Characterisation of PRRSV1 infection in bone marrow-derived dendritic cells

Student thesis: Doctoral thesis


The present thesis aims to characterize the attachment, replication and the induction of apoptosis during PRRSV infection in immature (i) and mature (m) bone marrow-derived dendritic cells (BMDC). Three PRRSV1 isolates (3249, 3262 and 3267) were used. The kinetics of replication were assessed by titrating cell culture supernatants in macrophages. The viral yield in iBMDC at 12 and 24 hpi was significantly higher than in mBMDC, and the replication of two isolates (3249 and 3262) peaked earlier in iBMDC (24 hpi) compared to mBMDC (48hpi). These results indicated that iBMDC were more efficient than mBMDC in supporting viral replication. This feature was not related to the proportion of CD163+ cells nor the levels of IFN-α in the cultures. In addition, the replication efficiency was strain-dependent. Isolate 3262 showed the lowest titres in both cell types at all times, consistently with the lowest proportions of 3262-infected cells in flow cytometry. The attachment and replication was further studied in association with the expression of three receptors, PoSn, CD163 and heparan sulphate. A three-colour confocal microscopy staining (PoSn, CD163 and PRRSV) on iBMDC showed that attachment occurred on the four subsets defined by PoSn and CD163. Removal of heparan sulphate from the cell surface did not fully avoid the attachment. These results indicated that attachment of PRRSV1 on BMDC might occur beyond the intervention of heparan sulphate, PoSn and CD163 and point towards the existence of other potential receptors. Next, a two-colour confocal microscopy labelling CD163/PRRSV or PoSn/PRRSV was performed. Replication was observed in cells that were apparently PoSn- and CD163-. As CD163 is the only recognized essential receptor for PRRSV, its expression together with the infection by isolate 3267 on iBMDC was further examined by flow cytometry. In that case, 8.4 ± 0.5% of apparently CD163- cells were labelled as infected. To further clarify this, a sorting experiment based on CD163 expression (CD163-, CD163lo and CD163hi) was done. The first sorting focused on “beyond doubt” CD163- cells. The second sorting grouped CD163- cells together with CD163lo. Unsorted iBMDC were used as controls. The “beyond doubt” CD163- cells were not infected by 40 hpi. When CD163- were sorted together with CD163lo, the proportion of infected CD163- cells was 0.6 ± 0.07% at 40 hpi and 1.6% ± 0.08% at 60 hpi. The proportion of infected cells at 60 hpi was higher than the initial number of CD163+ cells. These results can be explained by the generation of new CD163lo that were probably infected when expressing levels of this molecule below the sensitivity of the cytometer. Alternatively, the milieu created by CD163+ infected cells resulted in CD163- susceptible cells expressing yet unknown receptors for the virus. Regarding the induction of apoptosis, in PAM cleaved caspase-3 labelling was observed in both infected and bystander cells for all three isolates (confocal microscopy), while in BMDC bystanders were mainly labelled. This is indicative of different apoptosis triggering pathways for PAM and BMDC. Moreover, at m.o.i. 0.1, the caspase-3 signal in BMDC peaked later (48 hpi) than in PAM (24 hpi), which might allow more cycles of viral replication and result in higher viral yields in BMDC. Further examination of inoculated BMDC cultures for apoptosis/necrosis showed significant differences between isolates. Thus, 3249 and 3267 isolates apparently induced apoptosis/necrosis of BMDC but 3262 did not. Neutralization of IL-10 released by BMDC and induced by 3262 infection resulted in the occurrence of apoptotic cells, but this did not happen with a second IL-10-inducing isolate (2988). The above-mentioned results will be useful to understand the role of DC in PRRSV pathogenesis.
Date of Award23 Nov 2017
Original languageEnglish
Awarding Institution
  • Universitat Autònoma de Barcelona (UAB)
SupervisorLaila Darwich Soliva (Director) & Enrique Maria Mateu de Antonio (Director)


  • BMDC
  • CD163
  • PoSn

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