Abstract
Classical swine fever virus (CSFV) is the causative agent of a severe multi-systemic disease of pigs. While several aspects of virus-host-interaction are known, the early steps of infection remain unclear. For the closely related bovine viral diarrhea virus (BVDV), a cellular receptor is known: bovine complement regulatory protein CD46. Given that these two pestiviruses are closely related, porcine CD46 is also a candidate receptor for CSFV. In addition to CD46, cell-culture-adapted CSFV strains have been shown to use heparan sulfates as an additional cellular factor. In the present study, the interaction of field-type and cell-culture-adapted CSFV with a permanent porcine cell line or primary macrophages was assessed using anti-porcine CD46 monoclonal antibodies and a heparan-sulfate-blocking compound, DSTP-27. The influence of receptor blocking was assessed using virus titration and quantitative PCR. Treatment of cells with monoclonal antibodies against porcine CD46 led to a reduction of viral growth in both cell types. The effect was most pronounced with field-type CSFV. The blocking could be enhanced by addition of DSTP-27, especially for cell-culture-adapted CSFV. The combined use of both blocking agents led to a significant reduction of viral growth but was also not able to abolish infection completely. The results obtained in this study showed that both porcine CD46 and heparan sulfates play a major role in the initial steps of CSFV infection. Additional receptors might also play a role for attachment and entry; however, their impact is obviously limited in vitro in comparison to CD46 and heparan sulfates.
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Acknowledgments
We would like to thank Anja Petrov, Ulrike Kleinert, Kristin Trippler, and Jana Pietschmann for technical assistance, and Michaela Schmidtke for kindly providing DSTP-27.
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705_2014_2313_MOESM1_ESM.ppt
Supplementary Figure S1 Titration of supernatants from blocking assays on PBMC-derived macrophages with the field-type 2nd passage of CSFV “Roesrath” (A) and the cell-culture-adapted 26th passage of CSFV “Roesrath” (B). The results are presented as logTCID50/100 µl. Treatments were as follows: 1 = untreated cell control, 2 = anti-CD46 mabs, 3 = DSTP-27, 4 = anti-CD46 mabs and DSTP-27, 5 = irrelevant SWC3 mab (PPT 210 kb)
705_2014_2313_MOESM2_ESM.ppt
Supplementary Figure S2 Quantification of viral genomes in supernatants of blocking assays on PBMC-derived macrophages with the field-type 2nd passage of CSFV “Roesrath” (A) and the cell-culture-adapted 26th passage of CSFV “Roesrath” (B). Quantification was done by RT-qPCR. The results are presented as genome copies per well. Treatments were as follows: 1 = untreated cell control, 2 = anti-CD46 mabs, 3 = DSTP-27, 4 = anti-CD46 mabs and DSTP-27, 5 = irrelevant SWC3 mab. (PPT 1190 kb)
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Dräger, C., Beer, M. & Blome, S. Porcine complement regulatory protein CD46 and heparan sulfates are the major factors for classical swine fever virus attachment in vitro . Arch Virol 160, 739–746 (2015). https://doi.org/10.1007/s00705-014-2313-y
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DOI: https://doi.org/10.1007/s00705-014-2313-y