Abstract
(1) Background: Porcine deltacoronavirus (PDCoV) is a newly emerged enteric virus affecting pig breeding industries worldwide, and its pathogenic mechanism remains unclear. (2) Methods: In this study, we preliminarily identified the endocytic pathway of PDCoV in PK-15 cells, using six chemical inhibitors (targeting clathrin-mediated endocytosis, caveolae-mediated endocytosis, macropinocytosis pathway and endosomal acidification), overexpression of dominant-negative (DN) mutants to treat PK-15 cells and proteins knockdown. (3) Results: The results revealed that PDCoV entry was not affected after treatment with chlorpromazine (CPZ), 5-(N-ethyl-N-isopropyl) amiloride (EIPA)or ammonium chloride (NH4Cl), indicating that the entry of PDCoV into PK-15 cells were clathrin-, micropinocytosis-, PH-independent endocytosis. Conversely, PDCoV infection was sensitive to nystatin, dynasore and methyl-β-cyclodextrin (MβCD) with reduced PDCoV internalization, indicating that entry of PDCoV into PK-15 cells was caveolae-mediated endocytosis that required dynamin and cholesterol; indirect immunofluorescence and shRNA interference further validated these results. (4) Conclusions: In conclusion, PDCoV entry into PK-15 cells depends on caveolae-mediated endocytosis, which requires cholesterol and dynamin. Our finding is the first initial identification of the endocytic pathway of PDCoV in PK-15 cells, providing a theoretical basis for an in-depth understanding of the pathogenic mechanism of PDCoV and the design of new antiviral targets.
Highlights
To determine whether Porcine deltacoronavirus (PDCoV) can gain entry into PK-15 cells through this pathway, we treated the cells with chlorpromazine, an inhibitor of the clathrin-mediated endocytosis (CME) pathway, which inhibits the CME pathway by blocking the formation of clathrin and AP2 complexes at the plasma membrane; thereby, chlorpromazine inhibits the assembly of clathrin-coated pits [30]
EPS15 presents at clathrin-coated pits and involved in receptor-mediated endocytosis [31]
It is necessary for proper coated vesicle formation, so we sought to confirm whether EPS15 is involved in PDCoV entry
Summary
Porcine deltacoronavirus (PDCoV), belonging to the genus Deltacoronavirus within the family Coronaviridae, was first identified in swine in Hong Kong in 2012 [1], and can cause acute diarrhoea, vomiting, dehydration and death in new born piglets [2], sharing similar clinical symptoms with diseases caused by porcine epidemic diarrhoea virus (PEDV). The PDCoV strain was recently reported for the first time in plasma samples from three Haitian children with acute undifferentiated febrile illness [12]. All of these results indicate that PDCoV has potential cross-species transmission risk and potential public health risk
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