Re: To KF, Tong JH, Chan PK, et al . Tissue and cellular tropism of the coronavirus associated with severe acute respiratory syndrome: an in‐situ hybridization study of fatal cases. J Pathol 2004; 202: 157–163

We read with great interest the excellent article by To et al, which was published in the February issue of The Journal of Pathology [1]. Using in situ hybridization (ISH), To et al demonstrated the presence of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) in pneumocytes and in the surface enterocytes of the small intestine. Their findings are compatible with those of another study conducted in Taiwan [2]. This observation indicates the cellular tropism of the infectious agent and thus has an impact on our understanding of the disease. Supplementing their findings, we report here our recent study, which suggests that the SARS-CoV may not be exclusively located in pneumocytes, but also in pulmonary macrophages. The lung tissue that we studied was a necropsy specimen provided by one of us (CHH). The patient was a 36-year-old woman who died of SARS. Histological examination of the lungs revealed diffuse alveolar damage. The SARS viral genome had already been verified in the tissue by nested RT-PCR [2]. The full-length spike protein (S) cDNA, membrane protein (M) cDNA, and nucleoprotein (N) cDNA clones were kindly provided by the National Research Program for Genomic Medicine and the Genome Research Center, National Yang-Ming University, Taiwan. These inserts were amplified by PCR and labelled with digoxigenin using the DIG-High Prime kit (Roche, Germany). The tissue sections were hybridized with the mixed probes (S, M, and N), 3 ng/μl, at 42 ◦C overnight. BCIP/NBT was employed as a chromogen [3]. Under low magnification, scattered cells containing dark bluish signals, which represented the SARS-CoV, were visualized (Figure 1A, nuclear fast red counterstain) within the damaged alveolar spaces, small bronchioles, and even the vascular lumina. High magnification revealed that the virus-carrying cells harbouring the viral particles were frequently multinucleated and laden with anthracotic pigment (Figures 1B and 1C: empty arrows indicate anthracotic pigments; arrows indicate virus-carrying multinucleated cells; nuclear fast red counterstain). On the basis of our findings, we believe that some, if not all, positive cells are of the histiophagocytic lineage. First, the co-existence of viral signals and anthracotic pigment in the same cells indicates that Figure 1. (A) Virus-carrying cells are identified by dark bluish signals in lung (200×). (B and C) Viral signals (arrows) and anthrotic pigments (empty arrows) sometimes coexist in the same cells, indicating that these macrophages also harbour SARS-CoV (1000×)