Chin Med J 2008;121(17):1722-1731 1722

Medical progress

Pathogenesis of severe acute respiratory syndrome ZHANG Ding-mei, LU Jia-hai and ZHONG Nan-shan Keywords: pathogenesis; severe acute respiratory syndrome

evere 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, immuno- histochemistry, 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.18 A substantial number of SARS patients have diarrhea.11,24 In the intestine, little pathology is observed at the light

S

School of Public Health, Laboratory for Tropical Disease Control and Prevention, Sun Yat-sen University, Guangzhou, Guangdong510080, China (Zhang DM and Lu JH) Guangzhou Institute of Respiratory Diseases, Guangzhou Medical College, Guangzhou, Guangdong 510120, China (Zhong NS) Correspondence to: Prof. LU Jia-hai, School of Public Health, Sun Yat-sen University, Guangzhou, Guangdong 510080, China (Tel: 86-20-87330605. Fax: 86-20-87332438. Email: jiahailu@yahoo.com.cn); Prof. ZHONG Nan-shan, Guangzhou Institute of Respiratory Diseases, Guangzhou Medical College, Guangzhou, Guangdong 510120, China (Tel: 86-20-83337750. Email: nanshan@vip.163.com)

Chinese Medical Journal 2008; 121(17):1722-1731 1723

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.14,29 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 hemo- phagocytosis 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

Chin Med J 2008;121(17):1722-1731 1724

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.55 In 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 ACE2.39 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).59 In 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 immune- mediated injury.5 High levels of cytokines and

Chinese Medical Journal 2008; 121(17):1722-1731 1725

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 play important roles in intercellular signaling and chemotaxis. Based on their protein sequences, they are broadly divided into α-chemokines with a common C–X–C (cysteine–other–cysteine) structure of amino acid residues near the amino-terminus which interacts predominantly with neutrophils, and β-chemokines with a C–C (cysteine–cysteine) structure interacting with mononuclear cells.53 In the lung tissues obtained from SARS patients who died, chemokines CXCL10 and IL-18 were markedly activated. CXCL10 and a neutrophil chemokine, chemokine C–X–C motif ligand 8 (CXCL8), were also markedly increased.67,68 A number of chemokines, including CXCL10, CXCL9 and CCL2, were increased within 1 day after exposure to the virus.69 Most impressively, mRNA for CXCR3, the receptor for CXCL9 and CXCL10, was massively up-regulated in the lungs of SARS-CoV-infected mice.68 High-density oligonucleotide array analysis of gene expression changes in peripheral blood mononuclear cells from normal healthy donors inoculated in vitro with SARS-CoV. They show an early activation of the innate immunity pathway in the first 12 hours, including enhanced expression of CD14, Toll-like receptor 9 (TLR9), CC chemokines CCL4, CCL20, CCL22, CCL25, CCL27 and their receptors CCR4, CCR7 along with IL-8 and IL-17.70 Pneumocytes and macrophages in the patients’ lungs express P-selectin. In in vitro studies, A549 cells produced CCL2/monocyte chemoattractant protein 1 (MCP-1) and CXCL8/interleukin-8 (IL-8) after interaction with SARS-CoV and expressed P-selectin and vascular cell adhesion molecule-1 (VCAM-1). Moreover, SARS-CoV induced THP-1 cells to express CCL2/ MCP-1, CXCL8/IL-8, CCL3/MIP-1α, CXCL10/IP-10, CCL4/MIP-1β and CCL5/RANTES, which attracts neutrophils, monocytes and activated T cells in a chemotaxis assay.55 The SARS-CoV-infected DCs

showed low expression of antiviral cytokines-interferon α (IFN-α), IFN-β, IFN-γ and interleukin 12p40 (IL-12p40), moderate up-regulation of proinflammatory cytokines tumor necrosis factor α (TNF-α) and IL-6, but significant upregulation of inflammatory chemokine MIP-1α. It regulated activation of normal T cell to express and secret CCL5, CXCL10 and CCL2.58 CXCL10 also seemed to be significantly increased in lung tissue and lymphoid tissue of autopsied SARS patients, which was further confirmed by IHC with anti-IP-10 antibodies. An increased CXCL10 concentration has been found to be an independent predictor of adverse outcome.62,67 SARS-CoV, through a dynamic interaction with lung epithelial cells and monocytic cells, creates an environment conducive to immune cell migration and accumulation that eventually leads to lung injury.55 The lack of antiviral cytokine response against a background of intense chemokine upregulation could represent a mechanism of immune evasion by SARS-CoV.58 A two-wave model to explain how cellular infiltration may result in SARS was proposed: (1) pulmonary epithelial cells infected by SARS-CoV express adhesion molecules and produce high levels of CCL2/MCP-1 and CXCL8/ IL-8, which recruits macrophages and neutrophils, and (2) macrophages recruited by CCL-2/MCP-1 interact with SARS-CoV and produce a set of chemokines that attract more monocytes and neutrophils as well as activated T cells.55

VIRAL CYTOPATHIC EFFECTS

Direct viral effects are also likely to contribute to the serious pulmonary injury resulting from SARS-CoV infection. In particular, during the first 10 days of the disease, when virus replication is prevalent, viral effects seem to play an important role. The presence of multinucleated cells in SARS lungs may be the result of viral cytopathic effects.37 The virus is also capable of causing cytopathic effects in both monkey renal epithelial cells (Vero E6) and ciliated tracheobronchial epithelial cells in vitro.71,72 Apoptosis and syncytial formation are seen in infected Vero E6.73 In vivo studies have reported evidence of apoptosis in cells of the thyroid glands, spermatogenic cells, epithelial cells, pneumocytes, monocytes/macrophages, lymphocytes and hepatocytes.34,37,74,75 In vitro experiments indicate that over expression of certain non-structural proteins may induce apoptosis in several cell types.58,73,76,77 Over expression of 7a, a protein specifically encoded by SARS-CoV, can induce apoptosis via a caspase- dependent pathway in cell lines derived from different organs, including lung, liver and kidney. Expression of 7a may be one of the underlying mechanisms for the pathogenesis of SARS-CoV infection.77 SARS 3a protein also appears to be important in mediating apoptosis in some cell types.73 It is incorporated into the viron particle and may also act as one of the structural proteins.78 Through an unknown mechanism, host cells overexpressing SARS 3a have increased expression of fibrinogen mRNA.79 Furthermore,

Chin Med J 2008;121(17):1722-1731 1726

SARS-CoV structural proteins also have the ability to trigger apoptosis in vitro. SARS-CoV E proteins may be involved in T-cell apoptosis through inhibition of anti-apoptotic proteins Bcl-xL.80 Increased expression of transforming growth factor-β1 has been detected in infected alveolar and bronchial epithelial cells. Because transforming growth factor-β1 is an enhancer of Fas-mediated cell apoptosis, strong induction of this cytokine may also partially account for apoptosis of such cells.37

IMMUNE RESPONSE CHARACTER OF SARS PATIENTS

Lymphopenia and increasing viral load in the first 10 days of SARS suggested immune evasion by SARS-CoV.58 The innate immune system constitutes the first line of the immune defense against viruses and involves several cellular components and soluble factors. T lymphocytes and macrophages/monocytes are the key immune cells that are infected by SARS-CoV.8 In vitro infected peripheral blood mononuclear cells (PBMCs) have shown viral replication for up to 8 days.70 In PBMCs obtained from SARS patients, SARS-CoV has also been found to infect and replicate, although replication was self-limiting.81 Both T lymphocytes and macrophages/monocytes were found in the circulating blood, lymph nodes, lungs and spleens in SARS autopsies.28,82,83 These findings may provide a partial explanation for the lymphopenia and the widespread destruction of spleen and lymphoid tissue in the majority SARS patients. Infected immune cells may cause widespread dissemination to various organs. Because monocytes and T cells are involved in both the innate and adaptive immune system, the destruction of such cells may result in a compromised immune response.5 Lymphopenia with a rapid decrease in both CD4+ and CD8+ T cells is common during the acute phase of SARS and may be associated with an adverse outcome.26,84 Several other viral infectious diseases, such as measles, Ebola virus infections and respiratory syncytial virus infections, are also associated with severe lymphopenia.85-87 However, in these diseases, direct infection and subsequent destruction of lymphocytes are generally regarded as unlikely to account for the severe lymphocyte depletion. In measles, for instance, only a small proportion of the patient’s PBMCs are infected during acute infection,88 contrasting with the high infection rates of PBMCs in SARS. SARS resembles acquired immune deficiency syndrome (AIDS) with respect to the fact that both are viral diseases that result in immunodeficiency. However, SARS infects various immune cells and progresses rapidly whereas human immunodeficiency virus (HIV) mainly attacks CD4+ lymphocytes and is slowly progressive.8 In addition, SARS-CoV seems to impair the phagocytic capacity of macrophages, which may render SARS patients prone to secondary pulmonary infections. SARS-CoV also causes

phenotypic and functional maturation of dendritic cells in vitro, resulting in a moderate production of cytokines and an enhanced T-cell-stimulatory capacity.89 Activated T cells may exert cytotoxic activity in the lungs, further contributing to pulmonary injury. In viral infections in general, interferons are produced and secreted by infected cells. These cells cause adjacent cells to synthesize antiviral agents that tend to restrict viral dissemination. Contrary to other viruses, SARS-CoV is not capable of inducing significant interferon-α or -β gene expression in infected macrophages, PBMCs or in infected dendritic cells.58,90 Furthermore, in marked contrast to patients with influenza, there was a lack of expression of mRNA for the type-1 interferons, interferon α and β, in peripheral blood mononuclear cells of patients with SARS.91 Previous studies have found that convalescent plasmas from recovered SARS patients could be an effective treatment of SARS.92 Detection of serum IgG, IgM and IgA against SARS-CoV using immunofluorescent assays and by ELISA against nucleocapsid antigen occurs around the same time, day 14, that most patients sero-converted after onset of illness.93 IgG can be detected as early as 4 days after the onset of illness. The neutralization antibodies nearly parallel those for IgG and most of the neutralizing-antibody activity is attributed to IgG.93,94 In a study of 623 SARS patients, the neutralizing-antibody levels peaked at 20–30 days and were sustained for over 150 days.94 Our study on the long term retrospective neutralizing antibodies of 14 patients showed that the neutralizing antibody was maintained in 1:320–1280 for a long period, ranging from 17–181 days.95 Lu et al developed a candidate inactivated vaccine and inoculated horses, rabbits, pigs, Balb/c mice, monkeys and SD mice (some of the data not published).96 The results indicated that specific IgG antibodies and neutralizing antibodies could be elicited in vaccinated animals. And the neutralizing antibodies could specifically neutralize the SARS-CoV and protect Vero-E6 cells from SARS-CoV infection.97 The low response of pigs towards SARS-CoV has been identified in our study, which proposed a question that whether the pig potentially serve as a hiding host for SARS-CoV.95

There were 4 SARS cases that reemerged in Guangdong Province in 2004. In contrast to earlier infections in 2003, these 4 cases appeared with mild clinical manifestations, and caused a lower and transient immune response. The neutralizing antibody of the 4 reemerging cases rose to a peak at 11–13 days at 1:160–1:640, and then dropped rapidly within a short period of time.95 This suggested that SARS-CoV may have adapted to humans during the 2003 outbreak.98 These antibodies can neutralize the pseudotype particles bearing the S protein from different SARS-CoV strains, suggesting that these antibodies are broadly active and that the S protein is highly immunogenic.94 Indeed, the S protein, among the other structural proteins, such as M, E or N, is the only

Chinese Medical Journal 2008; 121(17):1722-1731 1727

significant SARS-CoV neutralization antigen and protective antigen target;99 with amino acids 441–700 as the major immunodominant epitope.100 The vaccine based on the S glycoprotein was also developed rapidly. Autoimmunity may also be involved in the pathogenesis of SARS. Both humoral and T-cell-mediated immune responses to animal coronaviruses have been identified to be capable of exacerbating the disease or causing new health problems; examples are seen with neurotropic mouse hepatitis virus,101 bovine coronavirus102 and cat coronavirus vaccines.97 Chou et al103 established a panel of twelve monoclonal antibodies (mAbs) by immunizing Balb/c mice with the inactivated SARS-CoV BJ01 strain. Seven of them were mapped to the specific segments of the recombinant spike, while none of the remaining five mAbs could recognize any of the recombinant S, N, M or E antigens. These mAbs may have recognized some of the antigen determinants that were unique to the inactivated virion. Wang and Lu characterized the carbohydrate binding activity of SARS-CoV neutralizing antibodies elicited by an inactivated vaccine. They detected undesired autoantibody reactivity specific for the carbohydrate moieties of an abundant human serum glycoprotein asialo-orosomocoid (ASOR).104 In another cohort of 22 SARS patients immune activity against antigens from lung epithelial cell lines and endothelial cell lines was found in some patients’ sera obtained approximately 1 month after infection.105 Moreover, high levels of these autoimmune activities in the sera were shown to be cytotoxic to lung epithelial cells and endothelial cells in culture.53 Autoimmunity may be partially attributable to the development of cross-reacting antibodies against specific SARS-CoV epitopes. IgG antibodies against the domain 2 of the spike protein have indeed been found to cross-react with pulmonary epithelial cells.106 Another mechanism that possibly explains autoimmunity is the exposure of autoantigens caused by cytokine-induced organ injury.105 Therefore, there has been a serious concern about the safety of inactivated vaccine since some antigens in the virions may elicit antibodies that enhance viral infection rather than neutralize it.

GENETIC SUSCEPTIBILITY Genetic factors also seem to play a causative role in the pathogenesis of SARS. For a group of Taiwanese patients, the HLA-B*4601 haplotype was associated with severity of SARS infection.107 This association has not been established for certain Hong Kong Chinese patients. Nevertheless, in the Hong Kong patients, a strong association was demonstrated between HLAB* 0703 (OR 4.08) and HLA-DRB1*0301 (OR 0.06) alleles and an increased susceptibility to SARS infection.108 Mannose-binding lectin (MBL) is a serum protein that can bind to the ligands of various pathogens, flagging them for immune destruction independently of a specific antibody response. MBL is capable of binding to the glycosylated SARS-CoV S protein and inhibiting

SARS-CoV infectivity in vitro.109 In a case-control gene-association study of 569 Hong Kong Chinese SARS patients and 1188 healthy controls, low-MBL-producing genotypes have been shown to be associated with increased risk (OR 1.5) of SARS.109 In contrast, CD209L homozygote individuals seem to have a significantly lower risk of SARS infection.110 The difference in genetic makeup between individuals is mostly accounted for by single base differences, single nucleotide polymorphisms (SNPs). In the context of predisposition to ARDS after trauma, insertion alleles at the promoter of plasminogen-activator-inhibitor-1 (PAI-1) were associated with high concentrations of PAI-1 in the plasma and a poor survival rate.111 In addition to PAI-1, other genetic polymorphisms, such as angiotensin-converting enzyme (ACE),112 CD14,113 surfactant protein,114 are also associated with predisposition, severity and outcome of ARDS. Although SASR-CoV utilizes ACE2 as its receptor and ACE2 is known to be an important protector of lung damage in experimental ARDS, no solid association between alleles of the two ACE genes (ACE1 and ACE2) and the severity of ARDS after SARS infection was found.115 There is considerable variability in the prevalence of immunogenotypes among different populations and the significance of detecting so-called “predisposing” alleles in clinical practice is questionable. More studies are needed to fully uncover the real genetic determinants for both predisposition to infection and the host–pathogen interaction after infection with the virus.53

CONCLUSION The pathogenesis of SARS appears to be multifactorial and complex. The most plausible and possible mechanism appears to consist of a direct injury to the target cells by the virus and an indirect injury mediated by subsequent immune system dysfunction. By droplet inhalation, SARS-CoV reaches the respiratory tract and invades the epithelial cells of the trachea, bronchi, bronchioles and alveoli. Virus infection and replication in target cells causes direct damage to the respiratory tract. Local inflammatory changes destroy the integrity of the blood–gas barrier and increase the permeability of the capillary blood vessels. Exudation of fibrin results in the formation of hyaline membranes. The infection and associated inflammation bring about acute injury of type II alveolar cells, decreasing the secretion of alveolar surfactant resulting in alveolar collapse. At the same time, SARS-CoV infects resident and circulating immune cells. The infected immune cells include mainly macrophages and T cells. Circulating immune cells disseminate the virus to other organs, including the spleen and the lymph nodes. The destruction of immune cells together with extensive damage to the splenic white pulp results in immunodeficiency. A weakened immune defense exacerbates the infection and replication of the virus in the lungs and viral damage to the respiratory alveoli,

Chin Med J 2008;121(17):1722-1731 1728

resulting in respiratory distress. The proposed mechanisms of SARS have significant implications for the prevention, diagnosis, therapy and future research on this newly emerged disease. Although much has been learned of SARS since its discovery, many questions with respect to the pathogenesis of SARS still remain unanswered. What’s more, there is a real possibility that SARS will re-emerge, for the existence of at least 2 known animal reservoirs, in civets and horseshoe bats that are commonly found in the wild as well as reared and consumed in southern China. Further investigations are still required.

REFERENCES 1. Kuiken T, Fouchier R, Rimmelzwaan G, Osterhaus A.

Emerging viral infections in a rapidly changing world. Curr Opin Biotechnol 2003; 14: 641-646.

2. Berger A, Drosten C, Doerr HW, Sturmer M, Preiser W. Severe acute respiratory syndrome (SARS)–paradigm of an emerging viral infection. J Clin Virol 2004; 29: 13-22.

3. 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: 1394-1399.

4. Marra MA, Jones SJ, Astell CR, Holt RA, Brooks-Wilson A, Butterfield YS, et al. The Genome sequence of the SARS- associated coronavirus. Science 2003; 300: 1399-1404.

5. Gu J, Korteweg C. Pathology and pathogenesis of severe acute respiratory syndrome. Am J Pathol 2007; 170: 1136-1147.

6. Lee N, Hui D, Wu A, Chan P, Cameron P, Joynt GM, et al. A major outbreak of severe acute respiratory syndrome in Hong Kong. N Engl J Med 2003; 348: 1986-1994.

7. Wong GW, Li AM, Ng PC, Fok TF. Severe acute respiratory syndrome in children. Pediatr Pulmonol 2003; 36: 261-266.

8. Guo Y, Korteweg C, McNutt MA, Gu J. Pathogenetic mechanisms of severe acute respiratory syndrome. Virus Res 2008; 133: 4-12.

9. Nicholls JM, Poon LL, Lee KC, Ng WF, Lai ST, Leung CY, et al. Lung pathology of fatal severe acute respiratory syndrome. Lancet 2003; 361: 1773-1778.

10. Zhan J, Chen W, Li C, Wu W, Li J, Jiang S, et al. Digestive system manifestations in patients with severe acute respiratory syndrome. Chin Med J 2003; 116: 1265-1266.

11. Leung WK, To KF, Chan PK, Chan HL, Wu AK, Lee N, et al. Enteric involvement of severe acute respiratory syndrome- associated coronavirus infection. Gastroenterology 2003; 125: 1011-1017.

12. Liu CL, Lu YT, Peng MJ, Chen PJ, Lin RL, Wu CL, et al. Clinical and laboratory features of severe acute respiratory syndrome vis-a-vis onset of fever. Chest 2004; 126: 509-517.

13. Tse GM, To KF, Chan PK, Lo AW, Ng KC, Wu A, et al. Pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (SARS). J Clin Pathol 2004; 57: 260-265.

14. Ding Y, Wang H, Shen H, Li Z, Geng J, Han H, et al. The clinical pathology of severe acute respiratory syndrome (SARS): a report from China. J Pathol 2003; 200: 282-289.

15. Cheung OY, Chan JW, Ng CK, Koo CK. The spectrum of pathological changes in severe acute respiratory syndrome (SARS). Histopathology 2004; 45: 119-124.

16. Hsiao CH, Wu MZ, Chen CL, Hsueh PR, Hsieh SW, Yang PC, et al. Evolution of pulmonary pathology in severe acute respiratory syndrome. J Formos Med Assoc 2005; 104: 75-81.

17. Hwang DM, Chamberlain DW, Poutanen SM, Low DE, Asa SL, Butany J. Pulmonary pathology of severe acute respiratory syndrome in Toronto. Mod Pathol 2005; 18: 1-10.

18. Jia HP, Look DC, Shi L, Hickey M, Pewe L, Netland J, et al. ACE2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia. J Virol 2005; 79: 14614-14621.

19. To KF, Tong JH, Chan PK, Au FW, Chim SS, Chan KC, 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.

20. Ren X, Glende J, Al-Falah M, de Vries V, Schwegmann- Wessels C, Qu X, et al. Analysis of ACE2 in polarized epithelial cells: surface expression and function as receptor for severe acute respiratory syndrome-associated coronavirus. J Gen Virol 2006; 87 (Pt 6): 1691-1695.

21. Franks TJ, Chong PY, Chui P, Galvin JR, Lourens RM, Reid AH, et al. Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore. Hum Pathol 2003; 34: 743-748.

22. Shieh WJ, Hsiao CH, Paddock CD, Guarner J, Goldsmith CS, Tatti K, et al. Immunohistochemical, in situ hybridization, and ultrastructural localization of SARS-associated coronavirus in lung of a fatal case of severe acute respiratory syndrome in Taiwan. Hum Pathol 2005; 36: 303-309.

23. Yilla M, Harcourt BH, Hickman CJ, McGrew M, Tamin A, Goldsmith CS, et al. SARS-coronavirus replication in human peripheral monocytes/macrophages. Virus Res 2005; 107: 93-101.

24. Choi KW, Chau TN, Tsang O, Tso E, Chiu MC, Tong WL, et al. Outcomes and prognostic factors in 267 patients with severe acute respiratory syndrome in Hong Kong. Ann Intern Med 2003; 139: 715-723.

25. To KF, Lo AW. Exploring the pathogenesis of severe acute respiratory syndrome (SARS): the tissue distribution of the coronavirus (SARS-CoV) and its putative receptor, angiotensin-converting enzyme 2 (ACE2). J Pathol 2004; 203: 740-743.

26. Wong RS, Wu A, To KF, Lee N, Lam CW, Wong CK, et al. Haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis. BMJ 2003; 326: 1358-1362.

27. Zhan J, Tang J, Zhang B, Tang Y, Wang JK, Li F, et al. The spleen as a target in severe acute respiratory syndrome. FASEB J 2006: 2321-2328.

28. Gu J, Gong E, Zhang B, Zheng J, Gao Z, Zhong Y, et al. Multiple organ infection and the pathogenesis of SARS. J Exp Med 2005; 202: 415-424

29. Lang ZW, Zhang LJ, Zhang SJ, Meng X, Li JQ, Song CZ, et al. A clinicopathological study of three cases of severe acute respiratory syndrome (SARS). Pathology 2003; 35: 526-531.

30. Chong PY, Chui P, Ling AE, Franks TJ, Tai DY, Leo YS, et al.

Chinese Medical Journal 2008; 121(17):1722-1731 1729

Analysis of deaths during the severe acute respiratory syndrome (SARS) epidemic in Singapore: challenges in determining a SARS diagnosis. Arch Pathol Lab Med 2004; 128: 195-204.

31. Xu J, Zhong S, Liu J, Li L, Li Y, Wu X, et al. Detection of severe acute respiratory syndrome coronavirus in the brain: potential role of the chemokine mig in pathogenesis. Clin Infect Dis 2005; 41: 1089-1096.

32. Chan KH, Poon LL, Cheng VC, Guan Y, Hung IF, Kong J, et al. Detection of SARS coronavirus in patients with suspected SARS. Emerg Infect Dis 2004; 10: 294-299.

33. Chu KH, Tsang WK, Tang CS, Lam MF, Lai FM, To KF, et al. Acute renal impairment in coronavirus-associated severe acute respiratory syndrome. Kidney Int 2005; 67: 698-705.

34. Chau TN, Lee KC, Yao H, Tsang TY, Chow TC, Yeung YC, et al. SARS-associated viral hepatitis caused by a novel coronavirus: report of three cases. Hepatology 2004; 39: 302-310.

35. Ding Y, He L, Zhang Q, Huang Z, Che X, Hou J, et al. Organ distribution of severe acute respiratory syndrome (SARS) associated coronavirus (SARS-CoV) in SARS patients: implications for pathogenesis and virus transmission pathways. J Pathol 2004; 203: 622-630.

36. Leung TW, Wong KS, Hui AC, To KF, Lai ST, Ng WF, et al. Myopathic changes associated with severe acute respiratory syndrome: a postmortem case series. Arch Neurol 2005; 62: 1113-1117.

37. He L, Ding Y, Zhang Q, Che X, He Y, Shen H, et al. Expression of elevated levels of pro-inflammatory cytokines in SARS-CoV-infected ACE2+ cells in SARS patients: relation to the acute lung injury and pathogenesis of SARS. J Pathol 2006; 210: 288-297.

38. Chan HL, Kwan AC, To KF, Lai ST, Chan PK, Leung WK, et al. Clinical significance of hepatic derangement in severe acute respiratory syndrome. World J Gastroenterol 2005; 11: 2148-2153.

39. Lau YL, Peiris JS. Pathogenesis of severe acute respiratory syndrome. Curr Opin Immunol 2005; 17: 404-410.

40. Guan Y, Zheng BJ, He YQ, Liu XL, Zhuang ZX, Cheung CL, et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 2003; 302: 276-278.

41. Li W, Shi Z, Yu M, Ren W, Smith C, Epstein JH, et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 2005; 310: 676-679.

42. Chen W, Yan M, Yang L, Ding B, He B, Wang Y, et al. SARS-associated coronavirus transmitted from human to pig. Emerg Infect Dis 2005; 11: 446-448.

43. Cheng VC, Lau SK, Woo PC, Yuen KY. Severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection. Clin Microbiol Rev 2007; 20: 660-694.

44. Wang XW, Li JS, Guo TK, Zhen B, Kong QX, Yi B, et al. Concentration and detection of SARS coronavirus in sewage from Xiao Tang Shan Hospital and the 309th Hospital. J Virol Methods 2005; 128: 156-161.

45. Li W, Moore MJ, Vasilieva N, Sui J, Wong SK, Berne MA, et al. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 2003; 426: 450-454.

46. Hamming I, Timens W, Bulthuis ML, Lely AT, Navis GJ, van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis. J Pathol 2004; 203: 631-637.

47. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, Luo S, et al. Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. Embo J 2005; 24: 1634-1643.

48. Liu S, Xiao G, Chen Y, He Y, Niu J, Escalante CR, et al. Interaction between heptad repeat 1 and 2 regions in spike protein of SARS-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors. Lancet 2004; 363: 938-947.

49. Yang ZY, Huang Y, Ganesh L, Leung K, Kong WP, Schwartz O, et al. pH-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through DC-SIGN. J Virol 2004; 78: 5642-5650.

50. Imai Y, Kuba K, Rao S, Huan Y, Guo F, Guan B, et al. Angiotensin-converting enzyme 2 protects from severe acute lung failure. Nature 2005; 436: 112-116.

51. Kuba K, Imai Y, Rao S, Gao H, Guo F, Guan B, et al. A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury. Nat Med 2005; 11: 875-879.

52. Itoyama S, Keicho N, Quy T, Phi NC, Long HT, Ha le D, et al. ACE1 polymorphism and progression of SARS. Biochem Biophys Res Commun 2004; 323: 1124-1129.

53. Lo AW, Tang NL, To KF. How the SARS coronavirus causes disease: host or organism? J Pathol 2006; 208: 142-151.

54. Soilleux EJ, Morris LS, Leslie G, Chehimi J, Luo Q, Levroney E, et al. Constitutive and induced expression of DC-SIGN on dendritic cell and macrophage subpopulations in situ and in vitro. J Leukoc Biol 2002; 71: 445-457.

55. Yen YT, Liao F, Hsiao CH, Kao CL, Chen YC, Wu-Hsieh BA. Modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro. J Virol 2006; 80: 2684-2693.

56. Jeffers SA, Tusell SM, Gillim-Ross L, Hemmila EM, Achenbach JE, Babcock GJ, et al. CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 2004; 101: 15748-15753.

57. Marzi A, Gramberg T, Simmons G, Moller P, Rennekamp AJ, Krumbiegel M, et al. DC-SIGN and DC-SIGNR interact with the glycoprotein of Marburg virus and the S protein of severe acute respiratory syndrome coronavirus. J Virol 2004; 78: 12090-12095.

58. Law HK, Cheung CY, Ng HY, Sia SF, Chan YO, Luk W, et al. Chemokine up-regulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells. Blood 2005; 106: 2366-2374.

59. van Kooyk Y, Geijtenbeek TB. DC-SIGN: escape mechanism for pathogens. Nat Rev Immunol 2003; 3: 697-709.

60. Huang IC, Bosch BJ, Li F, Li W, Lee KH, Ghiran S, et al. SARS coronavirus, but not human coronavirus NL63, utilizes cathepsin L to infect ACE2-expressing cells. J Biol Chem 2006; 281: 3198-3203.

61. Simmons G, Gosalia DN, Rennekamp AJ, Reeves JD, Diamond SL, Bates P. Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry. Proc

Chin Med J 2008;121(17):1722-1731 1730

Natl Acad Sci U S A 2005; 102: 11876-11881. 62. Jiang Y, Xu J, Zhou C, Wu Z, Zhong S, Liu J, et al.

Characterization of cytokine/chemokine profiles of severe acute respiratory syndrome. Am J Respir Crit Care Med 2005; 171: 850-857.

63. Peiris JS, Yuen KY, Osterhaus AD, Stohr K. The severe acute respiratory syndrome. N Engl J Med 2003; 349: 2431-2441.

64. Wong CK, Lam CW, Wu AK, Ip WK, Lee NL, Chan IH, et al. Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome. Clin Exp Immunol 2004; 136: 95-103.

65. Ward SE, Loutfy MR, Blatt LM, Siminovitch KA, Chen J, Hinek A, et al. Dynamic changes in clinical features and cytokine/chemokine responses in SARS patients treated with interferon alfacon-1 plus corticosteroids. Antivir Ther 2005; 10: 263-275.

66. Huang KJ, Su IJ, Theron M, Wu YC, Lai SK, Liu CC, et al. An interferon-gamma-related cytokine storm in SARS patients. J Med Virol 2005; 75: 185-194.

67. Tang NL, Chan PK, Wong CK, To KF, Wu AK, Sung YM, et al. Early enhanced expression of interferon-inducible protein-10 (CXCL-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome. Clin Chem 2005; 51: 2333-2340.

68. Glass WG, Subbarao K, Murphy B, Murphy PM. Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 2004; 173: 4030-4039.

69. Ng PC, Lam CW, Li AM, Wong CK, Leung TF, Cheng FW, et al. Chemokine response in children with SARS. Arch Dis Child 2005; 90: 422-423.

70. Ng LF, Hibberd ML, Ooi EE, Tang KF, Neo SY, Tan J, et al. A human in vitro model system for investigating genome-wide host responses to SARS coronavirus infection. BMC Infect Dis 2004; 4: 34.

71. Drosten C, Gunther S, Preiser W, van der Werf S, Brodt HR, Becker S, et al. Identification of a novel coronavirus in patients with severe acute respiratory syndrome. N Engl J Med 2003; 348: 1967-1976.

72. Sims AC, Baric RS, Yount B, Burkett SE, Collins PL, Pickles RJ. Severe acute respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of ciliated cells in viral spread in the conducting airways of the lungs. J Virol 2005; 79: 15511-15524.

73. Law PT, Wong CH, Au TC, Chuck CP, Kong SK, Chan PK, et al. The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells. J Gen Virol 2005; 86 (Pt 7): 1921-1930.

74. Xu J, Qi L, Chi X, Yang J, Wei X, Gong E, et al. Orchitis: a complication of severe acute respiratory syndrome (SARS). Biol Reprod 2006; 74: 410-416.

75. Wei L, Sun S, Xu CH, Zhang J, Xu Y, Zhu H, et al. Pathology of the thyroid in severe acute respiratory syndrome. Hum Pathol 2007; 38: 95-102.

76. Fielding BC, Tan YJ, Shuo S, Tan TH, Ooi EE, Lim SG, et al. Characterization of a unique group-specific protein (U122) of the severe acute respiratory syndrome coronavirus. J Virol 2004; 78: 7311-7318.

77. Tan YJ, Fielding BC, Goh PY, Shen S, Tan TH, Lim SG, et al.

Overexpression of 7a, a protein specifically encoded by the severe acute respiratory syndrome coronavirus, induces apoptosis via a caspase-dependent pathway. J Virol 2004; 78: 14043-14047.

78. Shen S, Lin PS, Chao YC, Zhang A, Yang X, Lim SG, et al. The severe acute respiratory syndrome coronavirus 3a is a novel structural protein. Biochem Biophys Res Commun 2005; 330: 286-292.

79. Tan YJ, Tham PY, Chan DZ, Chou CF, Shen S, Fielding BC, et al. The severe acute respiratory syndrome coronavirus 3a protein up-regulates expression of fibrinogen in lung epithelial cells. J Virol 2005; 79: 10083-10087.

80. Yang Y, Xiong Z, Zhang S, Yan Y, Nguyen J, Ng B, et al. Bcl-xL inhibits T-cell apoptosis induced by expression of SARS coronavirus E protein in the absence of growth factors. Biochem J 2005; 392 (Pt 1): 135-143.

81. Li L, Wo J, Shao J, Zhu H, Wu N, Li M, et al. SARS-coronavirus replicates in mononuclear cells of peripheral blood (PBMCs) from SARS patients. J Clin Virol 2003; 28: 239-244.

82. Zhan J, Deng R, Tang J, Zhang B, Tang Y, Wang JK, et al. The spleen as a target in severe acute respiratory syndrome. Faseb J 2006; 20: 2321-2328.

83. Ye J, Zhang B, Xu J, Chang Q, McNutt MA, Korteweg C, et al. Molecular pathology in the lungs of severe acute respiratory syndrome patients. Am J Pathol 2007; 170: 538-545.

84. Li T, Qiu Z, Zhang L, Han Y, He W, Liu Z, et al. Significant changes of peripheral T lymphocyte subsets in patients with severe acute respiratory syndrome. J Infect Dis 2004; 189: 648-651.

85. Geisbert TW, Jahrling PB. Exotic emerging viral diseases: progress and challenges. Nat Med 2004; 10 (12 Suppl): S110-S121.

86. Schneider-Schaulies J, ter Meulen V, Schneider-Schaulies S. Measles virus interactions with cellular receptors: consequences for viral pathogenesis. J Neurovirol 2001; 7: 391-399.

87. Openshaw PJ. Potential therapeutic implications of new insights into respiratory syncytial virus disease. Respir Res 2002; 3 Suppl 1: S15-S20.

88. Schneider-Schaulies S, Niewiesk S, Schneider-Schaulies J, ter Meulen V. Measles virus induced immunosuppression: targets and effector mechanisms. Curr Mol Med 2001; 1: 163-181.

89. Tseng CT, Perrone LA, Zhu H, Makino S, Peters CJ. Severe acute respiratory syndrome and the innate immune responses: modulation of effector cell function without productive infection. J Immunol 2005; 174: 7977-7985.

90. Cheung CY, Poon LL, Ng IH, Luk W, Sia SF, Wu MH, et al. Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis. J Virol 2005; 79: 7819-7826.

91. Reghunathan R, Jayapal M, Hsu LY, Chng HH, Tai D, Leung BP, et al. Expression profile of immune response genes in patients with Severe Acute Respiratory Syndrome. BMC Immunol 2005; 6: 2.

92. Cheng Y, Wong R, Soo YO, Wong WS, Lee CK, Ng MH, et al. Use of convalescent plasma therapy in SARS patients in

Chinese Medical Journal 2008; 121(17):1722-1731 1731

Hong Kong. Eur J Clin Microbiol Infect Dis 2005; 24: 44-46. 93. Hsueh PR, Huang LM, Chen PJ, Kao CL, Yang PC.

Chronological evolution of IgM, IgA, IgG and neutralisation antibodies after infection with SARS-associated coronavirus. Clin Microbiol Infect 2004; 10: 1062-1066.

94. Nie Y, Wang G, Shi X, Zhang H, Qiu Y, He Z, et al. Neutralizing antibodies in patients with severe acute respiratory syndrome-associated coronavirus infection. J Infect Dis 2004; 190: 1119-1126.

95. Liao JW, Lu JH, Guo ZM, Wang GL, Zhang DM, Chen LJ, et al. A retrospective serological study of severe acute respiratory syndrome cases in Guangdong province, China. Chin Med J 2007; 120: 718-720.

96. Zhang CH, Guo ZM, Zheng HY, Lu JH, Wang YF, Yan XG, et al. Humoral immune responses in rabbits induced by an experimental inactivated severe acute respiratory syndrome coronavirus vaccine prepared from F69 strain. Chin Med J 2004; 117: 1625-1629.

97. Zhang DM, Wang GL, Lu JH. Severe acute respiratory syndrome: vaccine on the way. Chin Med J 2005; 118: 1468-1476.

98. Chinese SMEC. Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science 2004; 303: 1666-1669.

99. Buchholz UJ, Bukreyev A, Yang L, Lamirande EW, Murphy BR, Subbarao K, et al. Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc Natl Acad Sci U S A 2004; 101: 9804-9809.

100. Lu L, Manopo I, Leung BP, Chng HH, Ling AE, Chee LL, et al. Immunological characterization of the spike protein of the severe acute respiratory syndrome coronavirus. J Clin Microbiol 2004; 42: 1570-1576.

101. Zhong X, Yang H, Guo ZF, Sin WY, Chen W, Xu J, et al. B-cell responses in patients who have recovered from severe acute respiratory syndrome target a dominant site in the S2 domain of the surface spike glycoprotein. J Virol 2005; 79: 3401-3408.

102. O'Connor A, Martin SW, Nagy E, Menzies P, Harland R. The relationship between the occurrence of undifferentiated bovine respiratory disease and titer changes to bovine coronavirus and bovine viral diarrhea virus in 3 Ontario feedlots. Can J Vet Res 2001; 65: 137-142.

103. Chou TH, Wang S, Sakhatskyy PV, Mboudjeka I, Lawrence JM, Huang S, et al. Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV). Virology 2005; 334: 134-143.

104. Wang D, Lu J. Glycan arrays lead to the discovery of autoimmunogenic activity of SARS-CoV. Physiol Genomics

2004; 18: 245-248. 105. Yang YH, Huang YH, Chuang YH, Peng CM, Wang LC, Lin

YT, et al. Autoantibodies against human epithelial cells and endothelial cells after severe acute respiratory syndrome (SARS)-associated coronavirus infection. J Med Virol 2005; 77: 1-7.

106. Lin YS, Lin CF, Fang YT, Kuo YM, Liao PC, Yeh TM, et al. Antibody to severe acute respiratory syndrome (SARS)- associated coronavirus spike protein domain 2 cross-reacts with lung epithelial cells and causes cytotoxicity. Clin Exp Immunol 2005; 141: 500-508.

107. Lin M, Tseng HK, Trejaut JA, Lee HL, Loo JH, Chu CC, et al. Association of HLA class I with severe acute respiratory syndrome coronavirus infection. BMC Med Genet 2003; 4: 9.

108. Ng MH, Lau KM, Li L, Cheng SH, Chan WY, Hui PK, et al. Association of human-leukocyte-antigen class I (B*0703) and class II (DRB1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome. J Infect Dis 2004; 190: 515-518.

109. Ip WK, Chan KH, Law HK, Tso GH, Kong EK, Wong WH, et al. Mannose-binding lectin in severe acute respiratory syndrome coronavirus infection. J Infect Dis 2005; 191: 1697-1704.

110. Chan VS, Chan KY, Chen Y, Poon LL, Cheung AN, Zheng B, et al. Homozygous L-SIGN (CLEC4M) plays a protective role in SARS coronavirus infection. Nat Genet 2006; 38: 38-46.

111. Menges T, Hermans PW, Little SG, Langefeld T, Boning O, Engel J, et al. Plasminogen-activator-inhibitor-1 4G/5G promoter polymorphism and prognosis of severely injured patients. Lancet 2001; 357: 1096-1097.

112. Marshall RP, Webb S, Bellingan GJ, Montgomery HE, Chaudhari B, McAnulty RJ, et al. Angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome. Am J Respir Crit Care Med 2002; 166: 646-650.

113. Gibot S, Cariou A, Drouet L, Rossignol M, Ripoll L. Association between a genomic polymorphism within the CD14 locus and septic shock susceptibility and mortality rate. Crit Care Med 2002; 30: 969-973.

114. Lin Z, Pearson C, Chinchilli V, Pietschmann SM, Luo J, Pison U, et al. Polymorphisms of human SP-A, SP-B, and SP-D genes: association of SP-B Thr131Ile with ARDS. Clin Genet 2000; 58: 181-191.

115. Chiu RW, Tang NL, Hui DS, Chung GT, Chim SS, Chan KC, et al. ACE2 gene polymorphisms do not affect outcome of severe acute respiratory syndrome. Clin Chem 2004; 50: 1683-1686.

(Received December 28, 2007)

Edited by WANG Mou-yue and LIU Huan