Supernatants from infected cells were clarified by centrifugation at 3,300 G, filtered on 0.8 m. a higher Th1 polarization and IFN secretion than FL13 and LV. Altogether, this work suggests an activation FRAX597 of cDC1 by Lena associated with a Th1 immune response polarization. order, the family, and the genus (ICTV 2017 Release). Two different species, PRRSV-1 and PRRSV-2 are now distinguished (1). PRRSV-1 have further been divided into 4 subtypes. PRRSV-1 subtype 1 (PRRSV-1.1) is present in all part of Europe, while PRRSV-1.2, 1.3, and FRAX597 1.4 are mostly present in Eastern Europe (2). PRRSV-1.3 such as Lena, are more pathogenic than PRRSV-1.1 as Lelystad virus (LV) (3C6). The infection by PRRSV-1.3 is characterized by higher body temperature, more sever clinical signs and lung pathology compared to PRRSV-1.1, whereas viremia and lung viral load are not consistently higher (5, 7). A lag of several weeks in the clearance of the PRRSV has been observed, mostly attributed to a delay in neutralizing antibodies appearance, although an inhibition of the cellular IFN response, less studied, might also be involved [for review see (8, 9)]. It has been reported that virulent PRRSV-1.3 induced a strong early inflammatory response associated with an enhanced adaptive cellular immune response that may participate to their higher pathogenicity (5). The main cellular targets of PRRSV are macrophages (10). Extracellular sialoadhesin (CD169/Siglec-1) mediates viral internalization via interaction with viral protein GP5/M heterodimer while CD163 receptor plays a role in viral internalization and disassembly interacting with GP2 and GP4 viral proteins (11). In addition to macrophages, other immune cells have been Rabbit Polyclonal to IPPK described to be permissive to PRRSV differentiation conditions might strongly impact the susceptibility of DC/macrophages to PRRSV (14). In 2013, Frydas et al. showed that virulent PRRSV-1.3 such as Lena were able, by PRRSV-1 and 2 respectively (17, 18). However, none of them clearly defined nor distinguished DCs and macrophages, leading to results that cannot be clearly interpreted in terms of DCs/PRRSV interactions. We recently identified porcine respiratory DC and macrophage subpopulations and classified them according to a nomenclature proposed by Guilliams et al. (19, 20). In accordance with knowledge in human and mice, we observed that porcine respiratory DCs presented migratory and na?ve T-cell stimulation capacities. Conventional DC1 preferentially inducing a T-helper (Th) 1 response, cDC2 a Th2 response and monocyte-derived DC (moDC) a Th17 response. Moreover moDC produced inflammatory cytokines such as IL1 and IL8, and their proportion increased upon viral infection (21). These populations represent differentiated respiratory DCs and macrophages which can be investigated for their interactions with PRRSV in their natural environment. In order to explore the role of PRRSV/DCs interactions in the induction of the immune response, we studied the infection of primary lung DCs and as well as the impact of PRRSV infection on DCs functionalities. Highly virulent Lena PRRSV-1.3 was tested and compared with two PRRSV-1.1, namely LV and the newly emerging pathogenic Flanders13 (FL13) (15). We found that primary lung DCs were not infected by any of these strains and that a strong cDC1/Type 1 immune response was activated by Lena, but not by FL13 and LV. Materials and methods Virus production and titration The 3 strains of PRRSV used in this study were kindly provided by Dr. Hans Nauwynck, (University of Ghent, Belgium). The highly pathogenic Lena PRRSV-1.3 was used for and infections. Lena has been FRAX597 isolated in Belarus in 2007 from a herd with mortality, reproductive failures and respiratory disorders (22). Lelystad virus was identified in the Netherlands in 1991 (23) and Flanders13 13V091 was isolated in Belgium in 2013 in farms experiencing uncommon long-lasting anorexia, fever and respiratory problems within the first 2 weeks after weaning during enzootic PRRSV infection. Lena viral stock for experiment was produced using SPF piglets AM. The production was tested negative for PCV2, swine Influenza, experiments, Lena, Fl13 and LV stocks were produced using fresh SPF primary alveolar macrophages. Supernatants from infected cells were clarified by centrifugation at 3,300 G, filtered on 0.8 m. Then 30 ml of supernatant were layered on.