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.

Lipids play a crucial role in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine virus that is endemic throughout the world. However, little is known about the effect of fatty acids (FAs), a type of vital lipid, on PRRSV infection. In this study, we found that treatment with a FA biosynthetic inhibitor significantly inhibited PRRSV propagation, indicating the necessity of FAs for optimal replication of PRRSV. Further study revealed that 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), a key kinase antagonizing FA biosynthesis, was strongly activated by PRRSV and the pharmacological activator of AMPK exhibited anti-PRRSV activity. Additionally, we found that acetyl-CoA carboxylase 1 (ACC1), the first rate-limiting enzyme in the FA biosynthesis pathway, was phosphorylated (inactive form) by PRRSV-activated AMPK, and active ACC1 was required for PRRSV proliferation, suggesting that the PRRSV infection induced the activation of the AMPK–ACC1 pathway, which was not conducive to PRRSV replication. This work provides new evidence about the mechanisms involved in host lipid metabolism during PRRSV infection and identifies novel potential antiviral targets for PRRSV.

As an emerging swine enteropathogenic coronavirus, porcine deltacoronavirus (PDCoV) not only causes serious diarrhea in suckling piglets but also possesses the potential for cross-species transmission, which has sparked growing interest when studying this emerging virus. We previously identified a novel accessory protein NS7a encoded by PDCoV; however, the function of NS7a was not resolved. In this study, we demonstrated that PDCoV NS7a is an interferon antagonist. Overexpression of NS7a notably inhibited Sendai virus (SeV)-induced interferon-β (IFN-β) production and the activation of IRF3 rather than NF-κB. NS7a also inhibited IFN-β promoter activity induced by RIG-I, MDA5, MAVS, TBK1, and IKKε, which are key components of the RIG-I-like receptor (RLR) signaling pathway but not IRF3, the transcription factor downstream of TBK1/IKKε. Surprisingly, NS7a specifically interacts with IKKε but not with the closely related TBK1. Furthermore, NS7a interacts simultaneously with the kinase domain (KD) and the scaffold dimerization domain (SDD) of IKKε, competing with TRAF3, and IRF3 for binding to IKKε, leading to the reduction of RLR-mediated IFN-β production. The interactions of TRAF3-IKKε and IKKε-IRF3 are also attenuated in PDCoV-infected cells. Taken together, our results demonstrate that PDCoV NS7a inhibits IFN-β production by disrupting the association of IKKε with both TRAF3 and IRF3, revealing a new mechanism utilized by a PDCoV accessory protein to evade the host antiviral innate immune response.

ABSTRACT. The purpose of this study was to assess the suitability of lactic acid bacteria (LABs) isolated from Muscovy duck as a potential probiotic. Isolates were identified by targeted polymerase chain reaction and assessed in vitro for probiotic characteristics such as autoaggregation; surface-charge; hydrophobicity; tolerance to acidic pH, bile salts and protease; and expression of genes involved in Caco-2 cell adhesion. The LAB isolates exhibited strong resistance to high bile concentration and acidic pH, produced lactic acid, and bacteriostatic (P < 0.05) were identified as bacilli compared with LAB isolates of cocci. Additionally, the LAB isolates showed high sensitivity to penicillin and tetracycline antibiotics, while they were resistant to ofloxacin, Macrodantin, and cotrimoxazole. The level of F-actin mRNA increased in the groups treated with CM3, Salmonella enterica, and CM3 + S. enterica (P < 0.0001, P < 0.05 and P < 0.05 ). The level of cell adhesion molecule (CAM) and E-cadherin (E-cad) mRNA expression was significantly lower in the treatment group (P < 0.05 for both) than in the control. The F-actin, CAM, and E-cad mRNA levels were significantly lower in the S. enterica and CM3 + S. enterica groups (P < 0.01) than in the CM3 group. Among these, RNA levels were higher in the CM3 + S. enterica than S. enterica group. These results indicate that the natural duck gut microflora is an excellent source for probiotic bacteria and can facilitate the establishment of criteria to select probiotic strains for the prevention of diarrhea.