Serological detection of Grapevine leafroll-associated virus 4 in grapevine growing areas of India using polyclonal antiserum raised against the recombinant coat protein

Grapevine leafroll disease (GLD), caused by a complex of serologically and genetically distinct or related viral species termed as Grapevine leafroll associated viruses (GLRaVs), is a serious threat to the grape production worldwide. Recently, Grapevine leafroll-associated virus 4 (GLRaV-4) has been reported in the Indian vineyards, mostly as a mixed infection with GLRaV-3 and GLRaV-1. In order to screen the planting material, a polyclonal antiserum against the in vitro expressed coat protein (CP) of GLRaV-4 was produced. The antiserum, purified IgGs and conjugated IgGs detected GLRaV-4 in crude saps of infected grapevines and purified coat protein when used in antigen coated plate enzyme-linked immunosorbent assay (ACP-ELISA), double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), Western blotting and dot immunobinding assay (DIBA). GLRaV-4 could be detected in 54% (39 out of 72) of symptomatic grapevine samples from different grape growing regions of India using DAS-ELISA. The validation of DAS-ELISA results was done by RT-PCR using GLRaV-4 CP-specific primers. To the best of our knowledge, this is the first report of a polyclonal antiserum against the in vitro expressed CP of GLRaV-4 and its successful application in diagnosis of the virus in grape vineyards of India.