Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens in swine. It is genetically unstable and difficult to control by vaccination. Our laboratory has made major contributions to a better understanding of the complex entry of PRRSV into its target cell, the macrophage. PRRSV enters through a receptor-mediated endocytosis and subsequently, a low pH is required to trigger proper viral uncoating. The fusion process remains to be unravelled. Preliminary work clearly demonstrated that the heterotrimeric GP2/3/4 complex together with the receptor CD163 is sufficient to induce fusion. Based on previous work, GP2 was proposed as fusion protein. In the present project, the fusion process of the viral GP2/3/4 complex with CD163 will be analysed and the role of GP2 as fusion protein will be confirmed. Mutagenesis analysis will identify GP2 residues involved in fusion. They will be mutated in PRRSV infectious clones using reverse genetics. Next, the putative cleavage of GP2 and the involved protease will be determined. A hallmark of the anti-PRRSV immunity is the delayed and weak neutralizing antibody response. We hypothesize that this is due to the fact that the antibodies are mainly raised against post-fusion GP2/3/4. Pigs will be vaccinated with inactivated PRRSV with a prefusion-stabilized GP2/3/4. The induction of neutralizing antibodies and virological protection upon challenge will be compared in pigs.