Development of Monoclonal Antibodies Reactive to a New Grapevine Leafroll-Associated Closterovirus

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Abstract
Monoclonal antibodies reactive to a previously uncharacterized grapevine leafroll-associated virus (GLRaV) coat protein were developed. A novel 37-kDa protein was found associated with grapevine leafroll disease in a mixed virus infection. The polypeptide was separated from the 38-kDa polypeptide associated with GLRaV-1 using a Tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Concentrated p37 preparations were used to immunize mice for the production of monoclonal antibodies (MAb). Serum collected from the immunized mice showed reactivity with the 37-kDa polypeptide but no reactivity with the 38-kDa polypeptide associated with GLRaV-1 using the Western blot assay. The polyclonal antiserum reacted to both native and heat-denatured virus preparations. Two sets of anti-p37 MAbs were developed. One set binds p37 and also cross reacts with other GLRaV proteins, such as p36 of GLRaV-4 and -5, while the second set recognizes p37 specifically. We obtained the partial nucleotide sequence of the putative p37 viral protein. The predicted amino acid sequence revealed the presence of three out of the five conserved amino acids present in closterovirus capsid and diverged duplicate capsid proteins. We propose the name grapevine leafroll-associated virus-8 for the new virus with a 37-kDa capsid protein.