Porcine alveolar macrophages and nasal epithelial cells internalize porcine epidemic diarrhea virus (PEDV) but do not support its replication in vitro

Abstract

Porcine epidemic diarrhea virus (PEDV) primarily targets enterocytes subsequent to fecal-oral exposure, resulting in severe gastrointestinal disease in neonatal piglets. However, recent evidence suggests potential alternative PEDV entry and replication routes via the respiratory tract. The present study delved into the possibility of an alternative pathway for PEDV infection in porcine alveolar macrophages (PAMs), 3D4/21 cells (3D4), and nasal turbinate epithelial cells, focusing on the inherent innate antiviral and anti-inflammatory immune responses to a cell-adapted non-S INDEL USA PEDV strain. CCL-81 cells were used as positive controls of infection, while non-infected CCL-81, PAMs, and 3D4 cells served as negative controls. Quantification of the viral load in cells and supernatants (SN) was carried out at multiple hours post-inoculation (hpi; 0, 6, 12, 24, 48, 72, and 96 hpi) using RT-qPCR, while infectious virus titers were assessed through TCID₅₀/ml on cell cultures and immunofluorescence (IF) staining. PEDV capture and internalization were examined using IF at 24 and 48 hpi, alongside the evaluation of the presence of viral particles and ultrastructural changes using transmission electron microscopy (TEM). Proinflammatory and antiviral cytokine levels in SN were measured using ELISA and Luminex. In both PAMs and 3D4 cells, PEDV RNA levels peaked at 12 hpi in cells and SN, then declined gradually without significant differences between cell types. Only few PAMs and 3D4 cells tested positive for PEDV IF, with no increase in positive cells between 24 and 48 hpi. TEM did not reveal viral particles or changes in cell organelles, and no proinflammatory or antiviral cytokine expression was detected in either cell type of macrophage cells. In parallel, nasal turbinate organoids (NTOs), cultivated as 2D monolayer and at an air-liquid interface (ALI), were exposed to PEDV, with RT-qPCR and IF conducted at 24 hpi. Despite the cultivation technique used, similar levels of PEDV RNA were detected in both the cells and the SN, with positive results observed for PEDV IF. Overall, while PAMs, 3D4 cells and nasal epithelium can capture and internalize PEDV, they do not support viral replication or trigger an antiviral or anti-inflammatory responses.