Porcine Deltacoronavirus Utilizes Sialic Acid as an Attachment Receptor and Trypsin Can Influence the Binding Activity.

Porcine deltacoronavirus (PDCoV) is a novel coronavirus that causes diarrhea in nursing piglets. Studies showed that PDCoV uses porcine aminopeptidase N (pAPN) as an entry receptor, but the infection of pAPN-knockout cells or pigs with PDCoV revealed that pAPN might be not a critical functional receptor, implying there exists an unidentified receptor involved in PDCoV infection. Herein, we report that sialic acid (SA) can act as an attachment receptor for PDCoV invasion and facilitate its infection. We first demonstrated that the carbohydrates destroyed on the cell membrane using NaIO4 can alleviate the susceptibility of cells to PDCoV. Further study showed that the removal of SA, a typical cell-surface carbohydrate, could influence the PDCoV infectivity to the cells significantly, suggesting that SA was involved in the infection. The results of plaque assay and Western blotting revealed that SA promoted PDCoV infection by increasing the number of viruses binding to SA on the cell surface during the adsorption phase, which was also confirmed by atomic force microscopy at the microscopic level. In in vivo experiments, we found that the distribution levels of PDCoV and SA were closely relevant in the swine intestine, which contains huge amount of trypsin. We further confirmed that SA-binding capacity to PDCoV is related to the pre-treatment of PDCoV with trypsin. In conclusion, SA is a novel attachment receptor for PDCoV infection to enhance its attachment to cells, which is dependent on the pre-treatment of trypsin on PDCoV. This study paves the way for dissecting the mechanisms of PDCoV-host interactions and provides new strategies to control PDCoV infection.

Porcine Deltacoronavirus Enters Porcine IPI-2I Intestinal Epithelial Cells via Macropinocytosis and Clathrin-Mediated Endocytosis Dependent on pH and Dynamin.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhea in suckling piglets and has the potential for cross-species transmission. Although extensive studies have been reported on the biology and pathogenesis of PDCoV, the mechanisms by which PDCoV enters cells are not well characterized. In this study, we investigated how PDCoV enters IPI-2I cells, a line of porcine intestinal epithelial cells derived from pig ileum. Immunofluorescence assays, small interfering RNA (siRNA) interference, specific pharmacological inhibitors, and dominant negative mutation results revealed that PDCoV entry into IPI-2I cells depended on clathrin, dynamin, and a low-pH environment but was independent of caveolae. Specific inhibition of phosphatidylinositol 3-kinase (PI3K) and the Na+/H+ exchanger (NHE) revealed that PDCoV entry involves macropinocytosis and depends on NHE rather than on PI3K. Additionally, Rab5 and Rab7, but not Rab11, regulated PDCoV endocytosis. This is the first study to demonstrate that PDCoV uses clathrin-mediated endocytosis and macropinocytosis as alternative endocytic pathways to enter porcine intestinal epithelial cells. We also discussed the entry pathways of PDCoV into other porcine cell lines. Our findings reveal the entry mechanisms of PDCoV and provide new insight into the PDCoV life cycle. IMPORTANCE An emerging enteropathogenic coronavirus, PDCoV, has the potential for cross-species transmission, attracting extensive attenuation. Characterizing the detailed process of PDCoV entry into cells will deepen our understanding of the viral infection and pathogenesis and provide clues for therapeutic intervention against PDCoV. With the objective, we used complementary approaches to dissect the process in PDCoV-infected IPI-2I cells, a line of more physiologically relevant intestinal epithelial cells to PDCoV infection in vivo. Here, we demonstrate that PDCoV enters IPI-2I cells via macropinocytosis, which does not require a specific receptor, and clathrin-mediated endocytosis, which requires a low-pH environment and dynamin, while a caveola-mediated endocytic pathway is used by PDCoV to enter swine testicular (ST) cells and porcine kidney (LLC-PK1) cells. These findings provide a molecular detail of the cellular entry pathways of PDCoV and may direct us toward novel antiviral drug development.

Development and utilization of an infectious clone for porcine deltacoronavirus strain USA/IL/2014/026.

We report the generation of a full-length infectious cDNA clone for porcine deltacoronavirus strain USA/IL/2014/026. Similar to the parental strain, the infectious clone virus (icPDCoV) replicated efficiently in cell culture and caused mild clinical symptoms in piglets. To investigate putative viral interferon (IFN) antagonists, we generated two mutant viruses: a nonstructural protein 15 mutant virus that encodes a catalytically-inactive endoribonuclease (icEnUmut), and an accessory gene NS6-deletion virus in which the NS6 gene was replaced with the mNeonGreen sequence (icDelNS6/nG). By infecting PK1 cells with these recombinant PDCoVs, we found that icDelNS6/nG elicited similar levels of type I IFN responses as icPDCoV, however icEnUmut stimulated robust type I IFN responses, demonstrating that the deltacoronavirus endoribonuclease, but not NS6, functions as an IFN antagonist in PK1 cells. Collectively, the construction of a full-length infectious clone and the identification of an IFN-antagonistic endoribonuclease will aid in the development of live-attenuated deltacoronavirus vaccines.