Multiple organ infection and the pathogenesis of SARS

Gu, Jiang; Gong, Encong; Zhang, Bo; Zheng, Jie; Gao, Zifen; Zhong, Yanfeng; Zou, Wanzhong; Zhan, Jun; Wang, Shenglan; Xie, Zhigang; Zhuang, Hui; Wu, Bingquan; Zhong, Haohao; Shao, Hongquan; Fang, Weigang; Gao, Dongshia; Pei, Fei; Li, Xingwang; He, Zhongpin; Xu, Danzhen; Shi, Xeying; Anderson, Virginia M.; Leong, Anthony S.-Y.

Procedures for histopathologic investigation of the autopsies for LM and EM were those performed routinely in laboratories as described previously (26, 27) . The technique of in situ hybridization was based on Zhang et al. (28) . In brief, a sequence of 154 bp was amplified by polymerase chain reaction from the SARS coronavirus genome sequence. These sequence data are available from GenBank/EMBL/DDBJ under accession no. AY274119. The RNA probe was prepared by in vitro transcription in the presence of digoxigenin-UTP. The in situ hybridization reaction was performed on formalinfixed and paraffin-embedded tissue sections with the SARS probe at 50 g/ ml. The hybridization cocktail was incubated at 55ЊC for 16 h. The alkaline phosphatase-labeled antidigoxigenin antibody (1:500) was incubated for 60 min, and colorization was achieved with nitroblue tetrazolium/5-bromo-4chloro-3-indolyl phosphate and counterstained with methyl-green. Immunoelectron microscopy was based on the technique of Goldsmith et al. (29) . The EM in situ hybridization technique was based on Vandell et al. (26) . In brief, the in situ hybridization reaction was performed on the tissue grids in a procedure similar to that for the LM. The labeled probe was detected with colloidal gold (10 nm)-labeled antibody to digoxigenin. Immunohistochemistry was performed using a range of monoclonal antibodies to identify the different cell types that were infected or affected by the virus (Table I) . Immunohistochemistry was performed as described by Lin et al. (30) , and antigen retrieval technique was performed as described previously (31, 32) . Real time RT-PCR was performed with the technique of Wu et al. (33) . In brief, total RNA was extracted from clinical samples using Trizol reagents according to the manufacturer's instructions. 1 g of total RNA was transcribed into cDNA with the superscript first-strand cDNA synthesis system. Taqman real-time quantitative PCR was conducted according to the manufacturer's instructions (Applied Biosystems). Three pairs of primers were designed and constructed (Genbank G130027616 [15121-15661]). The designs of the primers and the probes that were used for real-time PCR and in situ hybridization were those described previously (33) . They were 5Ј-GCGCAAG-TATTAAGTGAGATG-3Ј and 5Ј-GAAGTGCATTTACATTGGCTA-3Ј as the innermost pair; 5Ј-ACACTTGCTGTAACTTATCAC-3Ј and 5Ј-TCATAGAGCCTGTGTGTGAGG-3Ј as the outer pair; and 5Ј-CTAA-CATGCTTAGGATAATGG-3Ј and 5Ј-CAGGTAAGCGTAACTTAT-CAC-3Ј as the outermost pair. The molecular beacon technique was used (33) . The sensitivity and specificity of this standardized real-time RT-PCR assay for the detection of SARS-CoV in postmortem lung tissues were established previously (34) .