Pathogenesis of severe acute respiratory syndrome

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Severe acute respiratory syndrome (SARS) first emerged in Guangdong province, China in November 2002. During the following 3 months, it spread rapidly across the world, resulting in approximately 800 deaths. In 2004, subsequent sporadic cases emerged in Singapore and China. A novel coronavirus, SARS-CoV, was identified as the etiological agent of SARS.1,2 This virus belongs to a family of large, positive, single-stranded RNA viruses. Nevertheless, genomic characterization shows that the SARS-CoV is only moderately related to other known coronaviruses.3 In contrast with previously described coronaviruses, SARS-CoV infection typically causes severe symptoms related to the lower respiratory tract. The SARS-CoV genome includes 14 putative open reading frames encoding 28 potential proteins, and the functions of many of these proteins are not known.4 A number of complete and partial autopsies of SARS patients have been reported since the first outbreak in 2003. The predominant pathological finding in these cases was diffuse alveolar damage (DAD). This severe pulmonary injury of SARS patients is caused both by direct viral effects and immunopathogenetic factors.5 Many important aspects of the pathogenesis of SARS have not yet been fully clarified. In this article, we summarize the most important mechanisms involved in the complex pathogenesis of SARS, including clinical characters, host and receptors, immune system response and genetic factors. CLINICAL PATHOLOGY CHARACTERS OF SARS Patients with SARS-CoV infection have a wide spectrum of disease, varying from a self limiting illness to a fatal outcome.6,7 This disease consists of two phases, including prodromal influenza-like symptoms characterized by myalgia, malaise, chills and fever, and the onset of respiratory and gastrointestinal symptoms.8 Fever was the most common and the earliest symptom.9 The clinical picture is characterized by pulmonary inflammation and respiratory failure, resembling that of acute respiratory distress syndrome (ARDS). Upper-respiratory-tract symptoms are not prominent9 but gastrointestinal symptoms were common.10,11 A majority of the patients admitted to the hospital showed pulmonary X-ray abnormalities varying from bilateral interstitial infiltrates to focal consolidation.6,12 Autopsies of SARS cases indicated that the lungs were edematous and increased in weight.13,14 In some SARS cases organizing features, like dense septal and alveolar fibrosis, were demonstrated.15,16 The longer the disease persists, the more extensive becomes the fibrous organization of the lung tissue.17 Fibrin and collagen were found deposited in the alveolar space.18 Morphological changes identified were bronchial epithelial denudation, loss of cilia and squamous metaplasia. DAD is the most consistent finding in the lungs of SARS patients in the terminal stage.9 In SARS postmortem samples viral RNA has been localized by in situ hybridization to cells of the conducting airways and alveoli.19 The infection and release of virus was close to the pulmonary capillary bed, which might allow systemic spread of virus to distant organs, especially in the context of inflammation and alveolar capillary leak.20 In many cases, cellular infiltration has been observed, including macrophages, neutrophils and CD8+ T cells. Macrophages are a prominent component of the cellular exudates in the alveoli and lung interstitium.9,21 In addition, immunohistochemistry, in situ hybridization and electron microscopy examination of tissue upon autopsy or tissue biopsy showed that SARS-CoV replicates in pneumocytes and macrophages.22 The replication of SARS in macrophages suggests a passive role for macrophages as scavengers, rather than being the primary target.23 A disproportionate scarcity of inflammatory cells has been noted.5,13 Mononuclear infiltrates increased in the interstitium. Large multinucleated cells have been frequently observed in the lungs of SARS patients.5,14 The presence of hemophagocytosis supports the contention that cytokine dysregulation may account for the severity of the clinical disease. The lack of a prominent inflammatory response is also distinctive. Such changes reflect the combined effects of primary infection, host immune responses and therapeutic interventions. A substantial number of SARS patients have diarrhea.11,24 In the intestine, little pathology is observed at the light microscopy level, either in biopsies taken during early phases11 or in autopsy specimens.13,19 Severe depletion of mucosal lymphoid tissue in the small intestines and appendix has been described.25 Both extensive necrosis of the spleen and atrophy of the white pulp with severe lymphocyte depletion have also been found.14,26,27 A sharp decrease in the number of periarterial sheaths in the spleen have been demonstrated. CD4+ lymphocytes, CD8+ lymphocytes, CD20+ lymphocytes, dendritic cells, macrophages, and natural killer cells in the spleen showed a decrease of 78, 83, 90, 80, 39 and 48%, respectively. The average size of macrophages was found to be increased by more than 100%. T lymphocytes and macrophages in the spleen have been detected to be infected by SARS-CoV.27,28 Lymph nodes usually show atrophy and reduction of lymphocytes with loss of germinal centers.28 Focal necrotic inflammation of hilar lymph nodes has been found in some cases.29 Evidence of hemophagocytosis in lymph nodes was observed in a limited number of cases.30 High viral loads have been detected in lymph nodes, whereas viral isolation was negative.26 T lymphocytes and macrophages in lymph nodes have also confirmed SARS-CoV infection.28 Several observations suggest that SARS-CoV is also capable of causing an infection of the central nervous system. Cerebrospinal fluid, brain tissue specimens and neurons were detected to have SARS-CoV infection.28,31 Kidneys of autopsied SARS patients have shown focal necrosis and vasculitis of small veins in the renal interstitial tissue.14 High viral loads have been detected in the renal tissue specimens and the distal convoluted tubules, which suggest that urine may be an additional source of sewage contamination.32 In addition, monocytic infiltration, acute tubular necrosis and other nonspecific changes, such as glomerular fibrosis and nephrosclerosis, have been all observed.33 A high proliferative index has been demonstrated in hepatocytes in the liver in some cases. But viral particles are not detected by electron microscopy (EM).34 In both the liver and the kidney signals for SARS-CoV were detected by both immunohistochemical (IHC) and in situ hybridization (ISH),35 yet EM failed to reveal recognizable viral particles. This raises the question that whether the virus exists in a non-packaged form.25 Destruction of epithelial cells with significant changes in the follicular architecture was present in the thyroid glands. Both myofiber necrosis and atrophy were observed in the limited number of skeletal muscle tissue specimens of SARS autopsies examined.36 Edema of the walls of small veins and arteries has also been reported. The few available studies on adrenal glands described the presence of necrosis and vasculitis of the medulla with monocytic and lymphocytic infiltration. Edema of both myocardial stroma, as well as vascular walls, and atrophy of cardiac muscle fibers all have been demonstrated.14,22 SARS-CoV genomic sequences and antigens have also been detected in sweat glands and pancreatic islet cells.35,37 The presence of virus in the sweat glands suggests that SARS may be spread via contact with the skin.25 In some cases, evidence of reactive hemophagocytosis or bone marrow hypoplasia was present.29 Raised creatine kinase, thrombocytopenia, an increase in lactate dehydrogenase and a decrease in absolute lymphocyte counts are the most common laboratory findings. The majority of SARS patients showed a transient increase in serum alanine aminotransferase levels during the course of their disease.38 In some autopsy cases fatty degeneration were observed. These findings suggest that SARS is a systemic disease with widespread extrapulmonary dissemination, resulting in viral shedding in respiratory secretions, stools, urine and possibly even in sweat. The organ damage in patients with SARS could be due to both local viral replication and the immunopathologic consequences of the host response, hence it is important to delineate what human cells the SARS-CoV can infect and replicate in as well as the subsequent host immune response.35,39 HOST AND RECEPTORS SARS-CoV was isolated from Himalayan palm civets found in a live-animal market in Guangdong, China in 2003. The full-length genome sequences had 99.8% homology to the SARS-CoV genomic found in humans. So primarily, palm civets were suspected as the origin of the SARS outbreak in 2003. Subsequently, many other animals have also been found to be a host or to be infected by the virus.40 Bats and swine were also reported as natural carriers of SARS virus.41,42 Recently, horseshoe bats were designated as the natural reservoir for SARS-CoV-like virus and civets were identified as the amplification host. This highlights the importance of wildlife and biosecurity in farms and wet markets, which can serve as the source and amplification centers for emerging infections.43 SARS-CoV spreads via droplet and contact transmission and via the fecal-oral route.44 Through these routes, SARS-CoV can be transmitted from animal to human or from human to human. The primary target of SARS-CoV is epithelial cells in the respiratory and intestinal tract.18 Epithelia are primary barrier to infection by microorganisms entering their host via body cavities. Epithelial cells are organized in a polarized fashion that involves the separation of the plasma membrane into an apical and a basolateral domain. The polarity of these cells affects both the early and late stages of infection, i.e. viruses may enter into and exit from a cell either via the apical membrane facing the external environment or via the basolateral membrane directed to the internal milieu of the organism. An important determinant of the virus infection is the presence of suitable receptors on the cell surface that allow attachment to and penetration through the plasma membrane.20 Angiotensin-converting enzyme 2 (ACE2), a protector of lung damage, has been identified as the primary functional receptor for SARS-CoV.45 ACE2 is a membrane-associated aminopeptidase.46 A region of the extracellular portion of ACE2 that includes the first α-helix and lysine 353 and proximal residues of the N terminus of β-sheet 5 interacts with high affinity to the receptor binding domain of the SARS-CoV S glycoprotein.47 The N terminal half of the S protein (S1) contains the receptor binding domain whereas the C-terminal half (S2) is the membrane-anchored membrane-fusion subunit, which contains two heptad repeat regions (HR1 and HR2).48 After binding to ACE2 on the target cells, the transmembrane S protein changes conformation by association between the HR1 and HR2 regions to form a six helix oligomeric complex, leading to fusion between the viral and target-cell membranes. Apart from direct membrane fusion at the target cell surface, SARS-CoV might gain cell entry via pH-dependent endocytosis, which is also mediated by the S protein.49 In addition to being a cellular receptor, ACE2 may contribute to the pathogenesis of DAD in SARS through its role in the tissue rennin-angiotensin system (RAS).8 ACE2 is a negative regulator of the RAS and has a negative effect on the formation of angiotensin II. Angiotensin II appears to be one of the elements of the RAS that contributes to exacerbation of acute lung injury.50 With respect to SARS-related lung injury, binding of SARS-CoV Spike proteins to ACE2 has been found to reduce ACE2 expression, thus inducing acute lung edema.51 Based on animal experiments, ACE2 may protect against respiratory failure and down-regulation of ACE2 may cause acute lung injury. The insert/deletion genotype of the ACE gene was associated with DAD after SARS-CoV infection in a small cohort of 44 patients.52 ACE2 protein is reportedly present in type 1 and type 2 pneumocytes, enterocytes in all parts of the small intestine, the brush border of the proximal tubular cells of the kidney, as well as the endothelial cells of small and large arteries and veins and arterial smooth muscle cells.46 This localization of ACE2 explains the tissue tropism of SARS-CoV for the lung, small intestine and kidney. Theoretically, all tissues and cell types expressing ACE2 may be potential targets of SARS-CoV infection. However, notable discrepancies were found including virus replication in colonic epithelium, which has no ACE2, and no virus infection in endothelial cells, which have ACE2. Despite the fact that SARS-CoV can infect the lung and intestine the tissue responses in these two organs are different. Furthermore, studies in a new human cell culture model have indicated that the presence of ACE2 alone is not sufficient for maintaining viral infection.39,53 Other findings indicate that ACE2 expression positively correlated with the differentiation state of the epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected.18 It is apparent that the effect of SARS-CoV infection is different in different cell types and it is possible that the virus may utilize different receptors, or involve various co-receptors, in these different cells. C-type lectins, including CD209 and CD209L, were identified as alternative SARS-COV receptors.53 CD209, also known as dendritic cell-specific intercellular adhesion molecule-grabbing non-integrin (DC-SIGN), is mainly expressed in certain types of dendritic cells (DCs) and alveolar macrophages.54 However, in the lung tissue of SARS autopsies, CD209 has been localized to pneumocytes.55In vitro, CD209 was also inducible in lung epithelial and monocytic cells after SARS-CoV infection,55 which confirmed that SARS infection is capable of inducing CD209 expression. The glycosylated S protein has been shown to bind to the CD209 expressed on the DCs; these cells then mediate SARS-CoV infection in trans of cells that express human ACE2. CD209L, also known as L-SIGN or DC-SIGNR, is generally found in lymph nodes and liver sinusoidal cells. By IHC it has been demonstrated that CD209L is also expressed on type II pneumocytes and endothelial cells. CD209L can also bind to S protein and mediate virus entry.56,57 Although SARS-CoV does not replicate in DCs, these cells may act as a reservoir and distribute the virus to other cell types.58 This is an attractive concept and similar biological behaviours have been proposed for human immunodeficiency virus I (HIV I).59In vitro experiments have demonstrated that cells expressing CD209 or CD209L without ACE-2 are not, or are only partially, susceptible to SARS-CoV infection. This would imply that these molecules are much less efficient receptors than ACE2 as specific receptors and may therefore merely enhance infection of permissive cells.49,56,57 SARS-CoV infection of ACE2-expressing cells also seems to be dependent on the proteolytic enzyme cathepsin L. Cathepsin L is poorly expressed in endothelial cells which may explain the low infection rate of these cells despite the high expression of ACE2. SARS-CoV infection seems to be pH-dependent because the activation of cathepsin L is pH sensitive. Differential expression of cathepsin L in various cell types may explain the differences in viral distribution in relation to the ACE2 expression pattern.60,61 CYTOKINES AND CHEMOKINES Both cytokines and chemokines are soluble proteins with a key function in the innate immune system. Dysregulation of these proteins may result in immunemediated injury.5 High levels of cytokines and chemokines, triggered by the host immune response to SARS coronavirus (SARS-CoV), are believed to contribute to the progressive pulmonary infiltration of macrophages,9 polymorphonuclear leukocytes, T cells,62 eventual DAD and fibrosis.6 This assumption is supported by the clinical deterioration of many patients in the second week of the disease's course, despite decreasing viral loads.9,63 Increased serum levels of several cytokines were found in major SARS patients.51,57 Most cytokines showed only transient and short-lived activation in patients after SARS-CoV infection.64 Even in patients who developed DAD, most cytokine concentrations were not significantly increased.65 In contrast, circulating concentrations of several chemokines, including chemokine C-X-C motif ligand 9 or monokine induced by γ-interferon (CXCL9), chemokine C-X-C motif ligand 10 or interferoninducible protein-10 (CXCL10) and C-C motif ligand 2 or monocyte chemoattractant protein-1 (CCL2), were markedly increased in SARS patients.62,64,66 Recent studies have focused on the role of chemokines rather than cytokines in SARS infection.5 The chemokines are a family of small-molecule proteins that important in intercellular and Based on their protein are into with a common C-X-C of residues the which interacts with and with a C-C with In the lung tissues from SARS patients who chemokines and were markedly and a chemokine C-X-C motif ligand were also markedly A number of chemokines, including and were increased 1 after to the Most for the receptor for and was in the lungs of of gene expression changes in cells from in vitro with show an early activation of the innate in the first including expression of receptor 9 chemokines and their receptors with and and macrophages in the lungs express In in vitro cells chemoattractant protein 1 and after with SARS-CoV and expressed and vascular cell adhesion SARS-CoV induced cells to express and which and T cells in a The showed low expression of a and of cytokines necrosis and but significant of inflammatory chemokine It activation of T cell to express and and also to be significantly increased in lung tissue and lymphoid tissue of autopsied SARS which was confirmed by IHC with An increased has been found to be an of SARS-CoV, through a with lung epithelial cells and monocytic cells, an environment to immune cell and that to lung The lack of cytokine response against a of chemokine could a of immune by A model to explain cellular infiltration may result in SARS was pulmonary epithelial cells infected by SARS-CoV express adhesion molecules and high levels of and which macrophages and and macrophages by with SARS-CoV and a of chemokines that more and neutrophils as well as T viral effects are also to contribute to the pulmonary injury resulting from SARS-CoV infection. In during the first 10 of the disease, virus replication is viral effects to an important The presence of multinucleated cells in SARS lungs may be the result of viral The virus is also capable of causing effects in both renal epithelial cells and epithelial cells in and formation are in infected studies have reported evidence of in cells of the thyroid cells, epithelial cells, pneumocytes, lymphocytes and vitro experiments indicate that expression of certain proteins may in several cell expression of a protein by SARS-CoV, can via a dependent in cell from different organs, including lung, liver and kidney. of may be one of the mechanisms for the pathogenesis of SARS-CoV SARS protein also appears to be important in in some cell It is into the and may also act as one of the Through an host cells SARS have increased expression of Furthermore, SARS-CoV proteins also have the to in SARS-CoV proteins may be involved in through of proteins Increased expression of has been detected in infected alveolar and bronchial epithelial cells. is an of cell of this cytokine may also account for of such cells. OF SARS and viral in the first 10 of SARS immune by The innate immune system the first of the immune against viruses and involves several cellular and soluble factors. T lymphocytes and are the key immune cells that are infected by vitro infected cells have shown viral replication for to In from SARS SARS-CoV has also been found to infect and replication was Both T lymphocytes and were found in the circulating lymph nodes, lungs and in SARS These findings may a partial for the and the widespread of spleen and lymphoid tissue in the majority SARS immune cells may cause widespread to various and T cells are involved in both the innate and immune the of such cells may result in a immune with a decrease in both CD4+ and CD8+ T cells is common during the acute of SARS and may be associated with an Several other viral such as virus and respiratory virus are also associated with severe However, in these direct infection and subsequent of lymphocytes are generally as to account for the severe lymphocyte In for only a small of the are infected during acute with the high infection of in SARS immune syndrome with respect to the fact that both are viral that result in However, SARS various immune cells and rapidly whereas human immunodeficiency virus mainly CD4+ lymphocytes and is In addition, SARS-CoV seems to the of macrophages, which may SARS patients to pulmonary SARS-CoV also causes and functional of dendritic cells in vitro, resulting in a of cytokines and an T cells may in the to pulmonary injury. In viral in are and by infected cells. These cells cause cells to that to viral to other SARS-CoV is not capable of inducing significant or gene expression in infected macrophages, or in infected dendritic Furthermore, in contrast to patients with was a lack of expression of for the and in cells of patients with studies have found that from SARS patients could be an of of serum and against SARS-CoV and by against the that most patients after onset of can be detected as early as after the onset of The for and most of the is to In a of SARS the levels at and were for on the of 14 patients showed that the was in for a from developed a and and of the not The indicated that specific and could be in the could the SARS-CoV and protect cells from SARS-CoV The low response of SARS-CoV has been identified in which proposed a question that whether the serve as a host for were SARS cases that in Guangdong in In contrast to in these cases with clinical and caused a lower and transient immune The of the cases to a at at and then rapidly a of This that SARS-CoV may have to during the These can the particles the S protein from different SARS-CoV that these are and that the S protein is the S the other proteins, such as or is the only significant SARS-CoV and with as the major The on the S was also developed may also be involved in the pathogenesis of Both and immune responses to animal have been identified to be capable of the disease or causing new are with and coronavirus a of by with the SARS-CoV of were to the specific of the while of the could of the or These may have some of the that were to the and characterized the binding of SARS-CoV by an detected specific for the of an human serum In cohort of SARS patients immune against antigens from lung epithelial cell and endothelial cell was found in some approximately 1 after high levels of these in the were shown to be to lung epithelial cells and endothelial cells in may be to the of against specific SARS-CoV against the domain 2 of the protein have been found to with pulmonary epithelial that possibly explains is the of caused by organ has been a the of since some antigens in the may that enhance viral infection rather than also to a role in the pathogenesis of a of the was associated with severity of SARS This association has not been for certain Nevertheless, in the a association was demonstrated between and and an increased to SARS infection. is a serum protein that can bind to the of various for immune of a specific is capable of binding to the glycosylated SARS-CoV S protein and SARS-CoV in In a of SARS patients and have been shown to be associated with increased of In contrast, CD209L to have a significantly lower of SARS infection. The in genetic between is for by In the context of to after at the of were associated with high concentrations of in the plasma and a In addition to other genetic such as enzyme are also associated with severity and of Although ACE2 as its receptor and ACE2 is known to be an important protector of lung damage in no association between of the two ACE and and the severity of after SARS infection was is in the of different and the of in clinical is studies are to fully the genetic for both to infection and the after infection with the The pathogenesis of SARS appears to be and The most and possible appears to of a direct injury to the target cells by the virus and an injury mediated by subsequent immune system By droplet SARS-CoV the respiratory and the epithelial cells of the and infection and replication in target cells causes direct damage to the respiratory tract. inflammatory changes the of the barrier and increase the of the capillary of in the formation of membranes. The infection and associated inflammation acute injury of type II alveolar cells, decreasing the of alveolar resulting in alveolar the SARS-CoV and circulating immune cells. The infected immune cells mainly macrophages and T cells. immune cells the virus to other organs, including the spleen and the lymph The of immune cells with extensive damage to the white pulp in A immune the infection and replication of the virus in the lungs and viral damage to the respiratory resulting in respiratory The proposed mechanisms of SARS have significant for the and on this emerged disease. Although much has been of SARS since its many with respect to the pathogenesis of SARS is a that SARS for the of at 2 known animal in civets and horseshoe bats that are found in the as well as and in China. are