Animals challenge
All animal procedures and experiments were performed according to protocols approved by the Institutional Animal Care and Use Committee of Nanjing Agricultural University (Nanjing, China) and followed the National Institutes of Health guidelines.
A total of 30 newborn piglets were purchased successively from a high health status herd, which were confirmed seronegative to PEDV, TGEV, Porcine Reproductive and Respiratory Syndrome virus (PRRSV), Classical Swine Fever virus (CSFV), Porcine circovirus type 2 (PCV2) and Porcine Respiratory Coronavirus (PRCV). The animals were fed with milk every 3 h throughout the experiment to meet or exceed the National Research Council (NRC) requirements for nutrients and energy for newborn piglets.
For autotransfusion with virus-loaded RBCs, 12 newborn piglets with similar weight were randomly assigned to 2 groups: 6 control group (I); 6 PEDV infection group (II). Whole bloods were collected from precaval vein to isolate RBCs, respectively. Newborn piglets in control groups were infused autologous RBCs labeled with Dil via ear vein. For PEDV infection group, RBCs were infected with PEDV (MOI=0.1) for 1 h at 37 °C, then labeled with CM-Dil Dye (Invitrogen) and wash extensively to remove unbound virus and dye. The supernatants were collected and detected viral titer by plaque assays to ensure washing cleanly. In Both of I and II group, 2× 109 RBCs were suspended in 1 mL DPBS and infused via ear vein. After 1 h of transfusion, 3 piglets from control group and 3 piglets from PEDV infection group were euthanized and sacrificed to collect blood and organ samples, individually. The remained piglets were observed daily for diarrhea symptoms. All piglets were euthanized before dying or necropsy.
For PEDV intranasal infection, 18 newborn piglets with similar weight were randomly assigned to 2 groups: 9, control group (I); 9, PEDV intranasal infection (II). Newborn piglets in group II were infected 1 mL PEDV (107 PFU/mL) through nasal incubation. In group I, the same volume of PBS was incubated intranasally. Subsequently, three piglets from each group were euthanatized at 3 h, 12 h, and 24 h after virus inoculation to determine the dynamic changes of PEDV in nasal cavity.