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      Pathology, Molecular Biology, and Pathogenesis of Avian Influenza A (H5N1) Infection in Humans


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          H5N1 avian influenza is a highly fatal infectious disease that could cause a potentially devastating pandemic if the H5N1 virus mutates into a form that spreads efficiently among humans. Recent findings have led to a basic understanding of cell and organ histopathology caused by the H5N1 virus. Here we review the pathology of H5N1 avian influenza reported in postmortem and clinical studies and discuss the key pathogenetic mechanisms. Specifically, the virus infects isolated pulmonary epithelial cells and causes diffuse alveolar damage and hemorrhage in the lungs of infected patients. In addition, the virus may infect other organs, including the trachea, the intestines, and the brain, and it may penetrate the placental barrier and infect the fetus. Dysregulation of cytokines and chemokines is likely to be one of the key mechanisms in the pathogenesis of H5N1 influenza. We also review the various molecular determinants of increased pathogenicity that have been identified in recent years and the role of avian and human influenza virus receptors in relation to the transmissibility of the H5N1 virus. A comprehensive appreciation of H5N1 influenza pathogenetic mechanisms should aid in the design of effective strategies for prevention, diagnosis, and treatment of this emerging disease. H5N1 avian influenza was initially confined to poultry, but in recent years it has emerged as a highly fatal infectious disease in the human population. In 1997, the avian influenza A virus subtype H5N1 crossed the avian-human species barrier for the first time.1 Eighteen individuals were infected, six of whom died.2 In January 2003, avian influenza re-emerged among humans in Hong Kong,3 and since 2004 numerous human infections have also occurred in other Asian and non-Asian countries. To date, the World Health Organization has reported 348 laboratory-confirmed cases, 216 of which were fatal, resulting in a fatality rate of ∼60% (World Health Organization: http://www.who.int/csr/disease/avian_influenza/country/cases_table_2008_01_03/en/index.html; accessed January 2008). Human infections mainly resulted from poultry-to-human transmission. Recently, however, there have been reports of human-to-human transmission,4,5 increasing fears of a human pandemic. H5N1 influenza is still a relatively novel disease with poorly understood pathology and pathogenesis. During the period from the first known outbreak nearly a decade ago until the present, only a limited number of reports describing pathological findings in human H5N1 cases has been published. Nevertheless, recent studies combined with early findings have gradually resulted in a better understanding of the cell and organ pathology caused by the H5N1 virus, as well as the viral tissue tropism. These findings together with animal and in vitro experiments have also contributed to a basic understanding of the pathogenesis of this disease. On the molecular level, several viral genes and gene products have been identified that may be responsible for the high pathogenicity of H5N1 influenza viruses. Herein, we describe the pathology of H5N1 avian influenza by reviewing the major pathological findings reported in hitherto published postmortem studies of human H5N1 cases as well as some key findings of animal studies. In addition, the major pathogenetic mechanisms and etiological factors of H5N1 influenza are discussed. The various molecular determinants of increased pathogenicity of H5N1 avian influenza viruses that have been identified in recent years are also presented. Finally, we have taken a closer look at the role of avian and human influenza virus receptors in relation to the transmissibility of the H5N1 virus. Pathology Histopathology Thus far the results of only nine full autopsies including one autopsy of a fetus,3,4,6,7,8,9,10 three limited autopsies (only the lungs and spleen),10 and two cases of postmortem organ biopsies11,12,13 have been reported. The main histopathological findings for each of these reports are summarized in Table 1 and discussed below. The Respiratory Tract The lungs typically show diffuse alveolar damage.3,4,6,7,8,9,10 In cases with a short disease duration (<10 to 12 days), features of the exudative inflammatory phase of diffuse alveolar damage (edema, fibrous exudates, hyaline membranes) are predominant (Figure 1A).3,7,9,11 In cases with a longer disease duration, changes consistent with the fibrous proliferative phase (organizing diffuse alveolar damage) and the final fibrotic stage (interstitial fibrosis) have been observed.6,7,8,14 Hyperplasia of type II pneumocytes has been demonstrated in most autopsy cases.3,6,8,11 Viral inclusions or other cytopathic changes have not been observed in pneumocytes.6,8,11,14 Macrophages appeared to be the predominant cells within the alveoli,3,7 whereas in the interstitium T lymphocytes, with or without neutrophils, are present.3,6,7,11 Scattered histiocytes with hemophagocytic activity have been observed in the lungs of some cases.6 The following additional histopathological features have also been reported: 1) desquamation of epithelial cells into alveolar spaces4,8; 2) bronchiolitis8,9; 3) cystically dilated air spaces6; 4) hemorrhage,8,10,11 5) pleuritis9; 6) features of interstitial pneumonitis,4,7,8 and 7) apoptosis in alveolar epithelial cells and leukocytes.9 Two cases of possible superinfections caused by fungi have been reported.7,8 Because the above histopathological features are not unique to H5N1 influenza, it may be difficult to distinguish diffuse alveolar damage caused by H5N1 virus infections from diffuse alveolar damage caused by other microorganisms such as severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) or by other factors such as aspiration or oxygen toxicity. More specific tests such as in situ hybridization, reverse transcription-polymerase chain reaction (RT-PCR), and virus isolation are required to confirm H5N1 infection. Other Organs Reactive histiocytes with hemophagocytic activity have been noted in the spleen, lymph node, bone marrow, lungs, and liver,3,4,6,9,11,13 although in recent autopsies the appearance of histiocytes has been less prominent or even absent.7,8 Both hypercellular3,13 and hypocellular6 bone marrow have been reported. The spleen typically displays congestion and white pulp atrophy with depletion of lymphoid cells.4,6,7,14 Lymph nodes show, apart from hemophagocytosis,3,6 focal necrosis6 and loss of germinal centers7 in some cases. Apoptotic lymphocytes have been detected in both splenic and intestinal tissues.9 Acute tubular necrosis has been found in several cases.6,7,11,14 In liver tissue specimens, necrosis, activated Kupffer cells, cholestasis, and fatty changes have been observed.6,7,8,9 In most instances the brain is edematous without any significant histopathological change,6,7 whereas in two cases demyelinated areas and reactive histiocytes and foci of necrosis have been reported.6,8 In other organs no remarkable histological changes have been observed.3,7,8 Among the H5N1 cases reported, there was a 24-year-old woman who was pregnant at the time of death. Both the placenta and the 4-month-old fetus have been studied.7 The placenta showed foci of both syncytiocytotrophoblast necrosis and necrotizing deciduitis, and diffuse villitis. The fetal tissues did not display specific histopathological features, except for some edema and a few scattered interstitial neutrophils in the lungs.7 Virus Distribution A number of studies have been performed applying immunohistochemistry (IHC) with monoclonal antibodies to hemagglutinin (HA) and nucleocapsid protein (NP) and/or in situ hybridization with sense and anti-sense probes to HA and NP to detect viral antigens and genomic sequences in various organs of H5N1 cases.3,4,6,7,8,9 In addition, RT-PCR, strand-specific RT-PCR, and nucleic acid sequence-based amplification H5 detection assays have been performed to investigate virus tissue tropism.3,6,7,8,9 According to early studies H5N1 infection appeared to be confined to the lungs.3,6 However, the findings of recent studies indicate that the virus disseminates beyond the respiratory tract.7,8,9 The main findings of these studies are summarized below. The Respiratory Tract Viral antigens and genomic sequences have been found in epithelial cells of the trachea8 (Figure 1B) and alveoli (Figure 1C).3,4,7,8,9 Tracheal epithelial cells were identified as both ciliated and nonciliated cells by double labeling with antibodies to tubulin.7 The alveolar cells were identified as type II pneumocytes by double labeling with antibodies to surfactant protein (Figure 1D).7,8 RT-PCR and nucleic acid sequence-based amplification-based H5 detection assays have detected viral RNA in both the trachea7,9 and lungs.7,8,9 Positive-stranded RNA has been detected in lung7,8,9 and trachea7,9 tissue samples. Because the H5N1 virus is a negative-stranded RNA virus, presence of positive-stranded RNA (mRNA and complement RNA, both necessary for viral replication) in a specific tissue suggests active viral replication at that site. Brain Viral sequences (Figure 1E) and antigens have been found in neurons of the brain7 and H5N1 virus has been isolated from cerebrospinal fluid.15 RT-PCR has detected both negative- and positive-stranded RNA in the brain. Dissemination to the central nervous system may be blood-borne or may alternatively occur via the afferent fibers of the olfactory, vagal, trigeminal, and sympathetic nerves after replication in the lungs, as has been observed in a mouse model.16 Intestines Viral genomic sequences have been detected in epithelial cells of the intestines by in situ hybridization.7 RT-PCR has detected both negative- and positive-stranded RNA in the intestines.7,8 These findings are consistent with reports of viral shedding in stool samples, as detected by RT-PCR and viral isolation,15,17 and with frequently observed clinical symptoms related to the gastrointestinal tract.2,18,19 Because avian influenza viruses maintain sialidase activities, despite the low pH conditions in the upper gastrointestinal tract, infection of the intestines may be the result of ingestion of infected secretions.20 In contrast to viral sequences, viral antigens have not been detected in the intestines.7,8 The reason for this is at present undetermined. Other Organs In situ hybridization and IHC are both negative for the heart, spleen, kidneys, and liver. In contrast, RT-PCR and nucleic acid sequence-based amplification-based H5 detection assay are positive for these organs.7 The discrepancies between the in situ hybridization/IHC and RT-PCR results may be explained by either false-negative results of the in situ hybridization and IHC assays attributable to limitations in sensitivity or false-positive RT-PCR results attributable to viremia in blood perfusing the organs without actual viral replication in the tissues.21 Placenta and Fetus In the placenta of a female infected with H5N1 influenza virus, viral antigens and sequences have been found in Hofbauer cells (fetal macrophages) (Figure 1F) and cytotrophoblasts, but not in syncytiotrophoblasts.7 RT-PCR results have indicated viral replication in the placenta.7 In addition, in situ hybridization, IHC (Figure 1G), and real-time RT-PCR confirmed infection of the fetus, demonstrating that the virus is vertically transmissible from mother to fetus.7 Transplacental transmission of the virus may occur through mechanisms similar to those for transmission of the cytomegalovirus, a virus that is also known to infect mainly cytotrophoblasts and Hofbauer cells.22 Transmission may take place via transcytosis across syncytiotrophoblasts to cytotrophoblasts in chorionic villi.22 Alternatively, the virus may infect invasive cytotrophoblasts within the uterine wall after contact with maternal blood. These infected cells would subsequently transmit the virus via the cell columns to the anchoring chorionic villi.22 The virus may then be transmitted to Hofbauer cells, which would enter the fetal circulation and carry the virus to the fetus. Immune Cells and Blood Viral sequences and antigens have been detected in lymphocytes in lymph node tissue, as well as in Hofbauer cells (macrophages of the placenta), Kupffer cells (macrophages of the liver), and mononuclear cells in the intestinal mucosa.7 These findings are consistent with in vitro experiments demonstrating infection of macrophages by the H5N1 influenza virus23,24,25 and with ex vivo experiments showing H5N1 virus attachment to and infection of alveolar macrophages in human lung tissue.26,27 Viral RNA has been detected in blood samples of several H5N1 cases, all of them fatal.17 Viremia may occur in the course of the disease, as evidenced by virus isolation from serum and plasma samples of two fatal cases.15,28 Accordingly, extra-pulmonary dissemination may be the result of viremia or of infected immune cells transporting the virus to other organs. Histopathology and Virus Distribution in Animal Studies The available studies of human H5N1 autopsies have a number of limitations in terms of pathological findings. The majority of the individuals who died from H5N1 influenza had received various interventional therapies aimed at limiting tissue injury and viral replication. Second, none of these cases succumbed during the early phase of the infection (see Table 1), thus preventing histopathological and molecular pathological data from being obtained at the very early phase of the illness through autopsy. Animal studies have provided important supplementary information with respect to the natural course of H5N1 influenza. Several animal models including the mouse, ferret, cynolmolgus macaque, and cat have been used to investigate viral replication and histopathology in H5N1 infections. Histopathologically, early lesions in the lungs included features of focal peribronchiolar pneumonia (3 to 5 days after infection), whereas 6 to 8 days after infection the lungs showed extensive consolidation and bronchiolitis.29,30,31,32,33,34,35,36,37,38,39 In the majority of these animal models, tissue injury is also observed to varying degrees in extra-pulmonary organs, in particular in the brain.29,30,31,32,33,35,36 Similar to human cases, the virus appears to be capable of spreading beyond the lungs as has been evidenced by virus isolation and detection of viral antigens in various extra-pulmonary organs including the brain, liver, lymphoid tissues, heart, and kidneys.29,30,31,32,33,34,35,37 Infection with highly pathogenic H5N1 isolates in animal experiments has been associated with severe lymphopenia. In these experiments, H5N1 virus caused progressive depletion of lymphocytes, whereas infection with low pathogenic virus did not affect total white blood cell counts in mice.38,39 In human cases, lymphopenia has been associated with disease severity,2 and lower numbers of T lymphocytes have been detected in fatal cases compared to nonfatal cases.17 Several mechanisms have been implicated in the genesis of lymphopenia, including apoptosis and bone marrow suppression. Implications on Pathology H5N1 influenza appears to be a systemic infection in both human and animal cases. In humans the trachea, brain, and intestines may be infected in addition to the lungs. It appears that the virus may also spread to other organs, such as the kidneys and liver, as has also been demonstrated in animals. To gain a better understanding of viral distribution in humans, additional autopsy studies with molecular methods will be necessary. In view of adequate treatment of patients with avian influenza, it is important to realize that the disease affects multiple organs. Therapeutic regimes should therefore not only comprise optimal respiratory care but should also pay attention to adequate supportive care of other organs involved. Multiple-organ infection and vertical transmission of H5N1 have public health implications. The fact that the virus has been isolated from serum and feces makes it possible that infection can be transmitted through gastrointestinal contamination or infected blood. Needless to say that extreme care should be taken when handling body fluids from H5N1 cases. The finding that the virus is transmissible from mother to fetus is alarming and might reflect enhanced pathogenicity of the H5N1 virus. Care should be taken when handling delivery or abortion from H5N1-infected mothers. Pathogenesis Various factors are thought to be involved in the pathogenesis of H5N1 influenza (Figure 2), and a combination of these factors most likely determines the extent of tissue injury and disease outcome. The role of dysregulation of cytokines and chemokines has been studied extensively and may be one of the key mechanisms in the pathogenesis of H5N1 influenza, in addition to injury resulting from viral replication. Other factors, such as up-regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and reduced cytotoxicity of CD8+ lymphocytes are also believed to be involved in the pathogenesis, although their exact roles are less clear at present. We discuss these factors and related mechanisms below. Viral Replication It is generally thought that replication of the H5N1 virus results in cell and organ damage by either cytolytic or apoptotic mechanisms, similar to human influenza infections. There are clear indications of active viral replication in the respiratory tract. The virus has been isolated from throat and trachea aspirates, and postmortem lung tissues.3,17,18,40 Viral RNA has been detected in nasal, nasopharyngeal, and tracheal specimens.17,18,40,41 Viral RNA has been detected in nasopharyngeal aspirates ranging from 1 day up to 15 days after disease onset.40,42 Viral replication appears to be prolonged in H5N1 influenza because viral loads when plotted against time did not show a clear decline in a large group of H5N1 patients.17 In the same group of H5N1 cases, viral RNA levels in pharyngeal and nasal specimens were higher than in a group of patients infected with common human influenza viruses. In addition, the highest viral loads were detected in the fatal cases, suggesting a correlation between viral replication and negative disease outcome.17 Both in situ hybridization with anti-sense probes and RT-PCR detecting positive-stranded RNA have provided evidence of viral replication in the trachea and lungs (see Virus Distribution).7 In addition, ex vivo experiments have shown that H5N1 viruses can productively infect tissues of the nasopharynx and lungs.27 In animal models the H5N1 virus has also been detected in the upper respiratory tract and the lungs as early as 1 day after infection, replicating to extremely high levels.32,34,36,37,39 In contrast to the respiratory tract, little has been reported regarding H5N1 viral replication in extra-pulmonary organs. Positive-stranded RNA has been detected in the intestines, brain, heart, and placenta (see Virus Distribution). In addition, anti-sense probes have been found to hybridize in the intestines, brain, and placenta.7 These findings strongly suggest that active viral replication may occur in these organs. This would be consistent with findings in animal experiments in which high replication titers of human H5N1 isolates have been found not only in the respiratory tract, but also in several extra-pulmonary organs (see Histopathology and Virus Distribution in Animal Studies).29,30,31,32,33,34,35,37,39 In these studies viral replication peaked in the extra-pulmonary organs on days 3 to 6 after infection.31,32,37,39 Dysregulation of Cytokines and Chemokines Various studies indicate that aberrant production of proinflammatory cytokines and chemokines may play an important role in the pathogenesis of H5N1 influenza. Pathological features that are consistent with dysregulation of cytokines and chemokines, including hemophagocytotic activity, have been described in H5N1 autopsy cases (see Histopathology).3,4,6,9,11,13 In many H5N1 patients elevated serum levels of proinflammatory cytokines and chemokines have been detected.3,6,8,17 In a study cohort of 18 H5N1 patients, serum levels of most of the tested chemokines and cytokines were significantly higher than in the control group of H3N1 human influenza patients.17 In the same study group serum cytokine and chemokine levels have been found to correlate with viral loads in pharyngeal specimens, suggesting that high viral loads may induce hypercytokinemia and hyperchemokinemia.17 Suppression of viral replication by timely administration of antiviral agents may therefore help prevent hyperinduction of cytokines and chemokines. Serum cytokine and chemokine levels do not necessarily reflect local production of these regulatory proteins in the lungs.43 There are a number of studies investigating the local expression of cytokines and chemokines in the lungs of H5N1 cases. Immunohistochemistry has detected high expression of tumor necrosis factor-α (TNF-α) in the lungs of a H5N1 autopsy case in Hong Kong.3 In another investigation the lungs of a H5N1-infected case showed enhanced expressions of macrophage inflammatory protein-1α, regulated on activation normal T cell expressed and secreted (RANTES), interferon-γ, interferon-β, and interleukin-6, but neither of monocyte chemoattractant protein 1 (MCP-1) nor of TNF-α (R.S. Deng, unpublished observations) (Figure 1H). Furthermore, up-regulation of TNF-α has been detected in two autopsy cases by using RT-PCR.8,9 It should be noted that the availability of data regarding serum cytokine levels and immunohistopathological studies in humans is limited. In addition, interpretation of cytokines and chemokines in critically ill patients is not without difficulties. In view of these difficulties, in vitro and animal studies could provide additional information. Results of in vitro experiments support the role of an exaggerated immune response in the pathogenesis of H5N1 influenza. H5N1 avian influenza viruses induce significantly higher expression of several cytokines and chemokines in human macrophages and respiratory epithelial cells than human influenza viruses.23,24,44,45 In these experiments enhanced expression is reflected by increased production of cytokines and chemokines in the supernatants from infected cells.23,24,45 Animal experiments have also provided support for a possibly critical role of proinflammatory cytokines and chemokines in H5N1 pathology (see Nonstructural Proteins).46,47,48 Up-regulation of cytokines and chemokines, however, is not a unique feature of H5N1 influenza infection. In SARS, hyperinduction of the immune system is believed to be an important pathogenetic factor.49,50 Similarly, up-regulation of cytokines and chemokines is also a significant characteristic of the H1N1 human influenza virus that caused a major influenza pandemic in 1918 to 1919.51,52,53 High expression of cytokine and chemokine genes have been found in the lungs of mice and nonhuman primates infected with the reconstructed 1918 H1N1 influenza virus.52,53 H5N1 viruses may not only be capable of up-regulating cytokines and chemokines, but also be resistant to the anti-viral effects of interferons and TNF-α (see below). Up-Regulation of TRAIL and Apoptosis Up-regulation of functional TRAIL in macrophages infected with the H5N1 virus may be another important factor in the pathogenesis of H5N1 influenza virus infection.54 TRAIL is one of the many death receptor ligands that trigger apoptosis of cells by binding to death receptor ligand receptors expressed on target cells. Zhou and colleagues54 have demonstrated significantly higher expression of TNF-α and TRAIL in macrophages infected with H5N1 virus in vitro than in macrophages infected with human H1N1 influenza virus. In addition, T lymphocytes co-cultured with macrophages infected with H5N1 virus show increased induction of apoptosis compared to T lymphocytes co-cultured with macrophages infected with other influenza viruses. Enhanced sensitization of virus-infected T lymphocytes to death receptor ligand-induced apoptosis has also been demonstrated.54 Both sensitization and up-regulation of TRAIL may partially account for the lymphopenia and lung injury frequently observed in H5N1 patients.54 In addition, a delayed onset of apoptosis has been demonstrated in vitro in H5N1-infected macrophages compared with H1N1-infected macrophages.25 Prolonged survival of infected macrophages may further enhance the induction of apoptosis in T lymphocytes.25 It may also augment immune-mediated pathology because macrophages secrete cytokines and chemokines for a longer period of time. In human autopsies apoptosis has been detected in both alveolar epithelial cells and leukocytes in the lungs, as well as in spleen and intestinal tissues.9 Apoptosis may, therefore, be one of the pathogenetic mechanisms contributing to injury in the lungs and other organs. Apoptosis may occur as a result of direct viral replication or up-regulation of cytokines and chemokines.9 Reduced Cytotoxicity of CD8+ Lymphocytes As opposed to H1N1 and H3N2 viruses, the HAs of H5N1 viruses suppress perforin expression in cytotoxic T lymphocytes according to in vitro experiments.55 It has been suggested that this may result in impaired cytotoxic activity causing failure of clearance of H5N1 virus or HA (H5) protein-bearing cells, including antigen-presenting cells. Excessive production of interferon-γ caused by sustained antigenic stimulation of cytotoxic T lymphocytes may subsequently lead to up-regulation of proinflammatory cytokines in macrophages.55 Implications on Pathogenesis H5N1 viral replication appears to be prolonged with high levels of viral RNA, and the virus may disseminate to extra-pulmonary organs. Timely suppression of viral replication is the mainstay of therapy in H5N1 infection. Oseltamivir, a neuraminidase inhibitor is the principal antiviral agent of choice because many H5N1 isolates are resistant to amantadines (World Health Organization: Clinical management of human infection with avian influenza A (H5N1) virus. http://www.who.int/csr/disease/avian_influenza/guidelines/clinicalmanagement07.pdf; accessed January 2008). Given the potential role of up-regulation of cytokines and chemokines in the pathogenesis of H5N1 infection, it has been suggested that suppression of the exaggerated host immune response may also be a beneficial therapeutic strategy. Unfortunately, the data on treatment with corticosteroids, albeit limited, have thus far not shown any obvious clinical benefit in treatment of human H5N1 infection.41 Similarly, corticosteroids have failed to show any clear benefits in the treatment of other viral respiratory infections, including SARS.56 Further, no human trial with specific cytokine blockers for treatment of H5N1 influenza has been published to date. As to the use of cytokine blockers in animal experiments, only one study has been published describing reduced illness severity in human influenza virus-infected mice treated with anti-TNF antibodies.57 Altogether, there is insufficient data supporting the use of immunomodulating agents in the treatment of H5N1 influenza. There is an obvious need for more studies into pathogenetic factors as well as therapeutic intervention strategies including immunotherapy. Viral Genes and Gene Products Involved in the Pathogenesis of H5N1 Influenza Influenza viruses belong to the family of Orthomyxoviridae. There are three types of influenza viruses: type A, type B, and type C. Avian influenza viruses are all classified as type A influenza viruses.58 Influenza viruses can be subtyped based on the antigenicity of their two surface glycoproteins, HA and neuraminidase (NA).58,59 Sixteen HA and nine NA subtypes have thus far been identified.59,60 The influenza A virus genome consists of eight gene segments encoding 11 viral proteins (gene products) including HA, NA, polymerase proteins (PB1, PB2, PA, and PB1-F2), NP, nonstructural proteins (NS1 and NS2), and M1 and M2 proteins (Figure 2).61,62 These genes and/or gene products have various basic functions ranging from viral RNA synthesis to receptor binding (Table 2).58,59 Several of these genes and/or gene products have additional functions that may contribute to the pathogenesis of H5N1 influenza and enhance pathogenicity of H5N1 influenza viruses. Both the basic functions of influenza A viral proteins and the specific functions accounting for increased pathogenicity are summarized in Table 2 and discussed in the following sections. Figure 2 contains a schematic presentation of the viral genes and gene products that may be involved in the key pathogenetic mechanisms of H5N1 influenza. Hemagglutinin Hemagglutinin is a surface protein that functions as a receptor-binding site and is the target of infectivity-neutralizing antibodies.63 The HA protein attaches to sialic acid-containing receptors expressed on the host cell, and after proteolytic activation of the precursor HA molecule into HA1 and HA2, the virus fuses with the host cell.58 Early reverse genetics studies have demonstrated that the level of cleavability of HA determines the virulence of avian influenza viruses in poultry.64 Avirulent viruses usually possess HAs with a single arginine residue at the cleavage site that can only be cleaved by extracellular trypsin-like proteases present in the upper respiratory and gastrointestinal tracts, thus merely giving rise to local infections. In contrast, virulent viruses have HAs with multiple residues at the cleavage site that can be activated by ubiquitous intracellular proteases and may therefore cause systemic infections.65 The significance of multiple residues at the cleavage site has also been established for the virulence of H5N1 viruses, as evidenced by attenuation of disease in mice infected with a mutant H5N1 A/Hk/483/97 virus with a single arginine residue at the cleavage site.66 All H5N1 viruses isolated from human cases since 1997 have multiple basic amino acids at the cleavage site.1,12,45,66,67,68,69,70,71,72 However, the disease severity in human cases varies from mild to extremely severe, implying that there are other factors responsible for the virulence of H5N1 influenza viruses in humans.38 The destructive 1918 influenza virus resembles the H5N1 virus in having a HA that is highly cleavable. The HAs of both virus types can be cleaved in the absence of trypsin.65,73 However, the mechanisms for the high cleavability of HA differ for both virus types. In contrast to the H5N1 viruses, of which the HA is easily cleavable because of the presence of multiple basic amino acids at the cleavage site, both HA and NA appear to be involved in the HA activation of the 1918 H1N1 viruses through a yet unidentified mechanism.73 As discussed in Reduced Cytotoxicity of CD8+ Lymphocytes, the HA of H5N1 viruses may also be involved in the suppression of perforin in cytotoxic T lymphocytes. Neuraminidase The NA protein is a sialidase that cleaves the HA of progeny virions from the sialic acid-containing receptors on the surface of the host cells, thus separating the particles from the infected cells in which they were generated.59 The H5N1 viruses isolated during the 1997 outbreak have a deletion of 19 amino acids in the NA stalk region1,12,72,74 that is thought to play a role in the adaptation of the virus in the course of transmission from aquatic to terrestrial birds.74 The first 2003 human isolates had no NA stalk deletion,47,71 but more recent human and chicken isolates did show a deletion in the NA stalk, which is similar, although not identical, to that of 1997 H5N1 viruses.67,68,70,75 Viruses containing an NA stalk deletion are less capable of freeing virions from infected cells.74 This negative effect may, however, be counterbalanced by the facilitating effects on the release of viral particles conferred by an additional glycosylation site at the top of HA globular heads,74,76 frequently found in H5N1 viruses with an NA stalk deletion.67,70 Most H5N1 viruses are sensitive to neuraminidase inhibitors.67,77 It is remarkable that in three human H5N1 cases a histidine to tyrosine substitution at position 274 of the NA protein that confers resistance to oseltamivir (a neuraminidase inhibitor), has been reported.78,79 All instances were related to prophylactic or therapeutic use of oseltamivir and may have been caused by incomplete suppression of viral replication.78 Polymerase Gene Complex and Nucleocapsid Protein The polymerase complex is composed of three viral polymerase proteins (PB1, PB2, and PA) involved in viral RNA synthesis.59 The polymerase complex together with NP constitutes the ribonucleoprotein complex.58 The RNA gene segments are encapsulated by NP, facilitating their recognition by the polymerase complex.58 The polymerase gene complex is an important molecular determinant of virulence in animal models.66,80,81 Early studies have demonstrated that the amino acid at position 627 of PB2 determines virulence of H5N1/Hk/97 human isolates in mice. Glutamic acid at this position confers low pathogenicity, whereas lysine at this position confers high pathogenicity.66 Glutamic acid to lysine substitutions have also been detected in several viruses isolated from human cases in Vietnam and Thailand.68,69,70,82,83 H5N1/Vietnam/2004 virus isolates possessing lysine at position 627 have been shown to replicate better in a wider range of cell types including cells of the upper respiratory tract and at lower temperatures than similar isolates with glutamic acid at this position, thus possibly facilitating virus excretion by sneezing and coughing.83 It is noteworthy, however, that the presence or absence of lysine at position 627 does not appear to affect the clinical outcome in humans.17,69 Moreover, several viruses have been isolated from fatal human cases that lack this substitution.17,45,70,71,82 This would indicate that lysine at position 627 is not a prerequisite for high virulence in humans. Studies in mice and ferrets infected with reassortant viruses containing genes of A/Vietnam/1203/04 have demonstrated that polymerase complex genes, rather than HA or NA genes, account for the high virulence of this particular H5N1 strain.80 In this study, not only PB2 but also PB1 contributes to pathogenicity, as suggested by attenuated disease in mice inoculated with PB1 reassortants.80 To explain the molecular basis of adaptation of influenza viruses to a new host species, a model of species transmission has been designed using a low pathogenic avian influenza virus and its lethal mouse adapted descendant.81 In this model various mutations in the ribonucleoprotein complex were found to enhance pathogenicity.81 Remarkably, mutations similar to the ones detected in this mouse model have also been detected in mammalian and human strains that had only shortly been transmitted from poultry.17,81 This may indicate that the polymerase complex plays an important role in the adaptation to a new host.80,81 Such a role is further supported by findings regarding the 1918 influenza virus. Similar to the H5N1 virus, the 1918 influenza virus appears to be an avian-like virus rather than a reassortant.84 Only 10 amino acid changes have been found to differentiate the polymerase proteins of the 1918 human influenza virus from avian consensus sequences.84 It is thought that these changes were essential for the adaptation of the 1918 influenza virus to humans.84 Several similar changes, including the lysine residue at position 627, have also been independently detected in H5N1 viruses isolated from human cases. In a large-scale study of avian influenza isolates, the gene encoding for PB1-F2 was found to be the only gene under positive selection.85 PB1-F2 is a small mitochondrial protein that is encoded on an open reading frame of PB1.61 This open reading frame is highly conserved in avian influenza isolates.85 PB1-F2 sensitizes infected cells to apoptotic stimuli such as TNF-α through the interaction with the mitochondrial permeability transition pore complex.86 Increased cell death responses in mice infected with the reconstructed 1918 influenza virus have been linked to the PB1-F2 protein.52 It has been speculated that the PB1-F2 protein of the H5N1 virus has a similar role in the pathology in H5N1 infections. In addition, it may be possible that PB1-F2 also induces apoptosis of immune cells, which could result in diminished antigen presentation leading to an insufficient adaptive immune response.46 Recent recombinant virus studies have demonstrated that a single mutation in the PB1-F2 protein [serine (S) instead of asparagine (N) at position 66] of H5N1 (Hk/97) increases viral pathogenicity. Mice infected with this virus showed decreased survival rates, significantly higher viral loads in the lungs, delayed viral clearance, as well as elevated levels of cytokines in the lungs.46 Slower viral clearance, induced by the expression of the PB1-F2 protein, may cause immune-mediated injury, supported by the detection of increased levels of cytokines in the lungs.46 Nonstructural Proteins The NS proteins (NS1 and NS2) are viral proteins that play a significant role in viral replication.58 The NS1 protein is crucial for evading the innate immune response of the host by inhibiting the antiviral response mediated by type I interferons.87 The NS gene of certain H5N1 viruses may play an additional role in the pathogenesis of H5N1 influenza because it may confer resistance to the antiviral effects of TNF-α and interferons. In vitro experiments have shown that the replication of H5N1/97 viruses in porcine lung epithelial cells was not inhibited by pretreatment with either interferons or TNF-α.88 The presence of glutamic acid at position 92 of NS1 appeared to be a prerequisite for the resistance to antiviral cytokines.88 This in vitro resistance is supported by results of in vivo animal experiments in which pigs infected with a reassortant influenza virus (H1N1) bearing the NS gene of the H5N1/97 virus display a more severe disease compared to pigs inoculated with the parental H1N1 virus.72 Recent human and avian isolates, however, lack glutamic acid at position 92 of the NS1 protein.17,45,68,82 The NS gene of H5N1 viruses may also account for up-regulation of cytokines and chemokines. High concentrations of proinflammatory cytokines and low concentrations of an anti-inflammatory cytokine (interleukin-10) have been detected in lung homogenates of mice infected with a reassortant influenza virus encoding the NS gene of the H5N1/97 virus. Similar cytokine imbalances have not been found in mice inoculated with a reassortant influenza virus encoding the NS gene of the low pathogenic H5N1/2001 virus or an NS gene encoding a glutamic acid to asparagine substitution at position 92 of NS1.47 Furthermore, TNF-α concentrations in the supernatants from macrophages infected with recombinant viruses encoding the NS gene of the H5N1/97 are significantly higher than in those from macrophages infected with recombinant viruses containing the NS gene of nonrelated influenza viruses.24 It has been argued that both increased resistance to antiviral effects of cytokines and up-regulation of cytokine production may act synergistically to induce pulmonary injury.24 Two PDZ ligand (PL) sequence motifs in the NS1 gene of H5N1 viruses have recently been identified as potential co-determinants of virulence. Viruses isolated during the 1997 outbreak contain a Glu-Pro-Glu-Val (EPEV) motif at the carboxyl terminus of NS1, and 2003 to 2004 isolates contain a Glu-Ser-Glu-Val (ESEV) motif. These avian PLs bind in vitro to the PDZ domains of several human proteins that are crucial for cell signaling.85 Infection of human cells by viruses with avian PL motifs may therefore disrupt several PDZ-domain protein-mediated pathways, thus contributing to pathogenicity of H5N1 viruses.85 However, recently isolated, highly pathogenic viruses lack these motifs. The presence of EPEV or ESEV motifs at the carboxyl terminals of NS1 proteins thus appears not to be a prerequisite for virulence of H5N1 viruses.89 M1 and M2 Proteins The M gene encodes two proteins: M1 (matrix protein) and M2. The matrix protein lies underneath the viral envelope and plays a significant role in virus assembly.65 M2 is a small protein embedded in the viral envelope that functions as a H+ ion channel, thus controlling the pH in the Golgi complex during HA synthesis and virion disassembly.62 In a study of Thai and Indonesian isolates, the gene encoding the M2 protein was found to be one of the two gene segments (in addition to PB1-F2) under positive selection, indicating a possible role for M2 in the adaptation of the virus to a new host.69 Most viruses isolated from humans and/or birds in countries on the Indo-China peninsula (the so-called “clade 1 viruses”) contain a serine to asparagine substitution at residue 31 of the M2 protein, which is associated with resistance to amantadines.67,69 In contrast, only few viruses isolated from humans and/or birds in China, Indonesia, Japan, and South Korea (the so-called “clade 2 viruses”) possess such a substitution.7,77,80,90 Implications for Viral Genes and Gene Products Since the first avian influenza outbreak in 1997 several amino acid substitutions including the glutamic acid to lysine substitution at position 627 of PB2 and glutamic acid at position 92 of NS1 have been indicated as major contributors to virulence. In view of the observations that recent H5N1 viruses, including the ones isolated from fatal cases, lack these substitutions and that recent isolates have been found to posses other mutations, it becomes increasingly apparent that virulence cannot be attributed to a single gene or amino acid substitution. In fact virulence appears to be a polygenic trait with several genes co-operating together. The precise role of the recently discovered PB1-F2 protein in the pathogenesis of H5N1 influenza requires further exploration. Future studies will most likely identify other molecular determinants of pathogenicity. Receptor Specificity and Transmissibility of the H5N1 Virus Avian Versus Human Influenza Virus Receptors Human and avian viruses bind to different receptors. The HA protein of avian influenza viruses preferentially binds to sialic acids linked to galactose by α-2,3 linkage (avian influenza virus receptors), which are located on the intestinal epithelial cells of avians. In contrast, the HA protein of human influenza viruses primarily recognizes α-2,6-linked sialic acids (human influenza virus receptors), which are notably expressed on epithelial cells of the human trachea.91,92,93,94,95 Because of these differences in receptor specificity and distribution, as well as the limited replication in humans, avian influenza viruses were initially thought to be incapable of causing human infection. However, this presumption was proved incorrect when in 1997 the H5N1 avian influenza virus infected and killed several humans. Since then a number of studies have been performed aiming to explain the ability of avian influenza viruses to infect humans. Human influenza virus receptors are mainly expressed in the upper respiratory tract, whereas avian influenza virus receptors are primarily expressed in the lower respiratory tract (type II alveolar cells) (Figure 1I).26,27,96 However, avian influenza virus receptors have also been detected on epithelial cells in human tracheobronchial cell cultures and in human tissue sections of trachea and bronchi, albeit to a lesser extent than human influenza virus receptors.92,93,97,98,99,100 This may explain the capability of the virus to infect humans. In addition, H5N1 viruses are capable of infecting ex vivo nasopharyngeal tissues, despite a limited number of avian influenza virus receptors detected.26 Therefore, it has been suggested that the H5N1 virus may also use alternative binding sites on the epithelium to enter target cells.27 Conflicting results have been reported as to the cell type expressing avian influenza virus receptors in the trachea and bronchi. Some studies have found such receptors to be located mainly on basal cells101 or only on goblet cells,91,92 whereas others have detected their presence primarily on ciliated cells97,98,99,100 and only on a small proportion of nonciliated cells,98,99,100 including basal cells.99 In tracheobronchial cell cultures avian influenza viruses have been found to infect mainly ciliated cells.97,98,102 Alveolar macrophages appear to have none or very few avian influenza virus receptors.27,101 The receptor distribution in extra-pulmonary tissues has been less extensively studied. Thus far neurons and the epithelial cells of the pancreatic and bile ducts have been found to express avian influenza receptors.103,104 In addition, avian influenza virus receptors have been detected on endothelial cells in many organs throughout the body.101 Some studies have reported the presence of avian influenza virus receptors on the epithelial cells of the intestinal mucosa,105,106 whereas others did not find their presence on such cells.101 With respect to immune cells, avian influenza virus receptors have been detected on T cells87 and Kupffer cells of the liver.101 The receptor distribution pattern as detected by lectin histochemistry broadly resembles that of infected organs and cells as demonstrated by in situ hybridization. However, the abundant expression of avian influenza virus receptors found on the endothelial cells of various organs contrasts with the absence of virus in these cells. In addition, the widespread and abundant expression of avian influenza virus receptors in the lungs is not in line with the limited number of infected pneumocytes, as detected by in situ hybridization/IHC.7 At the same time the absence of avian influenza virus receptors on placental macrophages, alveolar macrophages, cytotrophoblasts, and intestinal epithelial cells is inconsistent with the detected infection of such cells. These discrepancies further support the assumption that other receptors, co-receptors, or mechanisms may play a role in the interaction between the virus and its target cells, thus warranting further investigation. Receptor Switch and Human-to-Human Transmission Most avian and human H5N1 isolates only bind to avian influenza virus receptors.67,70,71 Only a limited number of H5N1 human isolates have been identified that are capable of binding to human influenza virus receptors in vitro.35,71,96,97 It is thought that for efficient human-to-human transmission the HA protein of influenza viruses should preferentially recognize human influenza virus receptors.96,107,108 Previous studies with H1, H2, and H3 serotypes have demonstrated that minor mutations in the HA gene may cause receptor specificity to switch from recognizing avian influenza virus receptors to recognizing human influenza virus receptors.94,95,109,110 Similar mutations have been introduced on the framework of an A/Vietnam/2004 H5N1 virus.108 Mutations enabling H1 serotypes to recognize human receptors applied to H5N1 virus affect its affinity for avian receptors but do not result in human receptor specificity. In contrast, mutations enabling H2 and H3 receptors to recognize human receptors applied to H5N1 virus resulted in significant binding of the mutant virus to a natural, branched α-2,6-linked biantennary N-linked glycan and in a reduced binding to α-2,3-linked SA receptors. Viruses with these properties would be able to evade the virus-neutralizing effects of mucins containing α-2,3-linked SAs and bind more avidly to lung epithelial cells expressing α-2,6-linked biantennary N-linked glycans.108 Yamada and colleagues107 have recently provided further insight in possible mutations affecting the ability of the H5N1 virus to bind to human receptors. By performing reverse genetics studies and crystal structure determination, they have identified two mutations at position 182 and 192 of HA that enhance binding of H5N1 viruses to human influenza virus receptors. Despite the capability of a number of human H5N1 isolates to bind to human influenza virus receptors in vitro,35,71,96,107 these isolates do not spread efficiently from human-to-human in vivo. Animal experiments with reassortant viruses have also shown that the mere acquisition of human influenza surface proteins does not necessarily confer transmissibility of H5N1 virus.111 Inoculation with a reassortant virus containing genes for internal proteins of H5N1 A/Hk/486 and for surface proteins of human H3N2 A/vic/75 did not result in efficient transmission among ferrets in a respiratory droplet experimental design, even though viral replication was not compromised.101 In addition, four H5N1 human isolates (A/Vn/1203/04, A/Vn/JP36-2/05, A/Hk/213/03, and A/Turkey/65-596/06), including isolates capable of binding to human influenza virus receptors (A/Hk/213/03 and A/Turkey/65-596/06), have been studied in a direct contact model of ferrets.35 No transmission of either H5N1 A/Vn/1203/04 or A/Turkey/65-596/06 virus was detected. Although transmission of both H5N1 A/Hk/213/03 and A/Vn/JP36-2/05 viruses occurred, it appeared to be far from efficient. No secondary transmission from an infected contact ferret to a naïve contact ferret was demonstrated. On the basis hereof it appears that increased binding affinity for human influenza receptors alone is not sufficient for efficient transmission. Additional molecular determinants seem to be required. It has been suggested that certain biological properties such as the capacity to induce virus excretion from the upper respiratory tract (coughing and sneezing) may enhance efficient transmission.111 As mentioned above, the presence of lysine at position 627 is a molecular determinant associated with such a capacity and may, therefore, contribute to efficient spread among humans. However, it seems likely that various additional amino acid changes are required to give avian influenza viruses the capacity to spread among humans.83 In fact for the 1918 H1N1 influenza virus, a human influenza virus that is supposed to be derived solely from an avian source, various amino acid changes differentiate the human isolate from its avian consensus sequences.84,112 Implications on Receptors The widespread distribution of avian influenza virus receptors in various organs may explain the multiple organ involvement seen in H5N1-infected humans. However, there are a number of discrepancies between the cell types expressing avian influenza receptors and the cell types found to be infected by the H5N1 virus. Despite the relative lack of avian influenza virus receptors, viral replication has been demonstrated in the upper respiratory tract. Therefore, further research is needed to investigate the possible role of other receptors or mechanisms involved in the interaction between the virus and its target cells. The identification of other receptors could help the design of effective drugs treating H5N1 infection or preventing transmission. Recent studies have shown that not only the acquisition of the capacity to bind to human influenza virus receptors but also other genetic changes may be necessary for efficient transmission among humans. Continuous surveillance of the circulating H5N1 strains is of key importance as the emergence of amino acid substitutions similar to those demonstrated for the 1918 H1N1 virus might indicate that the virus could acquire pandemic potential in the near future. Final Remarks Since 1997 several studies have contributed to fundamental insights into the pathology and pathogenesis of human H5N1 influenza. Aside from the respiratory tract, other organs such as the intestines, the brain, and the placenta appear to be infection targets of the virus. The H5N1 virus is also capable of transplacental transmission to the fetus. Dysfunction of the immune system may be a key pathogenetic mechanism. At the molecular level, several viral genes and mutations in gene products have been suggested to be involved in increased virulence of H5N1 viruses. At the same time, however, it becomes increasingly apparent that what is known today about the virus and its pathogenicity is only the tip of the iceberg and that there are likely several additional pathogenetic mechanisms and molecular determinants of pathogenicity in H5N1 influenza yet to be identified. In light of the subsisting threat of a potentially devastating influenza pandemic, further investigations in these respects are urgently required.

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          Most cited references88

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          Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.

          Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein of influenza virus and the target for infectivity-neutralizing antibodies. The structures of three conformations of the ectodomain of the 1968 Hong Kong influenza virus HA have been determined by X-ray crystallography: the single-chain precursor, HA0; the metastable neutral-pH conformation found on virus, and the fusion pH-induced conformation. These structures provide a framework for designing and interpreting the results of experiments on the activity of HA in receptor binding, the generation of emerging and reemerging epidemics, and membrane fusion during viral entry. Structures of HA in complex with sialic acid receptor analogs, together with binding experiments, provide details of these low-affinity interactions in terms of the sialic acid substituents recognized and the HA residues involved in recognition. Neutralizing antibody-binding sites surround the receptor-binding pocket on the membrane-distal surface of HA, and the structures of the complexes between neutralizing monoclonal Fabs and HA indicate possible neutralization mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in its structure primes HA for subsequent activation of membrane fusion at endosomal pH (Figure 1). Priming involves insertion of the fusion peptide into a charged pocket in the precursor; activation requires its extrusion towards the fusion target membrane, as the N terminus of a newly formed trimeric coiled coil, and repositioning of the C-terminal membrane anchor near the fusion peptide at the same end of a rod-shaped molecule. Comparison of this new HA conformation, which has been formed for membrane fusion, with the structures determined for other virus fusion glycoproteins suggests that these molecules are all in the fusion-activated conformation and that the juxtaposition of the membrane anchor and fusion peptide, a recurring feature, is involved in the fusion mechanism. Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive factor attachment protein receptor (SNARE) protein complex of vesicle fusion allows a similar conclusion.
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            SARS: Systematic Review of Treatment Effects

            Introduction The severe acute respiratory syndrome (SARS) is a febrile respiratory illness primarily transmitted by respiratory droplets or close personal contact. A global outbreak of SARS between March 2003 and July 2003 caused over 8,000 probable or confirmed cases and 774 deaths [1]. The causative organism has been identified as a novel coronavirus (SARS-CoV) [2–4]. The overall mortality during the outbreak was estimated at 9.6% [5,6]. The overriding clinical feature of SARS is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ARDS). This complication occurred in approximately 16% of all patients with SARS, and when it occurred was associated with a mortality rate of 50% [7,8]. At the time of the SARS epidemic it was not known what treatments would reduce SARS-related illness and deaths. Because the urgency of the international outbreak did not allow time for efficacy studies, physicians in Canada and Hong Kong treated the earliest patients with intravenous ribavirin, based on its broad-spectrum antiviral activity [9,10]. Corticosteroids and immune-modulating agents were often prescribed empirically. Soon after SARS-CoV was identified as the causative agent, antiviral screening programs were initiated; these programs reported several antiviral agents that inhibited SARS-CoV replication in vitro. These results led to the experimental use of protease inhibitors and interferon alpha (IFN-α) in the treatment of patients. The most commonly used treatments for SARS are associated with adverse effects when used for other conditions (Table S1). In October 2003, the WHO established an International SARS Treatment Study Group, consisting of experts experienced in managing SARS. The group recommended a systematic review of potential treatment options to identify the targets for proper evaluation in trials should the disease recur [11]. This paper reports on this systematic review designed to summarise available evidence on the effects of ribavirin, lopinavir and ritonavir (LPV/r), corticosteroids, type I IFN, intravenous immunoglobulin (IVIG), or convalescent plasma in relation to (1) SARS-CoV replication inhibition in vitro; (2) mortality or morbidity in SARS patients; and (3) effects on ARDS in adult patients. Methods We prepared a protocol that defined our scope, inclusion criteria, and outcomes to be assessed. The interventions we included were defined by the WHO: ribavirin, LPV/r, corticosteroids, type I IFN, convalescent plasma, or IVIG. The types of study we included were: (1) in vitro studies, in which the authors examined inhibition of SARS-CoV viral replication, and data from an assay in human or animal cell line; (2) in vivo studies, which included randomised controlled trial (RCT), or prospective uncontrolled study design, or retrospective cohort design, or case-control design, or a case series, and patients treated for SARS, and ten or more patients; and (3) studies of ARDS that included RCT, or systematic review, and treatment for ARDS or acute lung injury, and 20 or more patients. In February 2005, we systematically searched the literature databases MEDLINE, EMBASE, BIOSIS, and the Cochrane Central Register of Controlled Trials (CENTRAL) for articles that included the selected treatments (Table S2). The full text of each identified study was retrieved and each was independently reviewed by two authors (LS and RB). Publications in Chinese were selected after review of the English abstract. Unpublished data were not sought, as the task of summarising existing published data was extensive and the International SARS Treatment Group indicated that much of the clinical data had already been published. We used the QUOROM checklist to help ensure the quality of this review (Table S3). Data from the full text of studies in English were extracted independently by two authors (LS and RB). Data from the Chinese literature were extracted with the assistance of a translator. Because the Chinese articles were reviewed by only one author, the consistency of the translated information with that from English articles was maintained by subsequent discussion with the translator to verify the extracted data. We established explicit criteria to assess the level of evidence for each human treatment study (Box 1). Since the treatments chosen for evaluation were often given in combination, evidence was classified by the treatment that was given to all patients in the cohort or given to some with the author's intention of studying its effects. If putative effects within a study included several drugs, then we extracted data for each intervention. The level of evidence was independently classified by two authors (LS and RB). Chinese studies were appraised and classified in the same way using translated information extracted from each report. Discrepancies were resolved by consensus. Box 1. Categories of Evidence Defined for In Vivo Studies of Treatments in SARS Patients “Inconclusive” if a study could not be used to inform a decision about treatment efficacy due to having either outcomes which were not reported consistently, an inconsistent treatment regimen, no control group or a control group which was a likely source of bias. A control group was considered a likely source of bias if there were differences in co-morbidities, sex, age and markers of severe disease compared to the treatment group. “Possible harm” if a study reported adverse effects of treatment that were consistent with adverse effects reported with the use of the drug in the treatment of other conditions. Evidence of direct causality was not required. A study could be classified as suggesting possible harm from the drug even if the study had methodological weaknesses. “Possible benefit” if a study had evidence of benefit for an important outcome measure which was recorded consistently (e.g., case fatality, need for mechanical ventilation, duration of hospitalization, frequency of ARDS) in patients treated in a defined way compared to a valid control group. A control group was considered valid if randomized, or if patient characteristics and illness severity were comparable to the treatment group. Evidence of direct causality was not required. “Definite harm” if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. “Definite benefit” if a study contained statistically significant evidence of harm demonstrated in a double-blind randomized trial, which did not contain serious methodological weaknesses. Results In vitro evidence was available in 15 studies. Clinical evidence of SARS treatment in humans was reported in 54 studies (37 in English, 17 in Chinese). Three studies addressed treatment of ARDS (Figure 1). Ribavirin In vitro. We found six studies that described the antiviral effect of ribavirin in vitro (Table S4); four showed an antiviral effect (Table S5). A synergistic antiviral effect between ribavirin and type I IFN (IFN-β1a or leukocytic IFN-α) was described in two studies performed in human cell lines and Vero cell lines [12,13]. In SARS patients. We found 24 studies that described ribavirin treatment in cohorts larger than ten patients (Table S6). Our formal assessment classified 20 studies as “inconclusive,” due to study design or because the effect of ribavirin could not be distinguished from the effects of other treatments (such as steroids and antiviral drugs). Four publications presented evidence of possible harm (14–17). Three of these studies, each of which included over 100 patients, documented a fall in haemoglobin levels after ribavirin treatment when compared to levels in patients before treatment [14–16]. Of patients treated with ribavirin, 49/138 to 67/110 (36%–61%) developed haemolytic anaemia, a recognised complication with this drug, although it is not possible to rule out the possibility that SARS-CoV infection caused the haemolytic anaemia, as there is no control group. One study noted that over 29% of SARS patients had some degree of liver dysfunction indicated by ALT levels higher than normal, and the number of patients with this complication increased to over 75% after ribavirin treatment (Table S7) [17]. In the Chinese literature six additional reports described patients with SARS treated with ribavirin (often with steroids). These six reports were determined to be inconclusive in the evaluation of treatment for SARS (Tables S8 and S9). LPV/r In vitro. Of three studies, two demonstrated that lopinavir inhibits cytopathic effects of SARS-CoV in fetal rhesus monkey kidney cells (Table S4). One study showed detectable but reduced activity in Vero-E6 cells [13], and one study concluded that neither lopinavir nor ritonavir had an effect [18]. A synergistic effect of lopinavir with ribavirin has been reported (Table S5). In SARS patients. We found two studies of LPV/r (lopinavir 400 mg with ritonavir 100 mg orally every 12 h) in cohorts larger than ten patients (Table S6). Patients also received ribavirin and corticosteroids. LPV/r use was compared among three groups of patients: those who received it as an early SARS treatment, those who received it as a late treatment, and those who did not receive it at all. When LPV/r was added as an initial treatment to ribavirin and corticosteroid therapy, the death rate was lower than among those who received ribavirin and corticosteroids (1/44 [2.3%] versus 99/634 [15.6%]; p < 0.05) [19]. A second study of this regimen reported fewer episodes of ARDS or death compared with historical controls who had not received LPV/r (1/41 [2.4%] versus 32/111 [28.8%]; p < 0.001) (Table S7) [20]. Both studies were determined to be inconclusive due to possible bias in the selection of control group or treatment allocation. No additional studies were identified from the Chinese literature. Corticosteroids In vitro. No studies were found on the cytopathic effect of corticosteroids alone against SARS-CoV. Corticosteroids act as immunomodulatory agents, and therefore studies to measure direct antiviral effects in vitro were not expected. In SARS patients. Fifteen articles examined corticosteroid treatment in ten or more patients. Of these cohorts 13 were also treated with ribavirin (Table S6). We determined that 13 of the 15 studies were inconclusive. Of these, in an uncontrolled and nonrandomised study, 95/107 (89%) of patients treated with high-dose methylprednisolone (0.5–1 mg/kg prednisolone on day 3 of illness, followed by hydrocortisolone 100 mg every 8 h, and pulse-doses of methylprednisolone 0.5 g IV for 3 d) after the first week of illness recovered from progressive lung disease (Table S7) [16]. Two studies contained evidence of possible harm from corticosteroids [21,22]. One measured SARS-CoV plasma viral load across time after fever onset in a randomized, double-blind, placebo-controlled trial; corticosteroid use within the first week of illness was associated with delayed viral clearance. The other study, which was case-controlled, found that patients with psychosis received higher cumulative doses of steroids than patients without psychosis (10,975 mg versus 6,780 mg; p = 0.017) [22]. In the Chinese literature, we found 14 reports in which steroids were used (Table S8 and Table S9). Twelve studies were inconclusive and two showed possible harm. One study reported diabetes onset associated with methylprednisolone treatment [23]. Another study (an uncontrolled, retrospective study of 40 SARS patients) reported avascular necrosis and osteoporosis among corticosteroid-treated SARS patients [24]. In ARDS patients. Three clinical trials examined the effect of corticosteroids on mortality in patients with established ARDS (Table S10). In two trials, high-dose methylprednisolone given for approximately 2 d was not effective for early ARDS [25,26]. One small RCT that used a regimen of lower dose methylprednisolone (2 mg/kg per day), tapered after 2 wk, showed possible evidence of ARDS improvement (Table S11) [27]. IFN Type I In vitro. Twelve in vitro studies with data on the antiviral effect of IFN type I have been reported, and all demonstrated an antiviral effect against SARS-CoV (six for IFN-α and ten for IFN-β) (Tables S4 and S5). Antiviral effects have been demonstrated in monkey (Vero; Vero-E6), fetal rhesus monkey kidney (fRhK-4), and human (Caco2, CL14, and HPEK) cell lines. Three reports presented evidence that IFN-β was superior against SARS-CoV compared to IFN-α and found rIFN-α2 virtually ineffective against SARS-CoV compared to other IFNs [28]. Synergistic effects were reported for leukocytic IFN-α with ribavirin [13], IFN-β with ribavirin [12,13] and IFN-β with IFN-γ [28,29]. In SARS patients. Two studies of IFN-α given with steroids and/or ribavirin were reported (Table S6). No significant difference was seen in outcome between IFN-α treatment group and those treated with other regimens. Results of both studies were inconclusive due to a lack of a consistent treatment regimen or suitable control group (Table S7). In the Chinese literature, one additional study reported the use of IFN-α as part of a regimen that included ribavirin and steroids [30]. We determined this study to be inconclusive because a variety of treatments given masked the effect of IFN-α alone (Table S8 and Table S9). Convalescent Plasma or Immunoglobulin In vitro. No studies were found on the cytopathic effect of this treatment on SARS-CoV. Convalescent plasma and IVIG act as immunomodulatory agents and therefore studies to measure direct antiviral effects in vitro were not expected. In SARS patients. Five studies of either IVIG or convalescent plasma treatment given in addition to steroids and ribavirin were reported for treatment of SARS (Table S6). These studies were inconclusive, because the effect of convalescent plasma or IVIG could not be discerned from effects of patient comorbidities, stage of illness, or effect of other treatments (Table S7). In the Chinese literature, two additional studies reported evidence on the effect of convalescent plasma as a treatment for SARS [30,31]. These studies were inconclusive (Table S8 and Table S9). Evidence collected on the benefit or harm of drugs used to treat SARS is summarized in Table 1. Discussion The rapid spread and subsequent control of SARS precluded controlled clinical treatment trials during the outbreak of 2002–2003. In this report we summarize the results of a systematic evaluation of the findings from published reports of treatments used for SARS during the epidemic. Publications from the Chinese literature were included to capture as much evidence as possible. We developed specific criteria (Box 1) to look for large, obvious effects of benefit, adverse or poor outcomes, or evidence of potential benefit that could be used to prioritise future research of SARS treatments. A summary of this evidence in SARS patients is shown in Table 1. Despite thirty reports of SARS-infected patients treated with ribavirin, there is no convincing evidence that it led to recovery. Haemolytic anaemia, a recognized side effect of this treatment, was observed in three studies. We would infer from these findings that any future use of ribavirin for SARS should be within the context of a controlled trial with close attention given to adverse effects. Corticosteroids were commonly prescribed to SARS patients with worsening pulmonary disease or progressing abnormalities on chest X-rays. Treatment regimens varied widely but can be classified into two groups, early treatment and rescue treatment given at a later stage of illness. It is difficult to make a clear recommendation about whether corticosteroids should be used to treat SARS-associated lung injury in any stage of illness, particularly as the drug is immunosuppressive and may delay viral clearance if given before viral replication is controlled [21]. Of added concern are infectious complications, avascular necrosis, and steroid-induced psychosis—recognized adverse effects of corticosteroid use. Fungal superinfection and aspergillosis have been noted in case reports and autopsy findings of SARS patients given corticosteroids at high doses or for prolonged periods [32,33]. This review has found evidence of avascular necrosis and steroid-induced psychosis in SARS patients. Seven studies of treatment with convalescent plasma or IVIG, three with IFN type I, and two with LPV/r were inconclusive by the criteria used in our analyses. Authors of four of the IVIG studies commented that patients seemed to improve upon treatment, but that more controlled trials of this approach are needed to provide evidence of an effect for SARS. Important caveats should be considered in this review. Most of the studies of SARS patients were descriptions of the natural course of the disease and had not been designed to reliably assess the effects of the treatments used. Patient characteristics such as age and presence of diabetes mellitus have been associated with severe disease and can confound treatment effects. A diagnostic test for early SARS illness was not validated or widely available, and in general, treatment was initiated once patients fulfilled a clinical and epidemiological case definition. It is possible that the inclusion of patients without laboratory confirmation of SARS-CoV infection in this review could cause an underestimate of any true effect of antiviral treatment on SARS. The variation in treatment regimens—particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids—is a major obstacle to a clear interpretation of the data in this review. The nonstandardised collection of clinical information limits the conclusions that can be drawn from a retrospective analysis. We suggest that, in the event of a future outbreak of SARS-CoV or another novel agent, attempts be made to develop treatment protocols and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings. As observational studies pose problems of interpretation, the need is great for good-quality randomised trials, despite the difficulties in organising such trials. Supporting Information Table S1 Rationale for Treatments and Recognized Adverse Effects (55 KB DOC) Click here for additional data file. Table S2 Method of Systematic Review (A) Search strategy, step 1: Select the treatments. (B) Search strategy, step 2: Narrow the scope. (C) Inclusion criteria and information sought from each study. (45 KB DOC) Click here for additional data file. Table S3 QUOROM Statement (50 KB DOC) Click here for additional data file. Table S4 Description of SARS-CoV Replication Studies: Assay Type and Outcomes Measured (79 KB DOC) Click here for additional data file. Table S5 Results from SARS-CoV Replication Studies: Inhibition of SARS-CoV Replication (84 KB DOC) Click here for additional data file. Table S6 Description of Studies within SARS Patients (186 KB DOC) Click here for additional data file. Table S7 Results of Treatment within SARS Patients (English literature) (183 KB DOC) Click here for additional data file. Table S8 Description of Studies of SARS Patients (Chinese Literature) (108 KB DOC) Click here for additional data file. Table S9 Results of Treatment within SARS Patients (Chinese Literature) (93 KB DOC) Click here for additional data file. Table S10 Description of Studies of ARDS or ALI (50 KB DOC) Click here for additional data file. Table S11 Results of Treatment of ARDS or ALI (48 KB DOC) Click here for additional data file.
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              Avian flu: influenza virus receptors in the human airway.

              Although more than 100 people have been infected by H5N1 influenza A viruses, human-to-human transmission is rare. What are the molecular barriers limiting human-to-human transmission? Here we demonstrate an anatomical difference in the distribution in the human airway of the different binding molecules preferred by the avian and human influenza viruses. The respective molecules are sialic acid linked to galactose by an alpha-2,3 linkage (SAalpha2,3Gal) and by an alpha-2,6 linkage (SAalpha2,6Gal). Our findings may provide a rational explanation for why H5N1 viruses at present rarely infect and spread between humans although they can replicate efficiently in the lungs.

                Author and article information

                Role: M.D.
                Role: M.D., Ph.D.
                Am J Pathol
                The American Journal of Pathology
                American Society for Investigative Pathology
                May 2008
                : 172
                : 5
                : 1155-1170
                From the Department of Pathology and Infectious Disease Center, School of Basic Medical Sciences, Peking (Beijing) University, Beijing, China
                Copyright © American Society for Investigative Pathology
                : 18 December 2007



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