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Highlights • IFN-activated macrophages differentiate PRRSV strains with varying virulence. • A collection of European PRRSV-1 strains were classified in this in vitro model. • Only the PRRSV-1 strain PR40 triggered significant release of TNF and IL-1β. Abstract The recent emergence of highly pathogenic porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) strains has caused severe economic losses. The biological elements defining virulence and pathogenicity are still unclear. In vitro characteristics using natural target cells of PRRSV provide important information to understand the basis of virulence at the cellular level, and provide a mean to reduce animal experimentations to achieve this goal. Here, we compared PRRSV strains from two geographically different regions, with varying in vivo characteristics, in terms of their interactions with monocyte-derived macrophages (MDMs). The strains included Lena and BOR59 from Belarus, and ILI6 from Russia, as well as PR11 and PR40, both from Italy. As a reference, we used a cell culture-adapted version of Lelystad, LVP. MDMs were pre-treated with IFNγ, IL-4 or IFNβ, in order to understand responses in polarized and antiviral MDMs. In general, independent of the geographical origin, the strains with high virulence infected a higher percentage of MDMs and replicated to higher titers. These virulence-dependent differences were most pronounced when the MDMs had been treated with IFNβ. Differentiation between intermediate and low virulent PRRSV was difficult, due to variations between different experiments, but LVP differed clearly from all field strains. IFNα and IL-10 were not detected in any experiment, but PR40 induced TNF and IL-1β. Taken together, these results validate the MDM model to understand pathogenicity factors of PRRSV and confirm the importance of the escape from type I and II IFN-mediated effects for PRRSV virulence. [ABSTRACT FROM AUTHOR]