Polyclonal antibodies to the putative coat protein of Citrus leprosis virus C expressed in Escherichia coli: production and use in immunodiagnosis

This work reports the in vitro expression of Citrus leprosis virus C (CiLV-C) putative coat protein (p29) and the production of a polyclonal antibody to be used as a tool for serological diagnosis of citrus leprosis. The ORF2/RNA1, corresponding to p29, was cloned in pET28a and transformed into Escherichia coli cells (BL21). Expression of p29 was induced in vitro and the protein was purified and used for immunization of rabbits to produce the polyclonal antibody. The anti p29 serum was shown to be highly specific to CiLV-C detection by immunological methods (Western blot, PTA-ELISA, tissue blot and in situ immunolocalization), without cross reaction with healthy citrus plants or other cytoplasmic and nuclear viruses transmitted by Brevipalpus mites. These results demonstrate that the antibody against CiLV-C p29 protein is highly specific for CiLV-C detection. In situ immunogold labeling assays on thin sections of sweet orange leaf cells infected by CiLV-C demonstrated that short, bacilliform particles present within cisternae of the endoplasmic reticulum were specifically labeled, confirming their viral nature. The dense cytoplasmic viroplasm was also heavily labeled indicating that it represents a site of p29 accumulation.