The present study shows that interaction of European genotype PRRSV with macrophages causes a clear reduction in macrophage phagocytic capacity, reducing both the number of macrophages phagocytosing beads and the number of beads taken up per cell. Using inactivated PRRSV (LV), we show that the observed downregulation of phagocytosis results from binding of PRRSV virions and their subsequent internalization, without the need for viral replication. This is supported by the fact that a higher number of internalized virions corresponds to a higher decrease in phagocytic capacity, both for Marc-145-grown and macrophage-grown virus. Finally, we suggest that the extracellular interaction of European genotype PRRSV with its receptor Sn, but not CD163, inhibits primary alveolar macrophage phagocytosis at the early stage of virus entry.
The fact that phagocytosis is inhibited 1 h after virus inoculation further supports our reasoning that the reduced phagocytic capacity is not merely due to virus-induced macrophage death, as often stated, and that the interaction of the PRRSV virion with its target cell is sufficient to reduce PAM phagocytosis. From previous research it is know that PRRSV has a replication cycle of approximately 12 h, after which the virus is released from the cell, thereby killing its target cell . Moreover, it was shown that early in infection, PRRSV promotes anti-apoptotic pathways in PAM, up to 8 h post infection. Thus, at 1 h post infection, no virus-induced cell death occurs and therefore the decrease in phagocytosis cannot be explained by virus-induced cell-death.
It is known that 1 h post infection, internalized virions can still be observed, whereas 5 h post infection, virions can no longer be detected , which indicates that all virions are uncoated. Starting from 6 h post infection, newly synthesized structural proteins can be detected . Consequently, this supports our finding that entry of the virus into the cell, but not production of progeny virus, causes the inhibition of phagocytosis.
Our study shows that a 1 h incubation of PAM with an Sn-specific mAb results in a decreased phagocytic capacity of PAM, whereas incubation with a CD163-specific mAb or pAb has no effect. Both Sn and CD163 are present at the cell surface and therefore available for antibody-binding. In addition, it was previously shown that incubation of PAM with any of these antibodies blocks infection of PRRSV, indicating that they can prevent interaction of the virus with their respective receptors ([28, 30]; H. Van Gorp, unpublished data). Moreover, at the highest administered dose, still no effect on PAM phagocytic capacity of the CD163-specific antibodies was observed. This concentration was 33 times higher than the dose at which phagocytosis was completely blocked with the Sn-specific mAb. Therefore, we conclude that the interaction of European genotype PRRSV with its receptor Sn, but not CD163, inhibits primary alveolar macrophage phagocytosis.
Recent studies investigating the functional role of porcine Sn (pSn) have demonstrated that pSn is an endocytic receptor that mediates endocytosis of PRRSV or antibodies upon binding [26, 31]. pSn was also shown to induce signalling  and reduce phagocytosis  in PAM upon antibody binding. Therefore, it was suggested that antibody binding to pSn activates certain molecular networks, which results in the downregulation of pathways involved in phagocytosis. This might also explain how the extracellular interaction of European genotype PRRSV with Sn downregulates phagocytosis. The results obtained in our study indicate that a certain number of virions need to interact with Sn before a certain ligand-receptor signalling threshold is reached and PAM get activated. In this scenario, the interaction of the virion with Sn serves as a trigger for further signalling, leading to a downregulation of PAM phagocytic capacity. Once this threshold is reached, administering higher moi, and therefore higher numbers of virions, results in a further downregulation of phagocytosis. Ligand-receptor signalling thresholds have been described for different viral ligand-receptor interactions, e.g. the interaction of human immunodeficiency virus (HIV) envelope glycoprotein (Env) with coreceptor CXCR4  and of viral RNA with TLR7 , which must be reached to trigger sufficient activation of a receptor and subsequent signalling. Moreover, our results show that the phagocytic capacity of PAM can be blocked completely when a sufficiently high antibody dose or number of European genotype PRRSV particles (an moi of 30 of Marc-145-grown PRRSV (LV) or an moi of 100 of macrophage-grown PRRSV (Lena)) is administered. This suggests that once Sn is triggered sufficiently through ligand binding, whether it is an antibody or European genotype PRRSV virions, phagocytosis is maximally inhibited.
Both for human and mouse Sn it is suggested that alternative splicing of the Sn gene produces a transcript variant encoding an isoform that is soluble rather than membrane-bound . It can be argued that these variants have no significant role in the downregulation of phagocytosis, since these variants are either excreted or present intracellularly in a non-membrane bound form, whereas surface-expressed Sn is needed to internalize PRRSV , subsequently downregulating phagocytosis. Moreover, no porcine splice variants have been reported, and no soluble Sn variants have so far been observed in vivo.
Interestingly, at an moi of 1, all macrophages have internalized several PRRSV virions, indicating that many non-infectious virions are present. This can be due to storage and thawing of the virus, but it can also be a direct result from the high mutation frequency reported for PRRSV [44, 45]. If these mutations occur at crucial sites in the genome, the progeny virus will still be able to enter its target cell, but will have lost its ability to replicate upon uncoating and genome release. This observation is of importance if the observed inhibition of phagocytosis upon European genotype PRRSV infection also occurs in vivo, since non-infectious progeny virus is also present in the lungs of pigs, and can contribute to the downregulation of PAM phagocytic capacity.
Several other viruses have been shown to affect phagocytosis. Respiratory syncytial virus and secondary dengue virus infections have been shown to increase macrophage phagocytosis [46, 47]. On the other hand, HIV, pseudorabies virus and influenza virus [48–50] infections are known to suppress phagocytosis, and in all cases this suppression is linked to an increased incidence of secondary infections. The way in which these viruses downregulate phagocytosis differs for each virus. For HIV for instance, one of the mechanisms causing the decreased phagocytosis in neutrophils and monocytes/macrophages is through the interaction of the cell with certain viral proteins, such as gp 120, p24 and Nef. It is difficult to comment on the relative moi at which phagocytosis is affected in these studies versus the moi at which European genotype PRRSV downregulates PAM phagocytosis. This is either due to different definitions of moi used in each study, such as egg infectious dose (EID50) or plaque forming units (PFU), or because persistently infected cells or cells isolated from infected individuals or animals were used and no moi was determined. It could be argued that the observed downregulation of PAM phagocytosis in our study occurs at relatively high moi, however, it is difficult to assess which moi are reached in the lungs of pigs in vivo. For PRRSV (Lena), titers of up to 107.8 have been reported in BAL (bronchoalveolar lavage) fluids , corresponding to an moi of 63, and it can be argued that the effective titers in the lungs will be even higher, due to freeze/thawing and storage procedures.
PRRSV is a major player in porcine respiratory disease complex (PRDC) (reviewed by [24, 51]). Field reports and clinical evidence demonstrate an increased incidence and severity of secondary infections associated with primary PRRSV infections. Strikingly, the direct effect of the virus on macrophage phagocytosis during the early stages of infection is poorly studied. Whereas some authors report that PRRSV infection has no effect on the uptake of Staphylococcus aureus and Escherichia coli, other authors report an impaired phagocytic capacity against Candida albicans, Salmonella typhimurium, and Haemophilus parasuis, which supports our findings. Importantly, all of these studies showed, like we did, that virus-induced cell death was not involved. One of the factors influencing the outcome of these experiments is the secondary pathogen, and specific isolate, used. Each pathogen interacts with PAM in a different way and each secondary infection might have an additional immunomodulatory effect on PAM. Therefore, in our study, polystyrene beads were used as a reproducible, inert model to study the effect of European genotype PRRSV on PAM phagocytosis. Additionally, different PRRSV isolates might influence the outcome of each experiment, which was not the case for the two isolates used in this study. Yet, preliminary results indicate possible divergent effects of North American genotype PRRSV isolates on PAM phagocytosis (data not shown), which we are investigating in an ongoing study.
In a previous study, Van Gucht et al.  showed that infection with PRRSV sensitizes the lungs for production of proinflammatory cytokines upon exposure to lipopolysaccharides (LPS) and demonstrated that overproduction of these cytokines led to respiratory disease. If the observed inhibition of phagocytosis upon European genotype PRRSV infection also occurs in vivo, higher amounts of LPS will be present in the lungs after infection, since removal of secondary or opportunistic bacteria, and thus bacterial endotoxins, from the alveolar space will be affected. This could lead to a further exacerbation of cytokine production and more severe respiratory disease. This might also explain why an uncomplicated PRRSV infection often fails to induce overt respiratory disease , although PRRSV is a primary agent in PRDC.
To our knowledge, the present study is the first to show a direct effect of European genotype PRRSV on PAM phagocytic capacity in vitro, linking it to the interaction of the virus with its target cell, and more specific with its attachment and internalization receptor Sn. If similar events occur in vivo, this contributes to the reported increase and severity of secondary bacterial infections following European genotype PRRSV infection and the prevalence of European genotype PRRSV in porcine respiratory disease complex (PRDC).