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Equine herpesvirus type 1 and 4 (EHV-1 and EHV-4) affects horses worldwide. Infection with EHV-4 usually remains restricted to the upper airways, while EHV-1 infection can result in neurological disorders and abortions following lymphocyte-associated viremia. One of the possible mechanisms allowing systemic dissemination of EHV-1 is the documented ability of EHV-1 gG, more specifically its hypervariable region, to interfere with the host’s immune response by binding to chemokines. In this study we tested the hypothesis that gG influences the ability of EHV-1 to cause systemic infection by constructing EHV-1 mutants in which the entire gG or only its hypervariable region upstream of the transmembrane region were exchanged with their EHV-4 counterparts. Based on the neurovirulent strain Ohio 2003 (OH- 03), we constructed four recombinant viruses by en-passant mutagenesis of OH-03 cloned as a bacterial artificial chromosome: A gG deletion mutant (vOH-ΔgG), a mutant in which EHV-4 gG was inserted in lieu of authentic EHV-1 gG (vOH-4gG), a mutant harboring EHV-4 gG with the chemokine-binding region of EHV-1 gG (vOH-4gGhyp1) and a mutant harboring the hypervariable region of EHV-4 gG in the EHV-1 backbone (vOH-hyp4). The mutant viruses were characterized in vitro by plaque size assays as well as single-step-growthkinetics and it could be shown that the various mutations in gG did not influence viral cell-tocell spread or replication in vitro. Next, chemotaxis assays were performed to analyze if the various gGs can interfere with neutrophil migration in vitro. Here, an increased chemotaxis of neutrophils could be observed when supernatants of cells infected with vOH-ΔgG or vOH- 4gG were used. In contrast, re-insertion of the predicted chemokine-binding region of EHV-1 glycoprotein G did not completely restore the ability to inhibit neutrophil migration as well as insertion of the hypervariable region of EHV-4 gG did not lead to complete loss of chemokine-binding function of gG. Finally, the different mutant viruses were tested in an in vivo infection model in BALB/c mice. Flow cytometric analyses were performed to determine the composition of immune cells in bronchoalveolar lavages of intranasally infected mice. The largest influx of neutrophils was noted for mice infected with the vOH-4gG virus, whereas here the gG deletion virus behaved more like the OH-03 virus and resulted in a more pronounced inhibition of chemotaxis in infected mice. Again, the two viruses with the exchanged hypervariable region did not show the expected influence on neutrophil migration to the site of infection, suggesting that it is not the hypervariable region alone that determines the immunmodulatory potential of gG.