RESEARCH C. Drosten, and published on their Web site on March 26, 2003: BNI OUT S2: ATG AAT TAC CAA GTC AAT GGT TAC (forward); BNI OUTAS: CAT AAC CAG TCG GTA CAG CTA C (reverse). The RT-PCR conditions were: 30 min at 50°C, 15 min at 95°C, followed by 50 cycles of 95°C for 10 s, 56°C for 10 s, 72°C for 20 s, and complet- ed at 72°C for 7 min, yielding an amplicon of 195 bp. The Qiagen OneStep RT-PCR kit was also used for the two-step RT-PCR with the following modifications: the template was incubated at 50°C for 30 min only with for- ward N primer followed by the incubation at 95°C for 15 min to inactivate the reverse transcriptase. Residual single- strand RNA template was removed by digestion at 37°C for 20 min with 2 U of Rnase H (Invitrogen, Burlington, ON). After both forward and reverse N primers and the Platinum Pfx DNA polymerase (Invitrogen) were added, the DNA synthesis was completed by using the above con- ditions for the N primers in a one-step RT-PCR. Randomly selected amplicons were sequenced with the respective primers to verify the identity of the bands. Sensitivity of the individual primer sets used in the RT-PCR assays was tested by spiking negative control lung tissues from chick- en and pig with virus inoculum before homogenization, titrating out the homogenate, and running the RT-PCRs in parallel on the same RNA extracts. SARS-CoV Antibody Detection Porcine serum collected before inoculation and during the final bleed was tested for antibodies against SARS by a standard plaque reduction neutralizing test, as previous- ly described (5). Briefly, mixtures of pre-titered (100 PFUs) SARS-CoV and serial twofold dilutions of animal sera were incubated at 37°C for 1 h and added to 6-well plates containing Vero E6 cell monolayers. After a 37°C incubation for 1 h, a nutrient-agar overlay was added, and the plates were placed in a CO2 incubator for approximate- ly 3 days. A second overlay, which contained neutral red as a vital stain, was then added. Plates were then checked periodically over the next few days for plaque formation. The highest serum dilution, which produced a plaque reduction of at least 90%, was defined as the titration end point. SARS ORIGINS Porcine Serum Cross-Reactivity with TGEV/PRCV Serum samples collected on the pre-inoculation bleed and terminal bleed were tested for neutralizing antibodies against SARS and TGEV/PRCV by using microtiter CPE blocking assay. Each of the above viruses was diluted to 100 50% tissue culture infective dose (TCID50)/well, mixed with doubling serial dilutions of test serum begin- ning at 1:5 (giving the first serum dilution 1:10), and incu- bated for 1 h at 37°C. The virus-serum mixtures were then added to 96-well microtiter plates (Costar) containing overnight confluent monolayers of Vero V76 cells or PT- K75 cells, for the SARS or TGEV CPE-blocking assay, respectively. The results were read after 3 days of incuba- tion at 37°C, 5% CO2. Results Preliminary tests to establish a sensitive cell system for virus replication were performed before animal inocula- tion and virus isolation. SARS-CoV replicated in Vero E6, Vero V76, and PT-K75 approximately to the same titer. QT-35 did not replicate SARS-CoV. Although CEKEC did not show any CPE, the virus replicated in those cells up to the approximate titer of 106, based on positive RT-PCR results on lysed cells and cell culture supernatant harvest- ed 54 h after inoculation. Animal Inoculation Neither clinical disease nor gross pathologic changes were observed in chickens or pigs. Repeated attempts to isolate SARS-CoV from swabs, blood, and organs on Vero V76 had negative results. No significant (drop in titer within 1 log) impact of tissue processing on the infectivity of virus during virus isolation was observed by using lung tissues from one control chicken and one pig under control conditions. The tissue was spiked with SARS-CoV before homogenization, and virus recovery was compared to the correspondingly diluted inoculum on Vero E6 and Vero V76 cells (Table 1). Additional attempts at virus isolation were carried out on PT-K75 cells and, with chicken sam- ples, on chicken embryo kidney epithelial cells. The results were again negative, as confirmed by RT-PCR on the inoc- ulated cells. RT-PCR assays were undertaken by using three sets of primers, one developed at the National Microbiology Table 1. Relative sensitivity of virus isolation and RT-PCR in tissue samples spiked with the SARS virus before homogenizationa,b Virus isolation (PFU/100 µ L) RT-PCR (100 µ L) Tissue samples Vero E6 Vero V76 NML primers N primers BNI primers Virus control -5.8 -6 -8 -10 -10 Chicken lung -5.6 -5.75 -7 -9 -9 Pig lung -5.5 -5.7 -7 -9 -9 aRT-PCR, reverse transcriptase–polymerase chain reaction; SARS, severe acute respiratory syndrome; N, nucleocapsid. bThe highest dilution in which the virus or the RNA were detected is given in log10. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 2, February 2004 181