EMERGENCE OF SARS Laboratory during the investigation of the Toronto out- break of SARS, which targeted the polymerase gene, a sec- ond (BNI) set also within the polymerase gene, and the third set targeting the nucleocapsid gene region. Due to the presence of 3′-coterminal nested mRNAs and genomic RNA (6,7) during coronavirus replication, nucleocapsid RT-PCR was expected to be more sensitive in samples containing replicating virus. The originally used NML primers were less sensitive than the other two sets of primers (BNI pol and N), and the samples were retested with these two sets of primers. Sensitivity of the RT-PCR employing the individual primer sets is illustrated in Table 1, as determined by using negative control lung tissues spiked with SARS-CoV. RT-PCR with the N and the BNI primers detected viral RNA equivalent to approximately 10-3/-4 PFU. RT-PCR amplicons were detected in blood samples from chickens and pigs at 2 (pig 9, chickens 114 and 115) and 3 (pigs 10, 11, 12, chickens 116, 117, 118) dpi using the NML polymerase primers. Positive results using a two- step RT-PCR assay, aimed at detecting negative strands of viral RNA, indicated that replicating virus was present in the above positive pig and chicken blood samples (Figure). By using N primers and the BNI primers, viral RNA was detected in blood of all inoculated chickens up to 7 dpi and in chicken 113 at 15 dpi (Table 2). No viral amplicons were generated from any of the har- vested organs or swabs when the NML polymerase primers were used; however, the N primers yielded amplicons from spleens of two pigs at days 7 and 13 after inoculation, and in a number of chicken organs. Lung, kidney, and trachea were positive in some birds at 13 to 16 dpi, while liver, spleen, and jejunum samples were all negative. These results were confirmed with BNI polymerase primers Figure. Amplification of severe acute respiratory syndrome–asso- ciated coronavirus (SARS-CoV) RNA in chicken blood, using one- step and two-step reverse transcriptase–polymerase chain reac- tion (RT-PCR) with nucleocapsid primers. Lane 1: 100-bp ladder, the bright band representing 600 bp; Lane 2: chicken 115, 2 days postinocuation (dpi), one-step RT-PCR; lane 3: chicken 115, 2 dpi, two-step RT-PCR (detecting negative-strand RNA); lane 4: chick- en 117, 3 dpi, one-step RT-PCR; lane 5: chicken 117, 3 dpi, two- step RT-PCR; lane 6: chicken 115, 4 dpi, one-step RT-PCR; lane 7: chicken 115, 4 dpi, two-step RT-PCR; lane 8: SARS-CoV–infect- ed cells; lane 9: negative control. Table 2. RT-PCR on blood samples from chickens using different primer setsa Primers dpi Chicken no. NML pol N BNI pol 2 113 - + + 114 + + + 115 + + + 3 116 + + + 117 + + + 118 + + + 4 113 - + + 114 - - + 115 - + + 5 116 - + + 117 - + + 118 - - - 6 113 - + + 114 - + + 115 - + + 7 116 - + + 117 - - - 118 - - - 13 114 - - - 14 116 - - - 15 113 - + + 16 117 - - - aRT-PCR, reverse transcriptase–polymerase chain reaction; dpi, days postinfection; pol, polymerase; N, nucleocapsid. (Table 3). Sequence analysis of selected amplicons con- firmed the SARS-CoV nucleotide sequence. No SARS-CoV–neutralizing antibodies (90% reduction of virus plaques on Vero E6 cells) were detected in pre- bleedings from pigs and chickens. The preexisting anti- bodies against PRCV/TGEV in pigs did not neutralize SARS-CoV and decreased during the experiment. Neutralizing antibody against SARS-CoV developed in pigs, with titers ranging from 1:10 to 1:160 at the time of euthanasia. The SARS antibody titers corresponded for both types of virus neutralization tests (the macrotiter plaque reduction assay and the microtiter CPE blocking assay). Table 4 summarizes the changes in SARS- and TGEV-neutralizing antibodies in pigs during the course of the experiment. No antibodies >1:10 were detected in chicken serum samples on the final bleed. Discussion After the experimental exposure of chickens and pigs to SARS-CoV, we detected coronavirus RNA in blood and several tissues from both species starting at 2 days after inoculation. Clearance of low or nonreplicating intra- venous inoculum from blood, including the viral RNA, occurs rapidly in a number of viruses (8,9). In light of the typical clearance rates and the estimated initial virus load 182 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 2, February 2004