EMERGENCE OF SARS plates (Costar, Corning, NY). Virus inoculum was then incubated on cells for 1 h at 37°C, 5% CO2, and removed. The cells were overlayed with 2% carboxymethyl-cellu- lose (Sigma, St. Louis, MO)/ Dulbecco modified Eagle medium (DMEM) (Wisent, St. Bruno, Quebec), and incu- bated at 37°C, 5% CO2, for 4 days. At the end of the incu- bation, the cells were fixed with 4% formaldehyde and stained with crystal violet. The experiment was repeated twice. Cells Vero E6 and Vero V76 (ATCC) cells were maintained in 10% fetal bovine serum (FBS)/DMEM medium (Sigma). Porcine turbinate PT-K75 (ATCC) cells were maintained in 10% FBS/ DMEM (Wisent). Quail QT-35 (ATCC) cells were maintained in 10% FBS/MEM-alpha medium (Mediatech Cellgro, Herndon, VA). For the preparation of primary chicken embryo kidney epithelial cells (CEKEC), kidneys were harvested from 18- day-old chicken embryos and digested with 3 U of pronase (Sigma)/mL in citrate buffer (1.5 mM KCl, 27 mM sodium citrate, 8 mM KH2PO4, 5.6 mM Na2HPO4, pH 7.3) by repeated incubation for 2 min at 37°C with stirring. Cells were collected into fetal bovine serum and washed exten- sively with phosphate-buffered saline before being seeded into 24-well plates (Costar). Cells were seeded at a densi- ty of 106 cells/cm2 in 1% FBS/Williams medium (Sigma) for the virus isolation. The cell suspension contained about 95% epithelial cells and 5% fibroblasts after 24 h of incu- bation, as previously determined by immunofluorescent assay; markers for epithelial cells (cytokeratin) and fibrob- lasts (vimentin) were detected. Experimental Infection The preimmune serum from chickens and pigs was col- lected 2 days before inoculation. Six-week-old pigs were inoculated simultaneously by four routes, intravenously, intranasally, ocularly, and orally, with 2 x 106 PFU of SARS-CoV per pig. Six-week-old chickens were inoculat- ed by the same routes with 106 PFU of SARS-CoV per chicken. Three pigs and three chickens were mock inocu- lated and served as negative controls. Both species were divided into two groups, and blood, nasal (nares), throat, and rectal (cloacal) swabs were collected on alternate days, starting at 2 days after inoculation (dpi) and ending at 7 dpi. On days 6, 7, 13, 14, 15, and 16 after inoculation, one pig and one chicken per day were euthanized. In addition to swabs and blood, samples from lung, trachea, liver, heart, spleen, kidney, tonsil (pig), and jejunum were col- lected at postmortem examination. All experimental work was carried out in BSL4 containment. Virus Isolation Virus isolation from porcine samples was attempted on Vero V76 and porcine turbinate cells PT-K75, seeded at a density of 2 x 105 cells/cm2 in 12-well plates (Costar) 24 h before inoculation. Samples were tested in duplicate twice, by plaque assay (described in Virus section) and monitor- ing cytopathic effect (CPE), followed by reverse transcrip- tase–polymerase chain reaction (RT-PCR) to detect virus replication. In addition, virus isolation from chicken sam- ples was attempted on chicken embryo epithelial kidney cells, seeded at a density of 106 cells/cm2 in 24-well plates (Costar), using CPE format followed by RT-PCR. The tissues were ground in a MiniMix blender (Topac, Hingham, MA) to prepare a 10% w/v suspension in Dulbecco’s PBS (Sigma) supplemented with antimicrobial drugs and stood for 1 h in the antimicrobial mix (strepto- mycin/vancomycin/nystatin/gentamycin). The suspension was centrifuged at 2000 x g, 4°C, 20 min. The supernatant was diluted 10-fold in the corresponding media for the individual cell types, and 400 µ L (in duplicates) was incu- bated on cells for 1 h at 37°C, 5% CO2. The inoculum was then removed and replaced with the appropriate media, supplemented with 5% FBS (Vero and PT-K75 cells) or 1% FBS (CEEKC). Plates were incubated for 5 days at 37°C, 5% CO2. Isolation from blood and swabs was per- formed as for tissues without the homogenization step. The sensitivity of virus isolation was determined by spiking negative control lung tissues from one chicken and one pig with virus inoculum before homogenization, titrating out the samples on Vero E6 and Vero V76 cells, and compar- ing the titers to the inoculum titer, using plaque assay described in the virus section. RT-PCR RNA was extracted from blood and tissue samples with the TriPure Extraction kit (Roche Diagnostics, Indianapolis, IN). Three sets of primers were used in a one-step RT-PCR assay employing the Qiagen OneStep RT-PCR kit (Qiagen, Mississauga, ON): 1. NML poly- merase primers: forward primer CAG AGC CAT GCC TAA CATG and reverse primer AAT GTT TAC GCA GGT AAG CG were used in the RT-PCR reaction (50°C for 30 min, 95°C for 15 min, followed by 50 cycles of 94°C for 15 s, 50°C for 30 s, 72°C for 30 s with 7-min extension at 72°C). The 389-nt amplicon is located within the RNA- dependent RNA polymerase gene (ORF 1b). 2. Nucleocapsid (N) primers: forward primer ATA ATA CTG CGT CTT GGT TC and reverse primer TGG CAA TGT TGT TCC TTG AG were used under the same reaction conditions as the first set of primers, yielding a 364-base pair (bp) long amplicon. 3. BNI polymerase primers and RT-PCR conditions were developed at the Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany, by 180 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 2, February 2004