Fig. 1.

Serological cross-reactivity of PEDV and PdCV virus-infected cells and virus-infected intestinal tissues. (A) Immunofluorescence assay (IFA) in PEDV or PdCV-infected ST cells. Confluent ST cells were infected with PEDV VBS2 strain or PdCV Michigan/8977/2014 strain at a multiplicity of infection (MOI) of 1.0. After 1 h absorption, the inoculum was removed, the cells were washed once with DMEM, and fresh DMEM (supplemented with 5 μg/ml trypsin 1:250) was added, and the infected cells were incubated at 37 °C. When cytopathic effects (CPE) were observed, the cells were fixed with 4.0% (v/v) paraformaldehyde-0.2% (v/v) glutaraldehyde in 0.1 M potassium phosphate buffer (PPB), pH 7.4, for 15 min, 22 °C, followed by washing 3 times with PBS. After permeabilization with 0.1% Triton X-100 in PBS for 15 min, the cells were washed with PBS, blocked with PBS containing 2% bovine serum albumin for 1 h, 22 °C. Then cells were incubated with PEDV or PdCV polyclonal antibody. After 3 washes with PBS, the cells were further incubated with FITC-labeled rabbit anti-pig secondary antibody overnight at 4 °C. After 3 washes with PBS, samples were examined under Olympus fluorescent microscope system at The Ohio State University. Photos at upper panels were taken under a light microscope (100×). Photos at lower panels were taken under an immunofluorescent microscope (100×). (B). Immunohistochemistry (IHC) analysis of small intestine sections from Gn piglets. PEDV VBS2 infected duodenal tissue at 72 h PI, PdCV infected Michigan/8977/2014 duodenal tissue at 72 h PI, or uninfected controls were stained with anti-PEDV serum or anti-PdCV serum. Black arrows indicate positive antigens. Hematoxylin, 300×. Five-micron sections of paraffin-embedded tissues were placed onto positively charged slides. After deparaffinization, sections were incubated with target retrieval solution (Dako, Carpinteria, CA) for antigen retrieval. After blocking, a primary anti-PdCV or PEDV serum antibody was incubated for 30 min, 22 °C followed by incubation with a biotinylated horse anti-pig IgG secondary antibody (Vector Laboratories, Burlingame, CA). Slides were further incubated with ABC Elite complex to probe biotin (Vector Laboratories) and then developed using a 3,3′-diaminobenzidine (DAB) chromogen Kit (Dako); hematoxylin was used as a counterstain.