Fig. 3.

Generation of rTGEV co-expressing GP5 and M proteins. (A) Schematic representation of PRRSVOlot91 GP5 domains. A detail of the domain containing the epitopes inducing non-neutralizing (IDE) and neutralizing (ECN) antibodies is shown. Two N-glycosylation sites, N46 (G1) and N53 (G2), are located within this domain. Three different mutants were generated, substituting Asn 46 and 53 by Ser, avoiding the glycosylation at these positions (N46S, N53S, and N46,53S). An additional mutant, lacking N46 glycosylation site and decoy epitope, was obtained (N46S-ΔIDE). In all cases, rTGEV viruses were recovered with high titers. (B) ST cells were infected with the rTGEVs and double immunofluorescence staining was performed. TGEV N protein specific monoclonal antibodies and a secondary antibody staining red were used to identify virus-infected cells. Expression of GP5 was detected with rabbit antiserum specific for a GP5 peptide coupled to a secondary antibody staining green (upper panels). Expression of M protein was detected with a rabbit antiserum specific for an M protein peptide, coupled to a secondary antibody staining green (lower panels). The percentage of infected cells expressing PRRSV antigens was estimated by the analysis 10 different microscopic fields.