Recombinant foot-and-mouth disease virus (FMDV) non-structural protein 3A fused to enhanced green fluorescent protein (EGFP) as a candidate probe to identify FMDV-infected cattle in serosurveys

Recombinant protein 3A-EGFP, a fusion construct between foot-and-mouth disease virus (FMDV) non-structural protein 3A and the enhanced green fluorescent protein (EGFP) was expressed in BL21-DE3 cells. The identity of the partially purified protein 3A-EGFP was confirmed by its reactivity with sera from cattle infected with FMDV and with a monoclonal antibody specific for FMDV-3ABC (MAb3H7) in Western blot assays. No reactivity was observed with sera from uninfected vaccinated animals. The performance of 3A-EGFP as an antigen in an indirect enzyme-linked immunosorbent assay (ELISA) was assessed and compared with that of a previously developed and validated capture ELISA that uses a 3ABC recombinant antigen (3ABC ELISA) and has been widely applied for serological surveys in Argentina. Parallel analysis of strongly and weakly positive reference sera from infected animals and 329 serum samples from uninfected vaccinated cattle showed that the 3A-EGFP antigen unequivocally identifies sera from FMDV-infected cattle with similar performance to its 3ABC counterpart. The 3A-EGFP ELISA is simpler and faster to perform than the 3ABC ELISA, since it does not require a capture step with a specific antibody. Moreover, the expression and storage of the recombinant 3A-EGFP is simplified by the absence of residual autoproteolytic activity associated to the 3C sequence. We conclude that the 3A-EGFP ELISA constitutes a promising screening method in serosurveys to determine whether or not animals are infected with FMDV.