RESEARCH SARS ORIGINS Table 3. Summary of RT-PCR results on chicken tissuesa Lung Trachea Heart Liver Chicken no. dpi N BNI N BNI N BNI N BNI 115 6 - - - - - - - - 118 7 - - - - - - - - 114 13 + + + + - - - - 116 14 - - - - - - - - 113 15 - - - - - - - - 117 16 + - + - - - - - aRT-PCR, reverse transcriptase–polymerase chain reaction; dpi, days postinfection; N, nucleocapsid. N - - - - - - Spleen BNI - - - - - - Kidney N BNI - - - - + - - - - - + + N - - - - - - Jejunum BNI - - - - - - Table 4. Overview of virus neutralization titers for pig preimmune and immune sera against SARS-CoV and TGEVa Pre-inoculation bleed serum antibody titer Final bleed serum antibody titer Pig no. VNT TGEV VNT SARS PRNT SARS VNT TGEV VNT SARS PRNT SARS 7 0 0 0 0 20 10 8 20+ 0 0 20 320 160 9 10 0 0 0 160 80 10 20 0 0 10 80+ 80 11 10 0 0 0 40 40 12 20 0 0 0 80 80 aDetermined by microtiter virus neutralization test (VNT) and plaque reduction neutralization test (PRNT). SARS-CoV, severe acute respiratory syndrome-coronavirus; TGEV, transmissible gastroenteritis virus. of 5 PFU/100 µ L (porcine blood), the detection of RNA, corresponding to a minimum of 10-3 PFU/ 100 µ L, in blood at 48 h after inoculation is likely not due to a non- replicating residual virus inoculum. Our data suggest that pigs and chickens of the age used in the experiment were infected with SARS-CoV and, to a very limited degree, supported virus replication. The unsuccessful attempts at virus isolation could be explained by a very low rate of virus replication perhaps combined with loss of infectivity during the sample collection and processing. Although the observed decrease in virus recovery (virus spiked control samples) is not significant, it may have played a role in case of the low virus load. The intravenous route likely does not represent the route of infection in a field situation, and the question of possible natural infection of chickens and pigs with SARS-CoV remains open. Neutralizing antibodies against SARS-CoV developed in the pigs within 2 weeks of inoculation. These antibodies did not cross-react with TGEV/PRCV in a TGEV neutral- ization assay (PRCV and TGEV are indistinguishable in the virus neutralization assays) (10). Likewise, the preinoculation serum samples with the highest TGEV-neu- tralizing antibodies did not neutralize SARS-CoV, and the neutralizing antibodies against TGEV decreased as the SARS antibody titers increased. Based on the serum neu- tralization tests, TGEV/PRCV and SARS-CoV do not appear to be antigenically closely related, an observation supported by the initial genomic analysis (1,2). The cross- neutralization with TGEV/PRCV was initially a concern after the publication of immunohistochemical assays on SARS-CoV–infected cells (11). Since virus-neutralizing antibodies often take approximately 3 weeks to develop in chickens, no conclusions were made with regard to the low or absent antibody titers in their sera at 2 weeks after inoc- ulation. In conclusion, the limited extent of virus replica- tion as indicated by RT-PCR, the failure to isolate the virus, and the lack of virus shedding indicate that neither pigs nor chickens are likely to play a role as an amplifying host. Acknowledgments We thank Gopi Nayar for the results of a competitive enzyme-linked immunosorbent assay for porcine respiratory coronavirus/transmissible gastroenteritis virus; June Larence for the preparation of the primary chicken embryo epithelial kidney cells; and Harvey Artsob for the critical review of the manuscript. Dr. Weingartl is head of the Canadian Food Inspection Agency’s Special Pathogens Unit at the Canadian Science Centre for Human and Animal Health. Her research interests are emerg- ing and zoonotic viruses of veterinary importance. References 1. Marra MA, Jones SJM, Astell CR, Holt RA, Brooks-Wilson A, Butterfield YSN, et al. The genome sequence of the SARS- associated–CoV. Science 2003;300:1399–404. 2. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP, et al. Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 2003;300:1394–9. 3. Kuiken T, Fouchier RA, Schutten M, Rimmelzwaan GF, van Amerongen G, van Riel D, et al. Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 2003;362:263–70. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 10, No. 2, February 2004 183