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.