Avian Interferon-Inducible Transmembrane Protein Family Effectively Restricts Avian Tembusu Virus Infection

Chen, Shilong; Wang, Long; Chen, Jieying; Zhang, Lanlan; Wang, Song; Goraya, Mohsan U.; Chi, Xiaojuan; Na, Yang; Shao, Wenhan; Yang, Zhou; Zeng, Xiancheng; Chen, Shaoying; Chen, Ji-Long

Cell Lines, Birds, Virus, and Infection ATMUV CJD05 strain was isolated from a chicken farm with acute egg-drop syndrome in Fujian, China . Duck embryo fibroblasts (DEFs) were prepared from 13-dayold mule-duck embryo as previously described (Shahsavandi et al., 2013) . DF-1 (immortalized chicken embryo fibroblast cell line) and 293T cells were obtained from American Type Culture Collection (Manassas, VA). DEF, DF-1, and 293T cells were cultured at 37 • C with 5% CO 2 in DMEM (Sigma, USA) supplemented with 10% fetal bovine serum (FBS, HyClone, Utah, USA), 100 units of penicillin G and 100 µg of streptomycin. DF-1 and DEF were infected with ATMUV CJD05 as previously described (Chen et al., 2016a) at the multiplicity of infection (MOI) of 1.0 and harvested at the indicated times after infection. Twenty five 7-day-old mule healthy ducklings were challenged with 0.4 mL of CJD05 (the 5th passage allantoic fluid virus, ELD 50 = 10 −6.0 /mL) per duckling by intramuscular injection. Each group of three randomly selected ducklings was sacrificed at 0, 12, 24, 48, and 72 h post-infection (hpi), and their spleen, kidney, bursa of fabricius, pancreas, and brain were harvested for detection of viral infection by indirect immunofluorescence assay. These tissues were also used for total RNA extraction to examine the mRNA expression of IFNs/IFITMs by quantitative real-time RT-PCR (qRT-PCR). The sera and spleen homogenates (30%w/v) of the ducklings were prepared for detecting of viral titers by 50% tissue culture infectious dose (TCID 50 ) assay during the infection. Other infected ducklings and 10 control ducklings (inoculated with 0.4 mL sterile PBS per duckling) were used to monitor clinical signs and rectal temperature daily for 10 days.