A B S T R A C TIonic calcium (Ca 2+ ) is a versatile intracellular second messenger that plays important roles in cellular physiological and pathological processes. Porcine deltacoronavirus (PDCoV) is an emerging enteropathogenic coronavirus that causes serious vomiting and diarrhea in suckling piglets. In this study, the role of Ca 2+ to PDCoV infection was investigated. PDCoV infection was found to upregulate intracellular Ca 2+ concentrations of IPI-2I cells. Chelating extracellular Ca 2+ by EGTA inhibited PDCoV replication, and this inhibitory effect was overcome by replenishment with CaCl 2 . Treatment with Ca 2+ channel blockers, particularly the L-type Ca 2+ channel blocker diltiazem hydrochloride, inhibited PDCoV infection significantly. Mechanistically, diltiazem hydrochloride reduces PDCoV infection by inhibiting the replication step of the viral replication cycle. Additionally, knockdown of CACNA1S, the L-type Ca 2+ voltage-gated channel subunit, inhibited PDCoV replication. The combined results demonstrate that PDCoV modulates calcium influx to favor its replication.

As one of the most significant etiological agents in pigs, porcine reproductive and respiratory syndrome virus (PRRSV) has adversely impacted the global swine industry since it was discovered in the 1980s. The mRNA-decapping enzyme 1a (DCP1a), a regulatory factor involved in removing the 5'-methylguanosine cap from eukaryotic mRNA, has recently been identified as an IFN-stimulated gene. However, the role of DCP1a in PRRSV infection is not well understood. In this study, overexpression and knockdown of porcine DCP1a (pDCP1a) showed that pDCP1a affected PRRSV infection. Interestingly, we found that PRRSV infection significantly downregulated pDCP1a expression at the protein level by cleaving pDCP1a. Furthermore, we demonstrated that PRRSV nonstructural protein 4 (nsp4), a 3C-like proteinase, is responsible for pDCP1a cleavage, and the cleaved site is at glutamic acid 238 (E238) of pDCP1a. The mutant pDCP1a-E238A, which cannot be cleaved by nsp4, showed higher anti-PRRSV activity, and the antiviral effects of two cleavage products (pDCP1a and pDCP1a) were significantly decreased compared with wild type pDCP1a. Unexpectedly, PRRSV infection or overexpression of nsp4 did not cleave monkey DCP1a, and monkey DCP1a showed a higher anti-PRRSV activity than pDCP1a. Taken together, this study reveals a new strategy evolved by PRRSV to dampen the host defense, complementing the known PRRSV-mediated immune evasion mechanisms.

Porcine deltacoronavirus (PDCoV), an emerging enteropathogenic coronavirus, causes serious diarrhoea 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, 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 the 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 that does not require a specific receptor and clathrin-mediated endocytosis that 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.