanical inoculation. The viral RNA was extracted from purified virus and used for amplification of N and NSs genes by RT- PCR using appropriate primers. The N and NSs PCR products were cloned into a pRSET vector and sequenced. The N gene of tomato tospovirus showed 98% identity with that of Groundnut bud necrosis virus (GBNV), alternate name Peanut bud necrosis virus (PBNV). Interestingly, though the virus was isolated from tomato plants, it showed only 82% identity with the N gene of GBNV-To isolate from Taiwan. The NSs gene of the virus under study showed 98% identity with GBNV. These results suggest that the tomato tospovirus in Karnataka is a strain of GBNV and is henceforth designated as GBNV-To (K). The N gene was overexpressed in Escherichia coli and the recombinant N protein was purified using Ni-NTA agarose affinity chromatography. The purified protein was used for the generation of poly- and monoclonal antibodies (mAbs). The polyclonal antiserum thus obtained had a dilution end-point > 1: 32,000 and nine unique mAbs were also obtained. These mAbs were used for epitope profiling of the tospovirus isolates from South India and for developing detection methods. The results showed that there are distinct GBNV strains in South India. A simple dot-blot assay was developed for detection of GBNV from infected field samples.