JMN designed the study. JB analyzed the data. KHC contributed to writing the paper. JMN, MW, and JSMP are the principal investigators, with overall responsibility for the design of the study and writing of the report. SLB coordinated the autopsy organization and collection of autopsy materials of SARS deaths in Hong Kong. LLMP designed the protein expression constructs and transfection studies. LLMP and JSMP were involved in the development of the RT-PCR assay. JMN and KHC were involved in the development of the SARS-CoV IHC assay. SP was involved in the treatment of the Canadian SARS cases and the coordination of obtaining autopsy specimens from those who died. All authors were involved in the correlative interpretation of the clinical, pathological, and molecular data.
The authors have declared that no competing interests exist.
Cellular localization of severe acute respiratory syndrome coronavirus (SARS-CoV) in the lungs of patients with SARS is important in confirming the etiological association of the virus with disease as well as in understanding the pathogenesis of the disease. To our knowledge, there have been no comprehensive studies investigating viral infection at the cellular level in humans.
We collected the largest series of fatal cases of SARS with autopsy material to date by merging the pathological material from two regions involved in the 2003 worldwide SARS outbreak in Hong Kong, China, and Toronto, Canada. We developed a monoclonal antibody against the SARS-CoV nucleoprotein and used it together with in situ hybridization (ISH) to analyze the autopsy lung tissues of 32 patients with SARS from Hong Kong and Toronto. We compared the results of these assays with the pulmonary pathologies and the clinical course of illness for each patient. SARS-CoV nucleoprotein and RNA were detected by immunohistochemistry and ISH, respectively, primarily in alveolar pneumocytes and, less frequently, in macrophages. Such localization was detected in four of the seven patients who died within two weeks of illness onset, and in none of the 25 patients who died later than two weeks after symptom onset.
The pulmonary alveolar epithelium is the chief target of SARS-CoV, with macrophages infected subsequently. Viral replication appears to be limited to the first two weeks after symptom onset, with little evidence of continued widespread replication after this period. If antiviral therapy is considered for future treatment, it should be focused on this two-week period of acute clinical disease.
The SARS coronavirus targets primarily the pulmonary alveolar epithelium. Viral replication seems limited to the first two weeks after symptom onset and restricted to the lungs.
The first SARS (severe acute respiratory syndrome) outbreak started in November 2002 in China, and over the next five months spread to a number of other cities around the world. During that time, 20%–30% of the infected people became seriously ill, and 10% died. SARS is caused by the SARS-CoV, which infects the lungs of patients and leads to breathing problems. Since the outbreak, scientists around the world have been working hard to understand the SARS virus and how it causes disease, and to develop drug treatments and vaccines.
For this study, researchers from Hong Kong and Toronto (both cities hit by the SARS outbreak) joined forces for a large and detailed study of the bodies of patients who had died from SARS. They hoped that this would help them to understand how the virus had killed the patients, and when and how certain treatments might be most effective. The researchers wanted to see whether there were differences between individuals who died from SARS at different times after they first fell ill. They were also interested in the distribution of SARS-infected cells in the lungs of these patients, and whether they could detect the SARS virus outside of the lung in other parts of the body.
They studied the organs of 32 patients who had died from SARS. The researchers had detailed clinical information on each of them. Some had died less than a week after they became ill, and others had died after more than a month. The researchers used a variety of modern scientific tools to visualize exactly where in the bodies the SARS virus had reached. They detected the virus in the lungs of most patients who died within two weeks of falling ill, but not in the lungs of any of the patients who died after more than two weeks of illness. Where detectable, the virus was mostly found in an area of the lung called the alveolar epithelium. Each of your lungs is made up of a network of breathing tubes, and each tube ends in a tiny pouch called an alveolus, which is surrounded by blood vessels. When you breathe in oxygen, it eventually passes into your bloodstream across the lining of these pouches, and this lining is the alveolar epithelium. The researchers also examined other organs (including heart, kidney, and liver) from two of the patients whose lungs had tested positive for the virus and three others who had shown no sign of virus in the lungs. They did not detect the virus in any of these samples.
These results suggest that the SARS virus causes illness and death by attacking cells in the alveolar epithelium. The virus multiplies mostly within this group of cells, and only for a limited time after infection. Antiviral drugs are likely to be only useful during this initial time window after infection. After less than two weeks, the body's immune system seems to be able to fight back and prevent the virus from further multiplying, but this does not lead to recovery in all patients. In fact, most of the patients in this study (25 out of 32) died more than two weeks after they became ill and without signs that the SARS virus was still multiplying in their body. This suggests that in patients who die, the initial damage to the lungs is so severe that they cannot recover, and this is not dependent on continued virus replication. The study also suggests that death from SARS is not due to the virus multiplying outside of the lungs.
The following Web sites provide information on SARS.
Pages from the Government of Hong Kong:
Health Canada pages:
Pages from the US Centers for Disease Control and Prevention:
World Health Organization pages:
MedlinePlus pages:
Severe acute respiratory syndrome (SARS) is a new disease that originated in the Guangdong province of China in November 2002, and subsequently spread to Hong Kong in February 2003 [
The causative agent has now been determined to be a novel coronavirus (SARS-CoV) that is genetically distinct from any previously identified coronavirus known to cause disease in animals or humans [
The pulmonary pathology observed in limited postmortem material has been reported together with the clinical symptomatology of SARS [
This study therefore had three purposes. The first was to investigate the presence of SARS-CoV by immunohistochemistry (IHC) and in situ hybridization (ISH) in lung autopsy specimens from patients who died at different time points of the disease. The second purpose was to determine the cellular distribution of SARS within the lung, and the third was to examine extrapulmonary tissues of patients who died of SARS to determine the extent of systemic distribution.
A total of 32 patients who died with SARS-CoV infection confirmed by PCR, serological, or viral culture tests were included in the study. The patients came from two geographical regions affected by SARS (Hong Kong [HK] and Toronto, Canada [TO]). For HK cases 1 and 10–13, whole lung specimens were perfused at autopsy with 4% neutral buffered formalin at 20–30 cm H2O pressure until the lungs were fully expanded, before immersion in formalin fixative. Multiple blocks were extensively sampled from all lobes of the lungs for HK cases 1, 6, 7, and 9–13; limited representative blocks were sampled from HK cases 2–5 and 8. For all Toronto cases, approximately half of each lung lobe was removed at autopsy, and a portion was snap-frozen for molecular studies. Multiple blocks of tissue were processed from each lobe for light microscopic examination. Stains for microorganisms and for collagen, and immunohistochemical stains for microorganisms, were performed as necessary. The clinical profiles were retrieved from the patients' records. HK case 1 was a patient who died in the quarantine period for SARS exposure during hospitalization for congestive heart failure. PCR of a throat swab was positive on the fifth day after symptom onset, and she developed fever and respiratory symptoms thereafter. HK case 13 was a patient who died unexpectedly during convalescence from SARS. All other patients died of SARS with progressive deterioration in lung function. All HK patients except cases 1 and 13 had received ventilatory support during life. The pulmonary histopathological features of six of the HK cases (2–6 and 8) have been previously reported [
BALB/c mice were immunized intraperitoneally with 0.1 ml of heat-killed SARS-CoV HKU39849-infected FRhK4 cell lysate (107 TCID50/ml). Injections of similar doses were repeated biweekly for 2 mo. Four days after the last booster, 108 spleen cells from an immunized mouse were fused with 107 of NSI myeloma cells with polyethylene glycol (PEG, molecular weight 4,000; BDH, Poole, United Kingdom) as the fusing agent. Hybridomas were screened for production of antibodies against SARS-CoV HKU39849-infected cells and recombinant SARS-CoV nucleocapsid protein by ELISA. Those that produced SARS-CoV nucleoprotein-specific antibodies were cloned twice by limiting dilution. Purified hybridomas were then injected intraperitoneally into mineral oil-primed mice for the production of ascitic fluid. Monoclonal antibodies (4D11 and 3E4) were purified from the ascitic fluid by precipitation with 50% ammonium sulfate and subcloned to ensure monoclonality.
The open reading frames of spike (S), envelope (E), and N genes of SARS-CoV were cloned into the AgeI site of a protein expression vector, pcDNA3A [
The primary antibodies used included those against cytokeratin (1:50, clone AE1/AE3 [Dako, Glostrup, Denmark]), CD68 (1:50; clone KP1, Dako), EMA (clone E29, Dako), thyroid transcription factor 1 (TTF1; clone 8G7G3/1, Zymed/Invitrogen), chromogranin (clone LK2H10 [Ventana, Tucson, Arizona, United States]), DC-SIGN (a gift from Professor J-L Virilizier, Pasteur Institute, Paris, France), LCA (clone T29/33, Dako), and SARS-CoV N (as described above) (1:400, clone 4D11). Antigen retrieval was performed by microwaving sections in 10 mM citrate buffer (pH 6.0) for 15 min and incubating with 1:200 4D11 antibody at 4 °C overnight. Secondary labeling was performed with biotinylated rabbit anti-mouse (Dako #E-0354) at 1:100 for 30 min at room temperature and was followed by incubation with streptavidin-ABC complex (Dako #K-0377) at 1:100 for 30 min at room temperature and color development by the 3-amino-9-ethylcarbazole (AEC) substrate kit (Vector Laboratories, Burlingame, California, United States; #SK-4200) at room temperature (15–30 min). For double labeling of lung tissue sections, the 4D11 antibody was labeled with FITC, and a TRITC anti-mouse secondary antibody was used. Sections were microwaved in 10 mM citrate buffer (pH 6.0) for 15 min, blocked with 10% normal donkey serum for 10 min at room temperature, incubated with the non-SARS monoclonal overnight, then incubated with TRITC-conjugated donkey anti-mouse antibody at 1:100 for 1 h at room temperature. The FITC-conjugated 4D11 antibody was incubated at 1:100 for 1 h at room temperature, followed by counterstaining of the nuclei with DAPI for 4 min and mounting with DAKO fluorescence mount (Dako #S-3023). Examination was performed with a Nikon Eclipse E-800 fluorescent microscope with a dual FITC/rhodamine filter.
We produced digoxigenin-labeled antisense riboprobes specific for the N, E, and S genes of SARS-CoV, and these were pooled and hybridized with 30 μg/ml proteinase K-treated lung sections for 30 min at room temperature. The hybridization buffer (250 μg/ml salmon sperm DNA, 125 μg/ml rat total RNA, 200 mg/ml yeast tRNA, 50% deionized formamide, 10% dextran sulfate, 1× Denhardt's solution, 1 mM EDTA [pH 8.0], 0.01% sodium pyrophosphate, 0.3 M sodium chloride, and 10 mM Tris-HCl [pH 8.0]) was mixed with the probes and hybridized overnight at 50°C. The hybridized probes were detected with mouse anti-digoxigenin at 1:100 in 10% normal rabbit serum for 1 h at room temperature, then biotinylated rabbit anti-mouse (Dako, E-0354) at 1/100 for 30 min. at room temperature, followed by detection with AEC substrate kit (Vector, SK-4200) at room temperature (up to 30 min). Prior to the tests, the sensitivity of the SARS-CoV probes were verified semiquantitatively by hybridization to separate cell block preparations of a SARS-CoV infected FRHK4 cell line harvested at 8 and 24 h.
SARS-CoV RT-PCR was completed on fresh (HK cases 1 and 10–13), fixed (HK cases 2–9), and snap-frozen (all TO cases) lung tissues from all patients. For the HK cases, total RNA was extracted by standard methods and reverse transcribed to cDNA with reverse transcriptase. PCR was performed with initial denaturation at 94 °C for 8 min followed by 40 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min using primers as previously described [
The HK and TO cases' clinical profile, treatment history, and SARS-CoV IHC, ISH, and RT-PCR results are summarized in
HK patient 1, who died 5 d after symptom onset during quarantine for SARS contact, showed moderate interstitial and alveolar edema with occasional epithelial desquamation and regeneration. A moderate number of macrophages with abundant foamy cytoplasm had accumulated in the alveolar spaces, but giant cells were not observed. In addition, features of aspiration pneumonia were present, involving small bronchi and adjacent parenchyma in all lobes of the lung. HK patients 2–12 showed established changes indicating diffuse alveolar damage (DAD) at different phases of disease progression that affected the lungs to varying extents. Lung pathology of the TO cases has been described previously [
Although most of the advanced histological changes revealing DAD can be attributed to ventilation, it should be pointed out that there were also changes of DAD present in five of the TO cases in which no intubation was carried out; thus, viral damage is therefore the most likely mechanism of these changes.
The transfected cells showed strong cytoplasmic staining for transfectants containing the N gene with no staining on the M or S gene transfectants (
SARS-CoV immunohistochemical staining of 293T cells transfected with spike (S), nucleocapsid (N), and spike, nucleocapsid, membrane, and envelope (SEMN) genes of SARS-CoV. Untransfected control is shown in the lower right photomicrograph. The monoclonal antibody 4D11 identifies the N protein. AEC stain with hematoxylin counterstain; magnification 100×.
Four cases showed positive staining for N protein by IHC (HK case 1 and TO cases 1, 4, and 5) (
No staining for N protein is present in the bronchus of HK case 1 (A), but there is focal positive staining of bronchiolar epithelium in this case (B). Positive staining is seen in epithelial cells and detached cells in the alveoli in TO case 1 (C), which on higher magnification morphologically resemble type 1 pneumocytes (D). In TO case 2, there is a thrombus present which contains mononuclear and spindle-shaped cells (E). Carbon-containing macrophages are also positive (F). Ten days after symptom onset, positive staining was reduced and is noted mainly in the exudates (G) with only very focal positive mononuclear cells seen (H). AEC stain with hematoxylin counterstain; magnification 100×.
In the four cases that stained positive for N protein by IHC, double labeling with the directly conjugated FITC SARS monoclonal antibody and antibodies for macrophages and epithelial cells showed that the infected cells were positive for EMA (anti-epithelial) and CD68 (anti-macrophage) (
Shown are tissue samples stained with SARS-CoV monoclonal nucleocapsid FITC and epithelial membrane antigen TRITC. At day 5 postinfection, there is positive staining of type 1 pneumocytes (A), with epithelial cell debris present in the alveolar lumen (B). In one case (TO case 1), there is positive staining for SARS-CoV of the type 1 pneumocytes with no staining of CD68 positive macrophages (C and D). HK case 1, however, shows FITC staining within the cytoplasm of CD68-positive cells (E).
The same four cases that showed positive staining for N protein by IHC (HK case 1 and TO cases 1, 4, and 5) were also positive by ISH for the pooled S, E, and N SARS-CoV genes. Multiple lung samples from HK case 1 showed strong cytoplasmic signals in alveolar cells, including desquamating pneumocytes and alveolar macrophages. Weak signals were detected in occasional macrophages scattered along the bronchial epithelium. No signals were detected in endothelial or stromal cells. In addition to the four cases that stained positive for SARS-CoV by IHN and ISH, a few SARS-CoV infected cells were also detected by ISH and IHC in an open lung biopsy taken 10 d after symptom onset from HK case 5; however, both ISH and IHC were negative on the autopsy lung sample taken from this patient after death (20 d after symptom onset).
Results of RT-PCR are listed in
SARS-CoV nucleocapsid protein and RNA were detected in lung autopsy samples from four of the seven patients who died within 2 wk after illness onset, all of whom had high (greater than 105 copies per gram) RT-PCR viral loads in the autopsy lung tissue. SARS-CoV nucleocapsid protein and RNA were also detected in an open lung biopsy from a patient on day 5 after symptom onset (before death), but not in the same patient's autopsy lung specimen collected on day 20 after symptom onset. Among those lung samples staining positive by ISH or IHC, the signals localized mainly to the alveolar epithelial cells, with lower intensity in alveolar macrophages, indicating that the former are the chief target cells of the virus. Only scattered positive cells were identified in the bronchiolar epithelium, and no significant staining of the bronchial epithelium was observed. No distinctive spread to regional lymph nodes was seen, and we were not able to determine colocalization with DC-SIGN–positive cells. Using an oligonucleotide probe with signal amplification, Nakajima et al. demonstrated SARS-CoV genomes in the alveolar epithelium and macrophages of a 46-year-old woman with SARS, but details of the clinical course of the disease were not stated [
Among the 25 patients who died later than two weeks after symptom onset, no ISH or IHC signals were detected in postmortem lung tissues, even in lesions that were morphologically earlier, such as alveolar edema and hyaline membrane or in lungs showing heterogeneous morphological progression, where relatively uninvolved areas were interspersed among more advanced lesions.
These findings suggest that there is a decrease of SARS-CoV replication and, consequently, a decrease in intracellular viral copy numbers in lung tissue after the first two weeks of disease, with the terminal event not dependent on continued widespread viral replication. It has also been noted that viral loads detected by RT-PCR or viral culture from nasopharyngeal aspirates, urine, and stool samples start to decrease 10–15 d after symptom onset, correlating with the time taken for the development of specific anti-SARS antibodies, which starts approximately 10 d from disease onset [
The identification of the cells infected by SARS-CoV in humans has yielded contradictory findings. To et al., using ISH combined with IHC, found dual labeling of only epithelial cells using AE1/AE3 and negative staining for macrophages (antibody CD68) [
Our data also suggest that pneumocytes may be infected first, followed by macrophages; TO case 1, who died 5 d after symptom onset, had staining of pneumocytes but no other cells, whereas all other cases, who died five or more days after symptom onset had both pneumocyte and macrophage staining. It is possible that the material used for the To et al. study came from early cases. Shieh et al. describe finding type 2 pneumocytes positive for SARS-CoV in one patient [
The spectrum of pulmonary lesions in the cases described in this study is characteristic of the acute and resolving stages of acute lung injuries, including DAD due to intrapulmonary or extrapulmonary causes such as bacterial and viral infection, trauma, shock, etc. Unlike the findings of Gu et al., who demonstrated ISH-positive cells in giant cells [
Previous studies have published viral loads per gram of tissue using quantitative PCR. This quantitative PCR method has been shown to be of value in determining viral load of viruses in peripheral blood, such as Epstein-Barr virus; but unlike blood, lung tissue is not homogeneous and, in an assessment of viral copies per gram of tissue, whether the tissue sample contains bronchus, scar tissue, edema fluid, or exudates is crucial. In addition, blood in tissues may lead to false-positive PCR results due to viremia rather than viral replication in the tissue. In this study, persons with high SARS-CoV viral loads in their lung tissue had good correlation with the IHC and ISH results [
This study has shown that alveolar epithelium and macrophages are the chief targets of SARS-CoV infection in the human lung, in agreement with the findings reported in a primate model of the disease [
We thank Kevin Fung for expert immunohistochemical and hybridization technical support. We also acknowledge staff of the Queen Mary Hospital, United Christian Hospital, Kwong Wah Hospital, Princess Margaret Hospital, Queen Elizabeth Hospital, and Tuen Mun Hospital, Hong Kong, for facilitating case collection in Hong Kong. We also acknowledge the Office of the Chief Coroner of Ontario, Canada for facilitating case collection in Toronto, Canada, and we acknowledge G. Farcas, T. Mazzulli, B. Willey, S. Asa, P. Faure, P. Akhavan, D. E. Low, and K. C. Kain for their help in coordinating and completing SARS-CoV RT-PCR on the Canadian SARS patients' tissues; and the Canadian SARS Research Network for facilitating clinical chart reviews. This work was supported by the Vice Chancellor's Fund for SARS Research 2003 granted by The University of Hong Kong (21395059 and 21395052) and the National Institute of Allergy and Infectious Diseases, United States (public health research grant A195357). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
coronavirus
diffuse alveolar damage
immunohistochemistry
in situ hybridization
severe acute respiratory syndrome
SARS coronavirus